Patent Application
20200315974
The present invention relates to a use of a pharmaceutical composition for regulating fibroblast growth.
Chinese patent ZL 02105541.6 discloses a pharmaceutical composition suitable for oral administration, comprising a homogenous mixture of edible oil, beeswax and β-sitosterol, wherein the beeswax in the composition forms microcrystals, the content of the beeswax is 0.5 to 50% and the content of the β-sitosterol is at least 0.1% by weight based on the total weight of the composition. In addition, the composition may also comprise other pharmaceutical ingredients, and is used to deliver other active ingredients to the gastrointestinal tract for treating various diseases.
Moreover, this pharmaceutical composition is mainly used to protect mucosal tissues from damage caused by irritants, and to promote the repair and regeneration of damaged or dysfunctional gastrointestinal mucosal tissues. It is particularly used for the treatment of gastrointestinal disorders such as gastritis, peptic ulcer, reflux esophagitis, dyspepsia and gastric cancer, as well as for the reconstruction of the physiological structure and function of mucosal tissues.
In the present application, “pharmaceutical composition”, “pharmaceutical composition according to the present invention” or “the present pharmaceutical composition” refers to a pharmaceutical composition comprising a homogenous mixture of edible oil, beeswax and β-sitosterol, wherein the beeswax in the composition forms microcrystals, the content of the beeswax is 0.5 to 50% and the content of the β-sitosterol is 0.1 to 20% by weight based on the total weight of the composition.
Fibroblasts are the main cells in the dermis of the skin, which are called fibrocytes at resting state. They are a kind of low-metabolism, inactive cells under normal condition. These “hibernated” cells are sparsely distributed in connective tissue, and the density of the cell is high merely in the soft tissue around the blood vessels. Fibroblasts have obvious functional diversity, and are key cells for organ and tissue fibrosis, scar formation and tissue aging.
About Organ and Tissue Fibrosis
The pathogenesis of organ and tissue fibrosis has evolved from the changes of histopathology in the past to the research of cell and molecular biology at present. Although the specific pathogenesises of fibrosis in different organs and tissues are somewhat different, they have many commonalities. Fibrosis is a slow, dynamic and complex process, involving the interaction and inter-adjustment of various factors and links such as cells, cytokines and extracellular matrix (ECM). The final outcome is the deposition of a large amount of ECM[1]. Organ and tissue fibrosis starts from the substantial cell damage caused by various reasons (inflammation, immunity, poison, ischemia, hemodynamic changes and the like), followed by inflammatory degeneration and necrosis of cells, which activates the corresponding macrophages (pre-inflammatory cells) to release various active factors such as cytokines and growth factors, the latter activates resting extracellular matrix producing cells (ECM-producing cells), which then transform into myofibroblasts (MFB). Myofibroblasts proliferate and secrete cytokines, and then act on macrophages by paracrine. Myofibroblasts synthesize large amounts of ECM components such as collagen, while ECM degradation is reduced, resulting in organ and tissue fibrosis. In such pathogenesis, the conversion of ECM-producing cells into myofibroblasts is a core event and a necessary step for organ and tissue fibrosis and scar formation, wherein it is determined that the cell type of ECM-producing cells is mainly fibroblast, and there are also other types of cells involved. Their role in the pathogenesis of fibrosis has been the focus of research in recent years. Normal fibroblast is at resting state, its metabolism and function are not active. However, under pathological conditions, fibroblast not only changes in morphology, but also shows obvious functional changes such as proliferation, secretion of cytokines, synthesis of large amounts of ECM, and production of protein degrading enzymes. The series of changes is called activation. The activation of ECM-producing cell is a key step and core link in the formation of fibrosis.
Studies on the activation of fibroblasts indicate that under normal conditions, they are a kind of non-activated cells with low metabolism. When the fibroblast is stimulated, it is transformed from proliferative cell into cell that proliferates and overproduces matrix, i.e. fibroblast activation occurs. The activated fibroblast undergoes functional and phenotypic changes and is transformed into myofibroblast that expresses α-smooth muscle actin (α-SMA), the ability of the latter to synthesize ECM is significantly improved. It is thought that fibroblast activation is a key step during the formation of fibrosis, factors such as growth factors and cytokines by autocrine and paracrine, direct cell-to-cell contact, the effect of ECM via integrin, and local tissue hypoxia may stimulate fibroblast activation.
Myofibroblasts transformed from fibroblasts are a group of smooth muscle cell-like cells[2], which have α-SMA in the cytoplasm. The myofibroblast has similar functions. The nuclear membrane is wrinkled, and the cytoplasm contains a large number of bundled filaments, actin, and expanded rough endoplasmic reticulum. The myofibroblast mainly expresses α-SMA, which is a marked antigen of active myofibroblast. The myofibroblast plays a biological role by secreting cytokines, chemokines, growth factors, ECM and proteases. The myofibroblast and fibroblast are very closely related. In summary, the activated fibroblast can undergo phenotypic changes and transform into myofibroblast; the myofibroblast has both the function and structural characteristics of fibroblast and smooth muscle cell, and can express α-SMA; the expression of α-SMA by fibroblast is a sign of its activation and damage.
A large number of studies have demonstrated that the pathogenesis of organ and tissue fibrosis is extremely complicated, involving the proliferation and activation of ECM-producing cell, the generation, release and regulation of various growth factors, cytokines and vasoactive substances, as well as the balance between ECM synthesis and degradation and the like. Therefore, the occurrence and development of organ fibrosis is a very complicated process involving the interaction and regulation of multiple factors such as cells, cytokines and ECM. Since the pathogenesis of fibrosis is extremely complicated and involves many factors, there should be more than one target for treatment. There are currently three most important targets for the treatment of fibrosis: ECM-producing cell, cytokine and ECM, and the most important of which is ECM-producing cell.
Recent studies have demonstrated that inhibiting the proliferation and activation of ECM-producing cell is the central strategy for anti-fibrosis. For example, it was found that γ-interferon, retinoic acids, cAMP inducers, endothelin receptor antagonists and certain antioxidants can inhibit the activation of hepatic stellate cell (an ECM-producing cell). However, these are mostly laboratory research results, which are still far from clinical application. Inducing the apoptosis of activated ECM-producing cell is an important target for anti-fibrosis. However, the method of selectively promoting the apoptosis of ECM-producing cell is not yet ideal at present.
About Scar Formation
Fibroblasts are the main cells that synthesize collagen in the body, and are closely related to scar formation. Among the various types of collagen synthesized by fibroblast, the type I and type III collagen are most closely related to scar formation. Various types of scar fibroblasts all have the characteristic of increased collagen synthesis, since the activity of collagen synthase is significantly enhanced. For example, the activity of collagen synthase proline hydroxylase in keloid is significantly higher than that in hypertrophic scar, and its collagen synthesis is 20 times that of normal skin and 3 times that of hypertrophic scar.
It is reported of using silver staining nucleolar tissue area method to determine the activity and proliferation of fibroblast. It was found that the cultured normal skin fibroblast, hypertrophic scar fibroblast and keloid fibroblast have significant differences in activity and proliferation, and the latter being the strongest. The proliferation activity of fibroblast is also different in different tissues. The test of proliferating cell nuclear antigen (PCNA) found that the PCNA positive rate of fibroblasts in normal skin was 38.8%±6.3%, while that of hypertrophic scar was 45.6%±10.6%, and that of keloid was as high as 75.1%±8.1%.
The keloid fibroblasts, hypertrophic scar fibroblasts and normal skin fibroblasts were cultured to observe the relationship between fibroblast and scar formation. The expression of PCNA, p16 protein (which can induce the cell to stop at G1 phase), type I collagen, type III collagen and corresponding procollagen genes were determined before and after cell contact. The proliferation and biosynthetic function of various fibroblasts were studied. The results showed that when the cells did not contact with each other, the keloid fibroblast, hypertrophic scar fibroblast and normal skin fibroblast all expressed PCNA, and hardly expressed p16. Once the fibroblasts contacted with each other, the normal skin fibroblast expressed less PCNA obviously or did not express PCNA, while p16 expression increased, and the cells appeared as a monolayer with directional arrangement, indicating that the normal skin fibroblast had contact inhibition and density inhibition. The keloid fibroblast still expressed high PCNA, while p16 expression was insignificant, and the cells appeared as a disordered arrangement with overlapping and stratification, indicating that the keloid fibroblast did not have contact inhibition and density inhibition. The hyperplastic scar fibroblast was in between. After cell contact, the synthesis of type I collagen and type III collagen in normal skin fibroblasts decreased significantly, accompanied by a significant decrease in the expression of corresponding procollagen genes. However after cell contact, the biosynthesis in keloid fibroblast and hypertrophic scar fibroblast was still vigorous, and type I and III collagen genes were still strongly expressed.
Many scholars have conducted in-depth studies on the growth and proliferation characteristics of scar tissue fibroblasts in vitro using cell culture techniques. The characteristics between normal skin fibroblast and keloid fibroblast were compared using this technique. It was found that there was no obvious difference in their morphological size and their growth kinetics. However, the division of keloid fibroblast may require less stimulation of growth factor.
The relationship between growth factor and scar is also very important. It is known at present that transforming growth factor β1 (TGFβ1) is a very important growth factor. Connective tissue growth factor (CTGF) is a polypeptide with platelet-derived growth factor (PDGF)-like activity, which is synthesized by skin fibroblasts after being activated by TGF. When skin fibrosis occurs, CTGF is positive. Abnormal expression of CTGF gene will cause skin sclerosis and fibrosis. In addition, TCiFβ1 is currently the only CTGF inducer confirmed. This also illustrates the role of TGF in fibrous pathology.
The understanding on the regulation of cell cycle provides a theoretical basis for specifically controlling the wound healing process. People now hope to control the pathological fibrosis process of scar by regulating the growth and proliferation of fibroblast.
The biggest physiological significance of fibroblast is tissue repair[3-5]. The normal function of fibroblast is one of the key factors for the successful completion of tissue repair. It is known that fibroblast is the effector cell of scar formation, and the abnormal proliferation of fibroblast is the main reason for the excessive proliferation and persistent existence of pathological scar. The current point of view has focused on the basic point of the imbalance of fibroblast proliferation/apoptosis (apoptosis refers to the natural death of the cell, which is initiated by the internal mechanism of the cell and is very important for maintaining physiological balance). It is demonstrated that the pathological healing of skin damage is due to the excessive proliferation of fibroblast. During the remodeling phase of skin repair, the proliferation and apoptosis of fibroblast present in the bud tissue are dysregulated. The proliferation activity of fibroblast is significantly higher than that of normal skin fibroblast, while the apoptosis of fibroblast lags behind the cell proliferation. This causes the accumulation of fibroblasts during the tissue repair process, resulting in the synthesis of more type I collagen, an increased ratio of type I/III collagen (opposite to the phenomenon of decreased ratio of type I/III collagen during wound healing process) and a pathological scar healing. Effective regulation of the balance between proliferation/apoptosis of fibroblast is the key to inhibit the formation of pathological scar.
The technical problem to be solved by the present invention is to regulate fibroblast growth and inhibit organ fibrosis by using the above known pharmaceutical composition.
In an aspect, the present invention provides a use of a pharmaceutical composition for regulating fibroblast growth, wherein the pharmaceutical composition is a pharmaceutical composition suitable for oral administration comprising a homogenous mixture of edible oil, beeswax and β-sitosterol, wherein the beeswax in the composition forms microcrystals, the content of the beeswax is 0.5 to 50% and the content of the β-sitosterol is 0.1% to 20% by weight based on the total weight of the composition.
The “regulating fibroblast growth” refers to inhibiting fibroblast growth and reproduction and inactivating fibroblast.
In another aspect, the present invention provides a use of a pharmaceutical composition in the preparation of medicaments for treating or preventing organ fibrosis, scar formation and/or tissue aging, wherein the pharmaceutical composition is a pharmaceutical composition suitable for oral administration comprising a homogenous mixture of edible oil, beeswax and β-sitosterol, wherein the beeswax in the composition forms microcrystals, the content of the beeswax is 0.5 to 50% and the content of the β-sitosterol is 0.1% to 20% by weight based on the total weight of the composition.
In a specific embodiment, the organ comprises heart, liver, lungs, kidneys and bone marrow.
In a specific embodiment, the organ is an organ from a mammal, and the mammal is preferably a human.
In a specific embodiment, the content of the β-sitosterol in the pharmaceutical composition is 0.5 to 20% by weight.
In a specific embodiment, the content of the β-sitosterol in the pharmaceutical composition is 1 to 10% by weight.
In a specific embodiment, the content of the beeswax in the pharmaceutical composition is 3 to 30% by weight.
In a specific embodiment, the content of the beeswax in the pharmaceutical composition is 5 to 20% by weight.
In a specific embodiment, the content of the beeswax in the pharmaceutical composition is 6 to 10% by weight.
In a specific embodiment, the edible oil in the pharmaceutical composition is corn oil, wheat germ oil, soybean oil, rice bran oil, rapeseed oil, sesame oil or fish oil.
In a specific embodiment, the pharmaceutical composition further comprises propolis, and the content thereof is 0.1 to 30% by weight.
In a specific embodiment, the pharmaceutical composition comprises water, and the content thereof is less than or equal to 1% by weight.
In a specific embodiment, the dosage form of the oral pharmaceutical composition is selected from the group consisting of tablet, pill, capsule, emulsion, gel, syrup and suspension.
In a specific embodiment, the pharmaceutical composition further comprises Scutellaria baicalensis or the extract of Scutellaria baicalensis, and the content of Scutellaria baicalensis or the extract of Scutellaria baicalensis containing 0.1 to 0.5% of baicalin is 2 to 5% by weight based on the total weight of the composition.
The extract of Scutellaria baicalensis is an extract of Scutellaria baicalensis with water, organic solvent such as oil and ethanol, or a combination of water and organic solvent. More preferably, the extract is an extract of 1 to 50% by weight of Scutellaria baicalensis in an edible oil, preferably sesame oil. The radix of Scutellaria baicalensis is preferred. Scutellaria baicalensis is one or more Labiatae plants selected from the group consisting of Scutellaria viscidula bunge, Scutellaria ainoena, Scutellaria rehderiana Diels, Scutellaria ikonnikovii Juz, Scutellaria likiangensis and Scutellaria hypericifolia.
In a specific embodiment, the pharmaceutical composition further comprises Cortex Phellodendri or the extract of Cortex Phellodendri, and the content of Cortex Phellodendri or the extract of Cortex Phellodendri containing 0.1 to 1% of obaculactone is 2 to 5% by weight based on the total weight of the composition.
The the extract of Cortex Phellodendri is an extract of Cortex Phellodendri with water, organic solvent such as oil and ethanol, or a combination of water and organic solvent. More preferably, the extract is an extract of 1 to 50% by weight of Cortex Phellodendri in an edible oil, preferably sesame oil. The cortex of Cortex Phellodendri is preferred. Cortex Phellodendri is one or more plants selected from the group consisting of Phellodendron chinense Schneid, Phellodendron amurense, Phellodendron chinense Schneid var. omeiense, Phellodendron Schneid var vunnanense and Phellodendron chinense Schneid var falcutum.
In a specific embodiment, the pharmaceutical composition further comprises 2 to 5% of Coptis chinensis or the extract of Coptis chinensis containing 0.1 to 1% of berberine by weight based on the total weight of the composition.
The extract of Coptis chinensis is an extract of Coptis chinensis with water, organic solvent such as oil and ethanol, or a combination of water and organic solvent. Preferably, the extract is an extract of 1 to 50% by weight of Coptis chinensis in an edible oil, preferably sesame oil. The radix of Coptis chinensis is preferred. Coptis chinensis is one or more Ranunculaceae plants selected from the group consisting of Coptis deltoidea C. Y. Cheng et Hsial, Coptis omeiensis and Coptis teeta Wall.
In a specific embodiment, the pharmaceutical composition further comprises 2 to 5% of Scutellaria baicalensis or the extract of Scutellaria baicalensis containing 0.1 to 0.5% of baicalin, 2 to 5% of Cortex Phellodendri or the extract of Cortex Phellodendri containing 0.1 to 1% of obaculactone, 2 to 5% of Coptis chinensis or the extract of Coptis chinensis containing 0.1 to 1% of berberine, 2 to 10% of Pericarpium Papaveris or the extract of Pericarpium Papaveris containing 0.1 to 1% of narcotoline, and 2 to 10% of earthworm or earthworm extract containing amino acid, by weight based on the total weight of the composition.
The extract of Pericarpium Papaveris is an extract of Pericarpium Papaveris with water, organic solvent such as oil and ethanol, or a combination of water and organic solvent. Preferably, the extract is an extract of 1 to 50% by weight of Pericarpium Papaveris in an edible oil, preferably sesame oil.
The earthworm extract is an extract of earthworm with water, organic solvent such as oil and ethanol, or a combination of water and organic solvent. More preferably, the extract is an extract of 1 to 50% by weight of earthworm in an edible oil.
The extraction of Scutellaria baicalensis, Cortex Phellodendri, Coptis chinensis, Pericarpium Papaveris and earthworm can be carried out according to the method described in Chinese Patent ZL 93100276.1 or Chinese Patent ZL 02105541.6.
In a specific embodiment, the pharmaceutical composition comprises 7% of beeswax, 1% of sterol, 0.5% of obaculactone, 0.3% of baicalin and 0.5% of berberine by weight based on the total weight of the composition.
In a specific embodiment, the beeswax has microcrystals with a length of 0.1 to 100 microns.
In a specific embodiment, at least two microcrystals of the beeswax in the pharmaceutical composition are polymerized into a microcrystal complex.
In a specific embodiment, the microcrystals of the beeswax are sufficiently uniformly dispersed in the edible oil.
It was determined in the skin graft of suckling mouse that the pharmaceutical composition of the present invention has a very significant inhibition effect on fibroblast. Fibroblasts growing out of the grafts were significantly inhibited by the pharmaceutical composition of the present invention, and changed in morphology. The cells turned black and shrinked until they died and dissolved. The effect of the pharmaceutical composition on fibroblasts that just grow out of the skin graft can also be observed under the microscope. In fact, the cells are inhibited at the early stage of growth without a chance of diffusion and growth. With respect to a large amount of fibroblasts growing in a short period of time, the cells will not be immediately inhibited or die due to the temporary balance of the pharmaceutical composition and fibroblast. After a certain period of time, the cells will eventually be inhibited by the pharmaceutical composition until they died, which is directly related to the amount of the pharmaceutical composition. With respect to fibroblasts that have grown and multiplied before the addition of the pharmaceutical composition, when the pharmaceutical composition is added, it takes time for the pharmaceutical composition to function, and the effect becomes more and more obvious over time.
It is determined in human fibroblasts that the pharmaceutical composition of the present invention can inhibit human fibroblast in vitro, and significantly inhibit human fibroblast growth.
The present invention is further illustrated by the following examples in combination with the figures, which should not be construed as a limitation to the present invention. Specific materials and sources thereof used in the embodiments of the present invention are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention. Materials that are the same as or similar to the following reagents and instruments in type, model, quality, properties or function can also be used in the embodiment of the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents and the like used in the following examples are commercially available.
The pharmaceutical composition was prepared according to the content disclosed in Examples 1 and 2 of Chinese Patent ZL 02105541.6.
Briefly, step 1: the refined sesame oil and Scutellaria baicalensis (100 kg: 5 kg) were added to a reaction tank and heated. Heating was stopped when the temperature reached 120° C., and the mixture was kept warm for 50 minutes with stirring. The mixture was filtrated to remove the dregs, the obtained extraction was the medicinal oil I.
Step 2: the medicinal oil I was added to another reaction tank and heated. When the temperature reached 85° C., the refined beeswax was added at a ratio of 93 kg of medicinal oil I:7 kg of beeswax, and stirred well. Heating was stopped when the temperature reached 120° C., kept stirring the warm mixture for 20 minutes to obtain the medicinal oil II.
Step 3: the medicinal oil II was grinded using a colloid mill with a pitch of 0.6 to 0.8 mm and an output speed of 15 Kg/15 min. Alternatively, the medicinal oil II could also be homogenized at 40±2° C. for 15 to 20 minutes using a homogenizer with a rotate speed of 6000 to 10000 rpm. The homogenate was stirred at 100 rpm, vacuumized to below 0.09 MP, cooled to 40±2° C., and kept warm for 50 minutes. When the temperature decreased to 20° C. and the vacuum degree reached 0.6 to 0.8 MP, the mixture was kept for 20 minutes to obtain the pharmaceutical composition.
According to Example 2 of Chinese Patent ZL 02105541.6, the active ingredients of the pharmaceutical composition prepared by the above method are shown as follows:
1. Materials and Methods
1.1 Instruments, Devices, Materials and Reagents
Quantitative imaging analytical flow cytometry (Amnis®ImageStreamxMarkII, Merk Millipore, USA, provided by High-throughput Single Cell Analysis Platform of Phoenix Project of Peking University); ultrapure water system (Milli-Q, Millipore, USA); two-stage reverse osmosis purified water system (Beijing Innogreen Technology Co., Ltd.); electronic scale (AB135-S, Mettler-Toledo, Switzerland); electronic scale (ES-1000HA, Changsha Xiangping Technology Development Co., Ltd.); high-speed refrigerated centrifuge (J20-XP, Beckman-Coulter, USA); desktop high speed refrigerated centrifuge (1-15K, Sigma, Germany); biological clean bench (BCN-1360B, Beijing HDL Apparatus Co., Ltd.); CO2 incubator (Forma3111, Thermo Fisher Scientific, USA); hybridization oven (Maxi14, Thermo Fisher Scientific, USA); particle ice machine (SIM-F124, Sanyo, Japan); electronic constant temperature water bath (CS501-3C type, Chongqing Sida Experimental Instrument Co., Ltd.), drying oven (Chongqing Sida Experimental Instrument Co., Ltd.); inverted microscope (Nikon TE2000U, Nikon, Japan); microscopic imaging system (Nikon DXM 1200, Nikon, Japan); ordinary optical microscope (BK1201, Chongqing Optical Instrument Factory); micro-sampler (1000 μl, 200 μl, 20 μl, 10 μl, Gilson, France); 6-well culture plate; 15 ml centrifuge tube; 1 ml and 0.5 ml Eppendorf centrifuge tube; dripper; sterilized small surgical instrument; sterilized small beaker; sterilized small flask with lid; sterilized culture dish (φ5 cm, (φ6 cm); 0.22 μm microporous membrane; needle filter; capped triangular flask; capped small test tube; needle; DMEM medium (GIBCO, Invitrogen Corporation, USA, packaged by Beijing Xinjingke Biotechnology Co., Ltd.); 7-AAD staining solution (provided by High-throughput Single Cell Analysis Platform of Phoenix Project of Peking University); super fetal bovine serum (Hangzhou Sijiqing Biological Engineering Material Co., Ltd.); type I mouse tail collagen (Cat. No. C8062, Solarbio Co.); PBS (self-made, stored at 4° C., and used in an ice bath); Hank's balanced salt solution (self-made, stored at 4° C., and used in an ice bath); 75% ethanol (self-made); 8% Na2S (self-made); penicillin sodium and streptomycin sulfate for injection; trypsin-EDTA cell digest solution (self-made); portable refrigerator.
1.2 Method[6-9]
1) Each well of two 6-well plates was coated with 60 μl of type I mouse tail collagen. The plate was laid flat, sealed with tape, and placed in a stainless steel box. The box was placed in the Maxi14 hybridization oven at 37° C. overnight, and stored at 4° C.
2) A Kunming suckling mouse (5 days old, purchased from Laboratory Animal Breeding Base of Academy of Military Medical Sciences) was placed on a foam board, and wiped with a cotton ball (dipped with 8% Na2S by tweezers) several times to remove hair, especially trunk hair.
3) The mouse was rinsed with deionized water, and killed by breaking its neck.
4) The mouse was placed in 75% ethanol for 2 min, 75% ethanol for 5 min, and turned over frequently.
5) The suckling mouse was taken out, and placed in a sterile plate after the ethanol was removed completely.
6) The skin, especially trunk skin, was cut as much as possible. The collected skin was placed in another sterile plate.
7) The sterile skin was spread in the plate with the dermis upward. Tissues such as subcutaneous fat were removed by ophthalmic tweezers.
8) The skin was placed in a sterile flask with lid, and added with 10 ml of cold PBS, 60 μl of penicillin and 60 μl of streptomycin. The skin was turned, stirred and rinsed well.
9) The above step was repeated 4 times with a total of 5 times.
10) The skin was washed with cold Hank's solution+two antibiotics twice.
11) The above suckling mouse skin (washed 7 times) was placed in the sterile flask with lid, added with 10?4) FBS DMEM medium (cold), and cut into skin grafts later.
12) The 6-well plate (coated with type I mouse tail collagen on the previous day) was washed with cold PBS 3 times. PBS was removed, and each well was added with 1 ml of 10% FBS DMEM medium. The 6-well plate was shaked and placed still.
13) The skin obtained in Step 11 was placed in a sterile plate, and cut into full-thickness skin grafts (about 1 mm2) by sterile ophthalmic curved scissors (note: the skin grafts were obtained, and moisturized by the culture medium). The plate was covered, and should not let it dry.
14) The 10% FBS DMEM medium was removed from the wells of the plate in Step 12 (note: some medium was remained on the bottom of the well as the nutrition for the pre-adherence of graft).
15) The skin grafts obtained in Step 13 were averagely placed into each well (A1, A2, A3, B1, B2, B3) of the two 6-well plates. About 10 pieces of skin grafts were placed into one well by straight tweezers under microscope. The skin graft was spread by straight tweezers under microscope, and pressed to adhere closely. Each skin graft was located in the center of the well, and the area of the spread graft in each well was equal (adjustment was carried out if there was a difference). After checking the adherence of each graft, the plate was covered and sealed with tape except for one side (for the entrance of CO2).
16) The freshly formulated 10% FBS DMEM medium (stored in a refrigerator) was incubated in a water bath at 37° C. for 10 min to avoid the expansion and contract of the skin graft, which would cause the skin graft falling from the bottom of the well.
17) The plate containing the skin grafts was incubated in an incubator at 37° C., 5% CO2 for 4 h. 4 ml of the above incubated 10% FBS DMEM medium was slowly added dropwise from the rim to each well. De-adherence of the skin graft was not observed.
18) On Day 5 of the experiment, all skin grafts in all wells were well adhered, and recorded by photograph. The test pharmaceutical composition was added to the wells. Specifically, 3 units (about 0.08 g per unit) of pharmaceutical composition, 4 units of pharmaceutical composition or equal amount of medium (as control) was added to the corresponding well, and 3 ml of 10% FBS DMEM medium incubated at 37° C. was added to each well at the same time.
19) The medium was changed regularly. Generally, 3 ml of original medium was removed, and an equal amount of 10% FBS DMEM medium incubated at 37° C. was added.
20) The growth of fibroblasts was observed and recorded regularly with the Nikon TE2000U inverted microscope during the whole culture process. Photos were recorded with the Nikon DXM1200 micro-camera and saved on a computer. Pictures at certain time points were selected and analyzed with Image-Pro Plus professional image software to draw the growth profile of fibroblast.
21) The experiment was stopped after a certain period of cultivation. Cells were collected from one control well, one well with 3 units of pharmaceutical composition and one well with 4 units of pharmaceutical composition. The cells were detected and analyzed with a flow cytometer. The skin grafts and supernatant were removed from the well. The well was rinsed with cold PBS 3 times. 0.5 ml of trypsin and 0.5 ml of EDTA cell digest solution were added to the well, shaked and reacted at room temperature for a certain time. The reaction was stopped when cell morphology change was observed under the inverted microscope. The well was added with 1 ml of 10% FBS DMEM medium to stop the reaction completely, followed by addition of 3 ml of cold Hank's solution. The bottom of the well was piped repeatedly until the cells were completely separated from the bottom of the well. The obtained cell suspension was added to a 15 ml centrifuge tube, and centrifuged at 4° C., 2000 rpm for 5 min. The supernatant was removed, and the volume of the residues was about 0.5 ml. The cells was suspended, added to an Eppendorf tube, and centrifuged at 4° C., 2000 rpm for 5 min. The supernatant was removed, and the volume of the residue was about 25 μl. It should be noticed that the above steps were carried out in an ice bath, and the obtained cells were labeled accurately. The cells were placed in the portable refrigerator with ice packs, and immediately tested at Peking University. At the flow cytometer room of Peking University, the cells were piped evenly, added with 10 μl of 7-AAD, mixed well and tested immediately. Detection parameters: single laser wavelength of 488 nm, 150 mW; SSC: 0.5 mV (785 nm); 40× objective lens.
2. Results
2.1 Test Results of Quantitative Imaging Analytical Flow Cytometry
2.1.1 Total Particle Count
All the particles in the cell suspension to be tested were counted to obtain the absolute number of particles. The maximum number of counted particles was set to 300,000. The counted particles included viable cells, dead cells and non-cells. The shape of each particle could be observed clearly through the image recorded by the instrument. Although the total particle count does not accurately reflect the true number of cells, it is of great reference significance. When analyzing the results, the non-cellular components contained therein should be fully considered. It can be seen from the test results that:
The cells were collected and tested by the flow cytometry to obtain a scatter diagram, which was analyzed by IDEAS software (published by Amnis and matched with the flow cytometer) to obtain the total number of particles. The results are shown in
2.1.2 Fibroblast Count
In addition to the total number of particles, the scatter diagram obtained by the flow cytometry was further analyzed by IDEAS software (published by Amnis and matched with the flow cytometer) to obtain the total number of cells, the number of fibroblasts, and the number of viable fibroblasts. In this Example, the parameters were reset, wherein the parameters included two important parameters of aspect ratio and area. The particles that did not meet the cell parameters were excluded to screen the particles that met the cell parameters out of the total particles. The results obtained represented the total number of different types of cells. The results are shown in
The non-fibroblasts were excluded to screen the cells that met the fibroblast parameters out of the total cells and obtain the total number of fibroblasts. The results are shown in
The final step of the flow cytometry test was to detect the number of viable cells among the total number of fibroblasts to reflect the effect of different analytes on fibroblast activity and life span. This was achieved by staining the fibroblasts with 7-AAD staining solution. The results are shown in
2.2 Drawing Cell Growth Profile with the Number of Fibroblasts at Different Time Points
Three areas with high, medium and low cell density were selected from each well of the plate. Three fields of vision with the same area were randomly selected from each area, with a total of 9 fields of vision with the same area. Cells with typical morphology were counted to obtain the total number of cells. Cells were counted by the professional image processing software image-Pro plus Version6.0. Cell count was carried out by clicking the “Measure” button in the menu, selecting the “Measurements . . . ” item in the drop-down menu, and selecting the “Create point feature” tool in the “Features” toolbar in the dialog box. A total of 12 time points (167 h, 186 h, 197 h, 210 h, 222 h, 234 h, 247 h, 258 h, 269 h, 281 h, 291 h and 300 h after the experiment) were selected in the experiment. Fibroblast count results are shown in Table 1:
The fibroblast growth profile is drawn based on the above results. For comparison, the growth profile is expressed as different line charts. The results show that there are a large number of cells in the control well, and the overall trend of the growth profile is upward, indicating that the cells increase over time, and the cell growth is not affected. There are a small number of cells in the pharmaceutical composition well, and the overall trend of the growth profile is downward, indicating that the cells decrease over time. In addition to the obviously inhibited cell growth, the viable cells decrease over time as well, indicating that the cell growth is inhibited. Specific results are shown in
2.3 Microscopic Examination at Different Time Points
A total of 14 detection time points for fibroblast growth (48 h, 66 h, 167 h, 186 h, 197 h, 210 h, 222 h, 234 h, 247 h, 258 h, 269 h, 281 h, 291 h and 300 h after the experiment) were selected to visually show the in vitro growth of fibroblast of suckling mouse skin graft under the effect of different biologically active substances. Similar to the results of the previous experiment, it can also be seen under the microscope that the pharmaceutical composition has a significant inhibition effect on fibroblast growth, while the fibroblasts in the control group without the pharmaceutical composition grow vigorously. The results under the microscope show that the pharmaceutical composition has a Very significant inhibition effect on fibroblast. Fibroblasts growing out of the grafts are significantly inhibited by the pharmaceutical composition, and changed in morphology. The cells turn black and shrink until they die and dissolve. The effect of the pharmaceutical composition on fibroblasts that just grow out of the skin graft can also be observed under the microscope. In fact, the cells are inhibited at the early stage of growth without a chance of diffusion and growth. With respect to a large amount of fibroblasts growing in a short period of time, the cells will not be immediately inhibited or die due to the temporary balance of the pharmaceutical composition and fibroblast. After a certain period of time, the cells will eventually be inhibited by the pharmaceutical composition until they die, which is directly related to the amount of the pharmaceutical composition. With respect to fibroblasts that have grown and multiplied before the addition of the pharmaceutical composition, when the pharmaceutical composition is added, it takes time for the pharmaceutical composition to function, and the effect becomes more and more obvious over time. Specific results are shown in Table 2 below:
1. Materials and Methods
1.1 Instruments, Devices, Materials and Reagents
Ultrapure water system (Milli-Q, Millipore, USA); two-stage reverse osmosis purified water system (Beijing Innogreen Technology Co., Ltd.); electronic scale (AB135-S, Mettler-Toledo, Switzerland); electronic scale (ES-1000HA, Changsha Xiangping Technology Development Co., Ltd.); high-speed refrigerated centrifuge (J20-XP, Beckman-Coulter, USA); desktop high speed refrigerated centrifuge (1-14K, Sigma, Germany); biological clean bench (BCN-1360B, Beijing HDL Apparatus Co., Ltd.); CO2 incubator (Forma3111, Thermo Fisher Scientific, USA); particle ice machine (SIM-F124, Sanyo. Japan); electronic constant temperature water bath (CS501-3C type, Chongqing Sida. Experimental Instrument Co., Ltd.), drying oven (Chongqing Sida Experimental Instrument Co., Ltd.); inverted microscope (Nikon TE2000U, Nikon, Japan); microscopic imaging system (Nikon DXM 1200, Nikon, Japan); ordinary optical microscope (BK1201, Chongqing Optical Instrument Factory); micro-sampler (1000 μl, 200 μl, 20 μl, 10 μl, Gilson, France); 12-well culture plate; 15 ml centrifuge tube; 1 ml and 0.5 ml Eppendorf centrifuge tube; dripper; sterilized small surgical instrument; sterilized small beaker; sterilized small flask with lid; sterilized culture dish (φ5 cm, (φ6 cm), 0.22 μm microporous membrane; needle filter; capped triangular flask; capped small test tube; needle; DMEM medium and 1640 medium (GIBCO, Invitrogen Corporation, USA, packaged by Beijing Xinjingke Biotechnology Co., Ltd.); fetal bovine serum (FBS, Hangzhou Sijiqing Biological Engineering Material Co., Ltd.); PBS (self-made, stored at 4° C.); 75% ethanol (self-made); trypsin-EDTA cell digest solution (self-made); portable refrigerator; vortex shaker.
1.2 Methods and Results
1.2.1 Early Human Embryonic Tissue
Source and transportation of the tissue: with the informed consent of the patient, early human embryonic tissue was obtained from the hospital through artificial abortion. The human embryonic tissue was placed in 10% FBS DMEM complete medium in an ice bath, and immediately processed in the laboratory.
Graft culture: A small amount of tissue was cut into small pieces. The tissue was washed with PBS once, then washed with a high concentration of double antibiotics-PBS five times. The tissue was rinsed with 15% FBS 1640 medium once, and cut into grafts (1 mm3). The grafts were cultured in a 12-well plate. Each well comprised several pieces of grafts, and was added with a small amount of 15% FBS1640 medium around. The plate was left to stand in the incubator at 37° C., 5% CO2 for 2.5 h. Each well was added with 2 ml of 15% FBS1640 medium. The grafts were divided into two groups: test group and control group. The test group was added with two units (0.1 g per unit) of the pharmaceutical composition prepared in Example 1, and the control group was added with the same amount of medium. The plate was incubated in the incubator at 37° C., 5% CO2. The cell growth in the test group and control group was observed after a certain period of time.
Results: On Day 70 of the cultivation, no cell growth was observed for the test group, while there was a large number of fibroblasts growth for the control group. Conclusion: The pharmaceutical composition inhibits human fibroblast.
Single cell culture: A small amount of tissue was cut into small pieces. The tissue was washed with PBS once, then washed with a high concentration of double antibiotics-PBS five times. The tissue was rinsed with 15% FBS 1640 medium once, and cut into grafts (1 mm3). The grafts were placed in a 50 ml centrifuge tube, and added with a mixed solution of 2 ml of 0.25% trypsin and 2 ml of 0.02% EDTA. The tube was shaked in a constant temperature shaker at 37° C. for 35 min, and vortexed for an appropriate time. The solution was filtrated through a stainless steel filter (80 mesh). The filtrate was added with an appropriate amount of PBS, and centrifuged at 1500 rpm for 7 min. The supernate was removed, and the residues were added with PBS, and centrifuged at 1700 rpm for 5 min. The supernate was removed, and the residues were added with 8 ml of 15% FBS1640 medium. The cells were counted, and cultured in a 12-well plate (2 ml of cell suspension per well). The cells were divided into two groups: test group and control group. The test group was added with two units (0.1 g per unit) of the pharmaceutical composition prepared in Example 1, and the control group was added with the same amount of medium. The plate was incubated in the incubator at 37° C., 5% CO2. The cell growth in the test group and control group was observed after a certain period of time.
Results: On Day 70 of the cultivation, no cell growth was observed for the test group, while there was a large number of fibroblasts growth for the control group. Conclusion: The pharmaceutical composition inhibits human fibroblast growth.
1.2.2 Gastric Fundus Cancer Tissue
Source and transportation of the tissue: with the informed consent of the patient, gastric fundus cancer tissue was obtained from the hospital through surgical excision. The gastric fundus cancer tissue was placed in 10% FBS DMEM complete medium in an ice bath, and immediately processed in the laboratory.
Gastric fundus cancer tissue graft culture: A small amount of cancer core tissue was cut into small pieces. The tissue was washed with PBS once, then washed with a high concentration of double antibiotics-PBS five times. The tissue was rinsed with 15% FBS 1640 medium once, and cut into grafts (1 mm3). The grafts were cultured in a 12-well plate. Each well comprised several pieces of grafts, and was added with a small amount of 15% FBS1640 medium around. The plate was left to stand in the incubator at 37° C., 5% CO2 for 2.5 h. Each well was added with 2 ml of 15% FBS1640 medium. The grafts were divided into two groups: test group and control group. The test group was added with two units (0.1 g per unit) of the pharmaceutical composition prepared in Example 1, and the control group was added with the same amount of medium. The plate was incubated in the incubator at 37° C., 5% CO2. The cell growth in the test group and control group was observed after a certain period of time.
Results: No fibroblast growth was observed for the test group (
1. Materials and Methods
1.1 Instruments, Devices, Materials and Reagents
The laboratory animals were 10-month-old Wistar male rats purchased from Institute of Laboratory Animal Sciences, CAMS. The rats were divided into two groups (59 rats for the test group, and 27 rats for the control group). After adapting the feeding environment for 3 days, the rats were subjected to differentiated feeding. The control group was continuely fed with the general nutrient feed produced by Institute of Laboratory Animal Sciences, CAMS, and the test group was fed with the nutrient feed added with the pharmaceutical composition prepared in Example 1. The first batch of rats (5 rats for the test group, and 5 rats for the control group) which were fed with the nutrient feed comprising the pharmaceutical composition and the normal nutrient feed were euthanized after 526 days of feeding. The second batch of rats (18 rats for the test group, and 10 rats for the control group) which were fed with the nutrient feed comprising the pharmaceutical composition or the normal nutrient feed were euthanized after 623 days of feeding. Samples of various tissues and organs (heart, liver, lungs, kidneys, and bone marrow) were sliced and stained (hematoxylin-eosin staining, HE staining).
Preparation method of the nutrient feed comprising the pharmaceutical composition: normal nutrient feed:pharmaceutical composition=9:1 (mass ratio). 9 parts of normal nutrient feed were added to a mixer. 1 part of the pharmaceutical composition was properly heated, stirred well, and poured into the mixer. The mixer was covered with a cap and started to run. The normal nutrient feed and the pharmaceutical composition were mixed evenly, and then screened through a fine sieve. The sieve was shaken vigorously to further mix the feed evenly. Finally, the mixture was processed into a biscuit feed with a feed processing machine. The feed was spreaded on a stainless steel plate, and dried in a steam drying room at 80° C. for 4 hours. The feed was cooled naturally after the steam stopped.
2. Results
With regard to myocardial fibrosis of aging rat, there are significant differences between the test group and the control group. Among the 17 samples of the test group, 7 samples have myocardial fibrosis, with an abnormal rate of 41%. Among the 16 samples of the control group, 13 samples have myocardial fibrosis, with an abnormal rate of 81%. The test group is superior to the control group, indicating that the pharmaceutical composition inhibits myocardial fibrosis (see
With regard to liver fibrosis of aging rat, there are significant differences between the test group and the control group. Among the 18 samples of the test group, 3 samples have fibrosis, with an abnormal rate of 17%. Among the 17 samples of the control group, 10 samples have fibrosis, with an abnormal rate of 59%. The test group is significantly superior to the control group. Bile duct fibrosis is a typical indicator of liver aging. The results indicate that the pharmaceutical composition has a significant effect of inhibiting liver fibrosis and promoting liver regeneration and recovery.
With regard to lung tissue fibrosis of aging rat, it is determined that the pharmaceutical composition significantly inhibits the lung tissue fibrosis of aging rat. The lung tissue fibrosis rate in the test group is 5/20=25%, while the lung tissue fibrosis rate in the control group is 10/14=71%.
With regard to renal parenchymal fibrosis of aging rat, there are significant differences between the test group and the control group. Among the 19 samples of the test group, 5 samples have fibrosis, with an abnormal rate of 26%. Among the 16 samples of the control group, 11 samples have fibrosis, with an abnormal rate of 69%. The test group is significantly superior to the control group.
The pharmaceutical composition inhibits the bone marrow fibrosis of aging rat. None of the 6 samples of the test group have bone marrow fibrosis, while all 6 samples of the control group have fibrosis. In the test group, myeloid cells are densely arranged, and erythroid precursor cells, myeloid (granulocyte) precursor cells and megakaryocytes can be easily found (see
The pharmaceutical composition was prepared according to the method of Example 1, wherein the refined sesame oil, Scutellaria baicalensis and Cortex Phellodendri (100 kg: 5 kg: 4 kg) were added to a reaction tank in step 1, and other steps were the same as in Example 1.
The pharmaceutical composition obtained in this example was subjected to the test methods of Example 2, 3 and 4. The results are similar to those of the pharmaceutical composition obtained in Example 1, indicating that this pharmaceutical composition can significantly regulate fibroblast growth, and inhibit human fibroblast and organ fibrosis of aging rat.
The pharmaceutical composition was prepared according to the method of Example 1, wherein the refined sesame oil, Scutellaria baicalensis and Coptis chinensis (100 kg: 5 kg: 4 kg) were added to a reaction tank in step 1, and other steps were the same as in Example 1.
The pharmaceutical composition obtained in this example was subjected to the test methods of Example 2, 3 and 4. The results are similar to those of the pharmaceutical composition obtained in Example 1, indicating that this pharmaceutical composition can significantly regulate fibroblast growth, and inhibit human fibroblast and organ fibrosis of aging rat.
The pharmaceutical composition was prepared according to the method of Example 1, wherein the refined sesame oil, Scutellaria baicalensis and Coptis chinensis (100 kg: 5 kg: 5 kg: 5 kg) were added to a reaction tank in step 1, and other steps were the same as in Example 1.
The pharmaceutical composition obtained in this example was subjected to the test methods of Example 2, 3 and 4. The results are similar to those of the pharmaceutical composition obtained in Example 1, indicating that this pharmaceutical composition can significantly regulate fibroblast growth, and inhibit human fibroblast and organ fibrosis of aging rat.
The pharmaceutical composition was prepared according to the method of Example 1, wherein the refined sesame oil, Scutellaria baicalensis, Coptis chinensis, Cortex Phellodendri, Pericarpium Papaveris and earthworm (100 kg: 5 kg: 4 kg: 4 kg: 5 kg: 5 kg) were added to a reaction tank in step 1, and other steps were the same as in Example 1.
The pharmaceutical composition obtained in this example was subjected to the test method of Example 2, and the following results are obtained:
1) Drawing cell growth profile with the number of fibroblasts at different time points
There are a large number of cells in the control well, and the overall trend of the growth profile is upward, indicating that the cells increase over time, and the cell growth is not affected. There are a small number of cells in the M well, and the overall trend of the growth profile is downward, indicating that the cells decrease over time. In addition to the obviously inhibited cell growth, the viable cells decrease over time as well, indicating that the cell growth is inhibited (see
The pharmaceutical composition obtained in this example was subjected to the test methods of Example 3 and 4. It is found that the pharmaceutical composition prepared in this example can inhibit human fibroblast and organ fibrosis of aging rat.
Case 1: Regeneration and Recovery of Gastric Scar
Case 1 (male, born on 25 Feb. 1953) has a history of gastric ulcer and chronic erosive gastritis. The patient was administrated with five units each time (0.1 g of the pharmaceutical composition per unit) of the pharmaceutical composition obtained in Example 1 three times a day. Before the administration, SB capsule endoscopy showed obvious scars on the gastric mucosa. After 9 months of administration, it was found that the mucosal scars were significantly reduced. After 4 years of administration, it was found that the mucosal scars were recovered (see
Case 2: Regeneration and Recovery of Liver Fibrosis
Case 2 (male, born on 6 Jul. 1950) has a history of chronic hepatitis and liver fibrosis for 30 years, with a spleen 7.4 cm below the ribs. The patient was administrated with five units each time of the pharmaceutical composition obtained in Example 1 three times a day. The patient was recovered to normal after three years. The corresponding biochemical function indexes are normal, which can be seen in Table 5 below:
Case 3: Regeneration and Recovery of Arteriosclerosis
Case 3 (female, born on 12 Feb. 1957) has a history of hypertension for 18 years and a history of diabetes for 17 years. The patient was administrated with five units each time of the pharmaceutical composition obtained in Example 1 three times a day. The coronary heart disease and arterial plaque are recovered at present.
Before the administration, the color B-ultrasound of double carotid arteries showed plaque on the left artery (soft plaque). After one year of administration, the ultrasound showed no obvious abnormalities in bilateral carotid arteries.
Before the administration, the coronary spiral CT showed coronary artery calcification and stenosis in the proximal segment of the left anterior descending branch (30%). After one year of administration, there were no abnormalities in coronary arteries diagnosed by imaging.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201711119842.9 | Nov 2017 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2018/115174 | 11/13/2018 | WO | 00 |
