Patent Application
20230391843
The present disclosure claims priority to Chinese Patent Application No. CN202110986914.X, entitled “Composition and Method for Controlling Heat Generation within Organism, and Use”, filed with China National Intellectual Property Administration on Aug. 26, 2021, the entire content of which is incorporated herein by reference.
The present disclosure relates to the field of biotechnology, and particularly relates to a composition, a method and a use, and particularly to a composition and a method for controlling heat generation within an organism, and a use.
Heat generation of brown and beige fat plays a key role in a development of maintenance of a body temperature. Although orientation and activation of the brown/beige fat are known, diversity and abundance of immune factors in adipose tissue are unclear.
The present disclosure provides a composition for controlling heat generation within an organism, including a molecule for adjusting binding of EBI-3 and p28 or binding of dimers of EBI-3 and p28 and receptors thereof, or a downstream signal pathway.
Optionally, the adjusting refers to inhibition or reduction of the binding of EBI-3 and p28, or inhibition or reduction of the binding of the dimers of EBI-3 and p28 and the receptors thereof, or down regulation of the downstream signal pathway, and the controlling refers to inhibition of heat generation, thereby reducing a body temperature of the organism.
Optionally, the molecule includes a biological macromolecule or small molecule compound, comprising an anti-EBI-3 antibody, an anti-p28 antibody, an anti-IL-27R antibody, an EBI-3 soluble fragment, a p28 soluble fragment and an IL-27R soluble fragment.
Optionally, the adjusting refers to promotion or enhancement of the binding of EBI-3 and p28, or promotion or enhancement of the binding of the dimers of EBI-3 and p28 and the receptors thereof, or up regulation of the downstream signal pathway, and the controlling refers to promotion of heat generation, thereby improving the body temperature of the organism.
Optionally, the molecule includes a recombinant IL-27 protein, a nuclear acid fragment like mRNA, virus or plasmid DNA that can express IL-27 protein in vivo, a modifier thereof, and polypeptide or a small molecule compound which can activate activity of IL-27R.
The present disclosure provides a method for controlling heat generation within an organism, in which a molecule for adjusting binding of EBI-3 and p28 or binding of dimers of EBI-3 and p28 and receptors thereof, or a downstream signal pathway is applied to the organism.
Optionally, the organism includes a human subject or tissue or cell thereof, a mammal or tissue or cell thereof, or a microorganism.
Optionally, the tissue or cell is in vitro tissue or cell.
The present disclosure further provides a method for screening a candidate compound capable of controlling heat generation within an organism, including a step of measuring a binding condition of EBI-3 and p28, or a binding condition of dimers of EBI-3 and p28 and receptors thereof, or a downstream signal pathway triggered by the binding before and after application of a to-be-tested compound.
Optionally, when the binding of EBI-3 and p28 is inhibited or reduced or the binding of the dimers of EBI-3 and p28 and the receptors thereof is inhibited or reduced or a downstream signal triggered by the binding is down-regulated after the to-be-tested compound is applied, the to-be-tested compound is identified as a candidate compound for inhibiting heat generation within the organism.
Optionally, when the binding of EBI-3 and p28 is promoted or enhanced or the binding of the dimers of EBI-3 and p28 and the receptors thereof is promoted or enhanced or the downstream signal triggered by the binding is up-regulated after the to-be-tested compound is applied, the to-be-tested compound is identified as a candidate compound for promoting heat generation within the organism.
Optionally, the method includes the following steps:
Optionally, the method includes the following steps:
Optionally, the method includes the following steps:
Optionally, the organism model includes an animal model or a cell model.
The present disclosure further provides use of the composition in any one of the above descriptions in treatment of a metabolic disease.
Optionally, the metabolic disease includes hypothermia.
The present disclosure further provides a method for treating a metabolic disease, in which the composition in any one of the above descriptions is administered to the subject in need.
Optionally, the metabolic disease includes hypothermia.
Various exemplary embodiments of the disclosure are described now, the detailed description should not be construed as limiting the disclosure but rather as a more detailed description of certain aspects, characteristics and implementations of the disclosure.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. In addition, for a numerical range in the disclosure, it is to be understood that the upper and lower limits of the range, and each intervening value therebetween, are specifically disclosed. Each smaller range between any stated value or any intervening value in a stated range and any other stated value or any other intervening value in the stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless stated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the disclosure belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the implementation or testing of the disclosure. All documents mentioned in the specification are incorporated by reference to disclose and describe methods and/or materials related to the documents. In case of conflict with any incorporated document, the specification will control. Unless state otherwise, “%” is a percent by weight.
As used herein, the term “heat generation” may be used interchangeably with the terms “heat production”, “heat generation effect” and “thermogenesis”. Thermogenesis used herein means a process in which mammals, including humans, regulate heat generation.
In the present disclosure, binding of an EB virus-induced gene 3 (EBI-3) and a subunit of cytokine IL-27 secreted by dendritic cells (p28) means that an EBI-3 subunit and a p28 subunit form a heterodimeric structure with a certain spatial arrangement by interaction which may be connection by a disulfide bond.
The term “receptor” as used herein refers to a biological macromolecule capable of being bound to the above-mentioned dimers and of causing a change in cell functions. The receptor contains at least an active site which recognizes and is bound to the dimer and a functional active site responsible for generating a response reaction, and thus initiates a series of biochemical reactions, ultimately leading to generation of a biological effect by a target cell. The binding between the receptor and the dimer in the disclosure results in activation of the receptor and creates a basic step of activating subsequent signaling by the receptor. Under physiological conditions, the binding between the receptor and the dimer is not mediated by covalent bonds, but rather is bounded to each other primarily by ionic bonds, hydrogen bonds, Van der Waals forces, and hydrophobic interaction.
The term “signal pathway” as used herein refers to a phenomenon that when a certain reaction occurs in a cell, a signal transmits information from an exterior of the cell to an interior of the cell, and the cell responds according to the information. The signal pathway in the disclosure particularly refers to a series of enzymatic reaction pathways which can transmit extracellular molecular signals into the cell through a cell membrane to exert an effect.
Some embodiments of the present disclosure provide a composition for controlling heat generation within an organism.
The present disclosure provides a composition for controlling heat generation within an organism, including a molecule for adjusting binding of EBI-3 and p28 or binding of dimers of EBI-3 and p28 and receptors thereof, or a downstream signal pathway.
In some embodiments, the adjusting refers to inhibition or reduction of the binding of EBI-3 and p28, or inhibition or reduction of the binding of the dimers of EBI-3 and p28 and the receptors thereof, or down regulation of the downstream signal pathway, and the controlling refers to inhibition of heat generation, thereby reducing a body temperature of the organism.
In some embodiments, the molecule includes a biological macromolecule or small molecule compound, comprising an anti-EBI-3 antibody, an anti-p28 antibody, an anti-IL-27R antibody, an EBI-3 soluble fragment, a p28 soluble fragment and an IL-27R soluble fragment.
In some embodiments, the adjusting refers to promotion or enhancement of the binding of EBI-3 and p28, or promotion or enhancement of the binding of the dimers of EBI-3 and p28 and the receptors thereof, or up regulation of the downstream signal pathway, and the controlling refers to promotion of heat generation, thereby improving the body temperature of the organism.
In some embodiments, the molecule includes a recombinant IL-27 protein, a nuclear acid fragment like mRNA, virus or plasmid DNA that can express IL-27 protein in vivo, a modifier thereof, and polypeptide or a small molecule compound which can activate activity of IL-27R.
Optionally, the composition according to the disclosure includes a molecule for adjusting IL27 signal or binding of IL27 to a receptor thereof or the downstream signal pathway. The downstream signal pathway includes, but is not limited to, JAK/STAT3, p38 MAPK, or the like. In the present disclosure, thermogenesis and energy expenditure are mediated by adipocytes which include, but are not limited to, brown/beige adipocytes and white adipocytes. In the present disclosure, the above-mentioned adipocytes act as IL27 target cells, not immune cells.
Some embodiments of the present disclosure provide a method for controlling heat generation within an organism, in which a molecule for adjusting binding of EBI-3 and p28 or binding of dimers of EBI-3 and p28 and receptors thereof, or a downstream signal pathway is applied to the organism.
In some embodiments, the organism includes a human subject or tissue or cell thereof, a mammal or tissue or cell thereof, or a microorganism.
In some embodiments, the tissue or cell is in vitro tissue or cell.
Some embodiments of the present disclosure provide a method for screening a candidate compound capable of controlling heat generation within an organism.
In the present disclosure, the screening method at least includes a step of measuring, using a reagent, a binding condition of EBI-3 and p28, or a binding condition of dimers of EBI-3 and p28 and receptors thereof, or a downstream signal pathway triggered by the binding before and after application of the to-be-tested compound. The above binding condition is further measured by a step of measuring a relevant parameter using a reagent, wherein the parameter includes a quantity of the dimer formed by EBI-3 and p28, or a metabolic phenotype, or a quantity of a thermogenic protein. The thermogenic protein includes, but is not limited to, UCP1, a peroxisome proliferator-activated receptor (PPARα) and a peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α). In the present disclosure, activation (also called as excitation sometimes) of the downstream signal pathway refers to further expression of a downstream target gene. Optionally, the reagent includes a sequence shown by SEQ ID NO: 1-30.
In some embodiments, when the binding of EBI-3 and p28 is inhibited or reduced or the binding of the dimers of EBI-3 and p28 and the receptors thereof is inhibited or reduced or a downstream signal triggered by the binding is down-regulated after the to-be-tested compound is applied, the to-be-tested compound is identified as a candidate compound for inhibiting heat generation within the organism.
In some embodiments, when the binding of EBI-3 and p28 is promoted or enhanced or the binding of the dimers of EBI-3 and p28 and the receptors thereof is promoted or enhanced or the downstream signal triggered by the binding is up-regulated after the to-be-tested compound is applied, the to-be-tested compound is identified as a candidate compound for promoting heat generation within the organism.
In some embodiments, the method includes the following steps:
In some embodiments, the method includes the following steps:
In some embodiments, the method includes the following steps:
In some embodiments, the organism model includes an animal model or a cell model.
The present disclosure further provides use of the composition in any one of the above descriptions in treatment of a metabolic disease.
The present disclosure further provides a method for treating a metabolic disease, in which the composition in any one of the above descriptions is administered to the subject in need.
In some embodiments, the metabolic disease includes hypothermia.
The present disclosure discloses functions of the IL-27(EBI-3/p28) signals in the improvement of in vivo heat generation. It is proved that IL-27 directly acts on the adipocytes, activates the p38 MAPK-PGC1 signals and stimulates generation of UCP1. Therapeutic administration of IL-27 may reverse hypothermia of EBI-3 KO mice induced by the low temperature.
The present disclosure provides a composition and a method for controlling heat generation within an organism, and a use. Research of the present disclosure finds that (EBI-3/p28) signals have an important function in an improvement of in vivo heat generation, and proves that IL-27 directly acts on adipocytes, activates p38 MAPK-PGC1 signals and stimulates generation of UCP1.
Interleukin-27Rα KO (B6N.129P2-Il27ratmMak/J, No.: 018078) and EBI-3 KO (B6.129X1-Ebi3tm1Rsb/J, No.: 008691) mice were purchased from Jackson Laboratory. Adipoq-cre mice were supplied by Professor Yong Liu. IL-27Rαf/f mice [herein] were prepared by our own laboratory. Animal experiments were conducted in accordance with ethical specifications and protocols approved by the institutional animal protection and utilization committee of Jinan University and the institutional animal care and utilization committee of Yale University. Groups of mice with matched genders and ages were used in all experiments, with 4 to 6 animals per cage. IL-27 treatment experiments and bone marrow chimera production were performed in random groups. The groups of mice were bred in a specific sterile animal facility with a controlled temperature and humidity, the temperature was 25° C., a cycle of light and dark was 12 h:12 h, and food and water were freely obtained.
Human serum samples were analyzed on a Bio-Plex system (BioRad) using a Bio-Plex-Pro™ Human Cytokine 17-Plex analysis kit (Cat. No. M5000031YV) and a Bio-Plex Pro™ Human Inflammation Panel 1 (Cat. No. 171-AL001M). A human IL-27 ELISA kit (434607) was purchased from Biolegend. Glucose (63005518) was purchased from Chinese Sinopharm Chemical Reagent Company. Blood glucose test strips (one-touch) were purchased from Johnson and Johnson. An anti-mouse pAKT monoclonal antibody (4060), an anti-mouse AKT monoclonal antibody (4685), an anti-mouse GAPDH monoclonal antibody (5174), an anti-mouse HSP90 monoclonal antibody (4877), an anti-mouse PPARγ monoclonal antibody (2435), an anti-mouse pSTAT3 monoclonal antibody (9145), an anti-mouse STAT3 monoclonal antibody (9139), an anti-mouse p-p38 MAPK (9211), an anti-mouse p38 MAPK (9212), an anti-mouse pATF2 monoclonal antibody (9221), and an anti-mouse ATF2 monoclonal antibody (35031) were purchased from Cell Signaling Technology. An anti-mouse UCP1 monoclonal antibody (ab10983), an anti-mouse PGC-1α monoclonal antibody (ab54481), an anti-mouse PPARα monoclonal antibody (ab8934), an anti-mouse PRDM16 monoclonal antibody (ab202344) and an anti-mouse IL-27Rα monoclonal antibody (ab5997) were purchased from Abcam. APC-CY7-anti-mouse CD45 (clone 30-F11) was purchased from BD. APC anti-mouse CD11b (clone M1/70) and PE anti-mouse F4/80 (clone BM8) were purchased from Sungene Biotech (Tianjin, China). Recombinant mice IL-27 (rmIL-27) (577408) were purchased from Biolegend. An STAT3 inhibitor SH-4-54 and a p38 MAPK inhibitor SB203580 were purchased from Selleck Chemicals. Brown/beige fat differentiation agent dexamethasone (D4902), rosiglitazone (R2408), indomethacin (I7378), isoproterenol (I5627), forskolin (F6886), 3-isobutyl-1-methylxanthine (IBMX) (I5879), a triglyceride detection kit (T2449) and insulin (I3536) were purchased from Sigma. A leptin detection kit (MOB00) and an adiponectin detection kit (MRP300) were purchased from R and D. An insulin detection kit (90080) was purchased from Crystal Chem.
Human serum was obtained as described above. Mouse serum was collected after high fat diet (HFD) feeding for 10 weeks. Human IL-27 and mouse insulin were detected using the ELISA kit according to an operation instruction.
Human serum was collected as described above. Detection of human serum inflammatory factors was performed on the Bio-Plex 200 flow liquid chip analysis system (BioRad) using the Bio-Plex-Pro™ Human Cytokine 17-Plex analysis kit (Cat. No. M5000031YV) and the Bio-Plex Pro™ Human Inflammation Panel 1 (Cat. No. 171-AL001M) according to instructions of the user manual.
A whole cell lysate or tissue lysate was extracted using an RIPA lysis buffer (Beyotime, China) supplemented with a complete protease inhibitor (Roche) and a phosphatase inhibitor Cocktail 2 (P5726, Sigma), and a supernatant was used for subsequent analysis. Proteins were diluted in a Loading dye (BL502A, Bio-sharp), heated at 95° C. for 10 min, and separated on 4-12% polyacrylamide gel. The proteins were transferred to polyvinylidene fluoride membranes and western blot experiments were performed with the commercial antibodies and methods-reagents mentioned in the drawing.
Adipose or liver tissue was fixed in 4% paraformaldehyde/1×PBS overnight at 4° C. and embedded in paraffin before sectioning. Sections were stained with hematoxylin and eosin (H and E) or oil red O (liver) and photographed under a bright field microscope.
UCP1 and immunohistochemistry were performed using a rabbit specific HRP/DAB (ABC) detection IHC kit (ab64261, Abcam) according to instructions of the manufacturer.
Cold stimulation experiments were performed in a temperature controlled cold room. Mice were placed individually in individual cages (without bedding) at 4° C. The mice had free access to food and water. Rectal core body temperatures were recorded every 2 hours using a digital thermometer and a rectal thermocouple probe (TH-212, HICHANCE, China). If the core body temperature fell below 20° C., the individual mice were euthanized and scored as events for survival analysis.
Energy loss and energy expenditure of the mice were measured using a comprehensive laboratory animal monitoring system (CLAMS, Columbus Instruments, Columbus, OH) metabolic cage in the environmental control rodent incubator of Yale University. The mice were bred individually and acclimated in metabolic chambers for 48 hours prior to data collection. The mice had free access to food and water. Each mouse was subjected to continuous monitoring of physical activity and food intake. During the experiment, concentrations of CO2/O2 were collected 4 times per hour for each mouse.
Total RNA was extracted from frozen tissue using a TRNzol general reagent (Tiangen, Beijing) and quantified using a Nanodrop 2000 ultraviolet-visible spectrophotometer (Thermo). The cDNA was prepared by 1 μg of total RNA through a reverse transcription polymerase chain reaction using a PrimeScript™ RT Reagent kit (TAKARA, Dalian). Real-time quantitative PCR of the cDNA was performed on a CFX96™ real-time PCR detection system (Bio-Rad) using a TB Green™ Premix-EX-Taq™ II kit (TAKARA). A fold change in expression was calculated by a ΔΔCt method using immunohistochemistry with mouse HPRT as an internal reference. Primer pair sequences were as follows.
IL-27Rα KO and WT mice which were 10 weeks old were used as bone marrow cell donors. Bone marrow was separated from hind limb femurs, erythrocytes were lysed, a product was filtered by a cell filter (40 μm), and cell pellets were washed 3 times with sterile PBS, counted and stored on ice for injection. The IL-27Rα KO and WT mice which were 10 weeks old were lethally irradiated (900 rads), 1×107 bone marrow cells were transplanted by ocular intravenous injection, the mice were placed in specific pathogen-free facilities and supplemented with sterile water and feed for 8 weeks to reconstitute immune systems, and then, metabolic research was performed.
Inguinal subcutaneous adipose tissue was minced and digested at 37° C. for 45 min in PBS containing collagenase II (1 mg/ml), and a tissue suspension was filtered by a 100 μm cell filter and centrifuged at 600 g for 5 min, such that a stromal vascular fraction (SVF) was granulated. After granulation, the product was further filtered by a 40 μm cell filter and placed on a collagen-coated plate. After overnight culturing, a supernatant containing non-adherent cells was removed. Precursor adipocytes were grown in DMEM containing 10% FBS and insulin (5 μg/ml) for fusion. The product was treated for 2 days with dexamethasone (1 μM), 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM), insulin (5 μg/ml), indomethacin (125 nM) and rosiglitazone (1 μM) to induce differentiation of the fused cells, and then independently treated for another 5 days with insulin (5 μg/ml) and triiodothyronine (T3, 1 nM). On day 7, the cells were pretreated overnight with and without IL-27 (100 ng/ml) and then treated with isoproterenol (10 μM) or forskolin (10 μM) for 4-6 hours. IL-27 (100 ng/ml) was added for treatment for 0-120 min on day 7 for protein phosphorylation analysis, or for 12-24 h for protein expression level detection. For signal inhibition experiments, either the STAT3 inhibitor (C188-9, 10 μM) or the p38 MAPK inhibitor (SB203580, 10 μM) was added to a medium 0.5 h prior to and during IL-27 treatment.
Epididymal adipose tissue was minced and digested as described above. SVF particles were used for the surface staining of the APC-CY7 anti-mouse CD45, APC anti-mouse CD11b, PE anti-mouse F4/80, the product was incubated for 15 min, and then the cells were washed with PBS and analyzed using a BD-FACSVerse flow cytometer (BD).
A development of IL-27Rα conditional knockout mice was performed using a gene targeting technology. Briefly, a 997 bps CKO region containing exon 3 and exon 4 of the IL27ra gene (GenBank NM_016671.3), a 5.2 kb 5′ homology arm and a 3 kb 3′ homology arm were amplified from BAC clones using a high fidelity Taq technology, and the resultant, a recombination site and a selectable marker were assembled into a targeting vector. The final targeting vector was determined by multiple times of digestion by restriction endonuclease and full sequence analysis and transformed into 129 s mouse embryonic stem cells by electroporation. A correct embryonic stem cell clone was identified and injected into a C57BL/6J mouse blastocyst. After a chimera mouse was unambiguously identified, the chimera mouse and a C57BL/6J mouse were backcrossed. Genotyping PCR confirmed germ line transmission. Then, mice with an IL-27Rα f/w genotype and B6 adipoq-cre mice were bred. Mice with a desired genotype were selected for the experiment. Genotyping primers at flox or wt were detected:
8-week-old WT mice were fed with HFD for 32 weeks and randomly divided into 2 groups. The mice were injected intraperitoneally with rmIL-27 (100 μg/kg) or PBS for 15 consecutive days, and then, metabolic analysis was performed. 12-month-old EBI-3 KO mice were randomly divided into 2 groups, and injected intraperitoneally with rmIL-27 (100 μg/kg) or PBS for 7 consecutive days. The mice were then subjected to a cold stimulation experiment at 4° C.
Mapping and statistical analysis were performed using Graphpad Prism software (version 7). All statistical tests were fully described in the graph legend and met the criteria for normal distribution with similar variances. A sample size was not predetermined using a statistical method. Comparison between the two groups was performed using a t-test. For evaluation data of relevant samples, repeated measurement analysis of variance (AVOVA) was performed. For an evaluation of more than two groups, one-way analysis of variance and multiple comparison were used. For an evaluation between two independent variables, a two-way analysis of variance with multiple comparison was used. Survival analysis was tested using a log rank. Linear regression analysis was adopted for correlation analysis. The data was expressed by a mean±s.e.m., unless stated otherwise. P<0.05 was considered to be statistically significant, and denoted as *p<0.05, **p<0.01, **p<0.001, and NS meant “not significant”.
Browning of white adipose tissue predisposes mice to increase energy expenditure. In order to examine a systemic metabolic status, in the present disclosure, the energy expenditure and nutrient consumption of the IL-27Rα KO mice and the WT control group having the normal diet or HFD treatment were detected. The energy expenditure of the IL-27Rα KO mice fed with the normal diet was significantly reduced without changing the food intake (shown in
The IL-27Rα KO mice were sensitive to cold-induced hypothermia under the normal diet condition (shown in
Next, the cell types responsible for IL-27 mediated signaling were dissected. First, 4 groups of chimera mice (WT>WT, WT>KO, KO>WT and KO>KO) were generated from the bone marrow of the IL-27Rα KO or WT mice, and these mice were subjected to HFD treatment. Surprisingly, only the chimeras made with IL-27Rα KO (no matter what the donor cells were) were highly susceptible to HFD-induced fat accumulation and cold stimulation (shown in
To investigate the molecular mechanism of action of IL-27 on adipocytes, primary beige adipocytes were directly treated with IL-27 in vitro. The expression of UCP1 was significantly increased after IL-27 treatment, and meanwhile, the expression of the peroxisome proliferator-activated receptor α (PPARα), the peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and the key transcriptional co-activator mediating energy metabolism was up-regulated (shown in
Furthermore, supplementation of the EBI-3 KO mice with IL-27 improved UCP1-mediated heat generation and avoided low temperatures in cold stimulation (
In summary, the results indicate that the IL-27 signal is indispensable for the full adaptive heat generation. Direct targeting of IL-27 to adipocytes induces activation of p38 MAPK, thereby driving activation of ATF2 and subsequent expression of PGC-1α and UCP1. In conclusion, these findings provides new insights and extends understanding of the mechanism of in vivo heat generation.
While the present disclosure has been described with reference to the exemplary embodiments, it is to be understood that the disclosure is not limited to the disclosed exemplary embodiments. Various modifications or variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present disclosure. The scope of the claims should be based on the broadest interpretation to encompass all modifications and equivalent structures and functions.
The present disclosure provides the composition and method for controlling heat generation within an organism, and the use, and the present disclosure discloses the functions of the IL-27 signals in adjustment of heat generation. Molecular mechanism research proves that the IL-27 signals directly act on adipocytes, activate p38 MAPK-PGC1 signals and stimulate generation of UCP1. Therapeutic administration of IL-27 reverses hypothermia of EBI-3 KO mice induced at the low temperature. Therefore, the present disclosure reveals a key role of the IL-27 signals in an energy metabolism process, provides a feasible solution for controlling the body temperature, and has a remarkable industrial practical performance and high market value.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110986914.X | Aug 2021 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2022/114928 | Aug 2022 | US |
| Child | 18321062 | US |
