| ObjectiveIL-37 is a newly discovered member of the IL-1 family in human but not in mice.It is now considered to be a negative regulator of innate immunity.Human IL-37 is composed of five different subtypes of IL-37a,b,c,d and e,these subtypes are produced by alternative splicing.Although their expression profile remains to be revealed,it is believed that each isotype is expressed in a tissue-specific manner.Among all these isoforms,IL-37b is the largest isomer composed of exons 1,2 and 4-6.It plays an anti-inflammatory role mainly through the induction or constitutive expression of innate immune cells(such as macrophages and dendritic cells).Using transgenic mouse models,several studies have demonstrated that human IL-37b can resist LPS induced endotoxemia,and dextran sulfate(DSS)-induced colitis or obesity induced inflammation.Unlike IL-37b,IL-37d is encoded by exons 1 and 4-6,which may have unique expression characteristics in tissues or cells.We have shown that IL-37d attenuates endotoxemia or colitis in mice by inhibiting the production of IL-1β,IL-6 and TNF-α in a Smad3 dependent manner or by IL-1r8 mediated NLRP3 expression in macrophages.Although IL-37d has similar effects to IL-37b,its involvement and function in other physiological and pathological processes are still unknownThe prevalence of obesity is a global public health problem,which increases the risk of various life-threatening diseases,including type 2 diabetes,atherosclerotic cardiovascular disease and cancer.Obesity is caused by excess energy,which is mainly stored in white adipose tissue(WAT)in the form of fat.In addition to energy imbalance,metabolic inflammation is fully involved in the pathology of obesity,and then aggravates the metabolic disorder.Obesity can lead to the transition of Wat from homeostasis to inflammation.In this process,with the development of obesity,type 2 immune cells including wat homeostasis(including type 2 innate lymphoid cells(IL C2s),activated M2 macrophages,eosinophils and regulatory T(Treg)cells will be reduced;instead,a large number of immune cells,such as classic activated M1 macrophages,effector helper T(th)1 cells and cytotoxic T(T)cells,will be replaced The accumulation of lymphocyte(CTL)in obese wat mediates type 1 inflammation.It is worth noting that ILC2 population plays a key role in maintaining wat homeostasis and inducing wat to overcome obesity,which can be directly mediated by the production of enkephalin,or indirectly mediated by maintaining eosinophils,M2 macrophages and Treg cells.Interleukin(IL)-33 can trigger the expansion or activation of type 2 immune cells(including ILC 2s or Treg cells)in Wat.Interleukin(IL)-33 is up-regulated after inflammatory stimulation and inhibits its tumorigenic 2(ST2)receptor on these cells,thus providing protection against obesity induced type 1 inflammation.Unlike full-length ST2,soluble ST2(sST2)lacks transmembrane and cytoplasmic domains and is a bait receptor of IL-33,which can block IL-33/ST2 signal transduction.Recently,some studies have shown that sST2 disrupts the Treg/ILC 2 homeostasis in Wat,thus aggravating obesity related insulin resistance in mice.Therefore,sST2 may be a promising target for regulating type 2 immune response in the treatment of obesity.In our previous study,we found that IL-37d was specifically expressed in human adipose tissue and adipose tissue-derived stem cells(ADSC).Therefore,it is very important and necessary to clarify the exact role and potential regulatory mechanism of IL-37d in obesity.We tried to further clarify the mechanism of IL-37d regulating obesity and whether it is related to type 2 immune response.In order to clarify the mechanism of IL-37d regulating obesity and insulin resistance.This paper intends to study from the following aspects:first,IL-37d promotes type 2 immune response in adipose tissue of mice by regulating IL-33/ST2;second,the mechanism of IL-37d regulating IL-33/ST2.MethodsI.IL-37d promotes type 2 immune response in mouse adipose tissue by regulating IL-33/ST21.Construction of IL-37d&IL-33ko gene miceThe IL-37d transgenic mice produced by the company were hybridized with IL-33ko gene knockout mice,and the IL-37d&IL-33ko gene mice were verified by PCR and DNA agarose electrophoresis combined with gene sequencing.2.IL-33 knockout reverses the inhibition of IL-37d on obesity and insulin resistance.The IL-37d transgenic mice,wild-type mice and IL-37d&IL-33ko gene mice were fed with normal diet or high-fat diet for 23 weeks,respectively.During the period,the changes of the mice’s weekly body weight and food intake were tested.When the high-fat model is established until the average weight of the mice exceeds 40g,the mice are tested for glucose tolerance and insulin tolerance:according to the weight of each mouse,the mice are given intraperitoneal injection of glucose or insulin to detect the blood glucose of the mice within 120 minutes3.IL-33 knockout reverses the inhibition of IL-37d on fatty liverTake the dissected normal diet or high-fat diet IL-37d transgenic mice,wild-type mice and IL-37d&IL-33ko gene mice livers,freeze the liver sections and use the oil red O staining method to detect the degree of fatty liver.After liver tissue homogenization,the supernatant was taken to detect the content of triglyceride(TG).4.IL-33 knockout reverses IL-37d’s promotion of type 2 immune response to adipose tissueTake the dissected normal diet or high-fat diet IL-37d transgenic mice,wild-type mice and IL-37d&IL-33ko gene mice subcutaneous fat and epididymal fat,culture the fat tissue in vitro for 24 hours,and take the culture supernatant for use ELISA method was used to detect the levels of type II cytokines IL-4,IL-5,and IL-13 in the supernatant.5.IL-33 knockout reverses IL-37d’s inhibition of adipose tissue inflammationObtain the epididymal fat of normal diet or high-fat diet IL-37d transgenic mice,wild-type mice and IL-37d&IL-33ko gene mice after dissection,the tissues were dehydrated and embedded,then paraffin sectioned,and stained with immunohistochemistry Method to detect the expression level of F4/80.II.The mechanism of IL-37d regulating IL-33/ST21.Inhibition of IL-37d on soluble ST2 induced by high fatTake the subcutaneous fat and epididymal fat tissue of high-fat-induced WT or IL-37d transgenic mice,extract adipose tissue RNA with Trizol reagent,reverse transcription of RNA into DNA with a reverse transcription kit,and detect soluble ST2 by real-time fluorescent quantitative PCR method Subcutaneous fat and epididymal adipose tissue were cultured in vitro for 24 hours and the culture supernatant was used to detect the soluble ST2 content in the supernatant by ELISA.2.Inhibition of IL-37d on soluble ST2 induced by TNF-αTake subcutaneous fat derived from WT or IL-37d transgenic mice,extract mesenchymal stem cells(S-ADSC)derived from subcutaneous fat tissue,and stimulate with TNF-α(10ng/ml)or PBS control for 24 hours to extract total cell RNA.Reverse transcription kit was used to reverse transcription of RNA into DNA,real-time fluorescent quantitative PCR method was used to detect the mRNA level of soluble ST2;cell culture supernatant was taken,and ELISA method was used to detect the content of soluble ST2 in the supernatant.3.IL-37d inhibits soluble ST2 by inhibiting the NF-κB pathwayTake the subcutaneous fat and epididymal fat tissue of high-fat-induced WT or IL-37d transgenic mice,extract adipose tissue RNA with Trizol reagent,reverse transcription of RNA into DNA with a reverse transcription kit,and detect NF-by real-time fluorescent quantitative PCR method The mRNA levels of IL-6,IL-1β and TNF-α regulated by the κB pathway;subcutaneous fat and epididymal fat tissue were lysed with RIPA lysis solution,the protein was extracted after high-speed centrifugation,and the level of P65 phosphorylation was detected by western blot.4.IL-37d pair inhibits the NF-κB pathway by binding to the receptor IL-1R8Take the subcutaneous fat of IL-37d transgenic mice,extract the mesenchymal stem cells(S-ADSC)derived from subcutaneous adipose tissue,use small interfering RNA to interfere with IL-1R8,use TNF-α(10ng/ml)or PBS control to stimulate 24 After hours,extract total cellular RNA,reverse transcription of RNA into DNA with a reverse transcription kit,use real-time fluorescent quantitative PCR to detect the level of soluble ST2 mRNA;use RIPA lysate to lyse subcutaneous fat and epididymal fat tissue,and extract protein after high-speed centrifugation,Use western blot to detect P65 phosphorylation level.ResultI.IL-37d can inhibit obesity and insulin resistance caused by high fat1.Expression of IL-37d in various tissues of IL-37d transgenic miceDNA was extracted from the liver,subcutaneous fat,visceral fat,epididymal fat,brown fat,and skeletal muscle of IL-37d transgenic mice.PCR and DNA electrophoresis methods were used to detect that the above organs all expressed IL-37d.2.Loss of IL-33 reverses the inhibition of IL-37d on obesity and insulin resistance caused by high fatThe IL-37d transgenic mice,wild-type mice and IL-37d&IL-33ko gene mice were fed normal diet or high-fat diet for 23 weeks.The weight of IL-37d transgenic mice fed high-fat diet was significantly lower than that of wild-type mice.The body weight of IL-37d&IL-33ko gene mice is higher than IL-37d transgenic mice but lower than wild-type mice.This result proves that IL-33 deletion partially reverses the inhibitory effect of IL-37d on obesity.When the high-fat model was established until the average weight of the mice fed high-fat diet exceeded 40g,the mice were tested for glucose tolerance and insulin tolerance.The results showed that IL-37d transgenic mice performed better than wild-type mice.Good insulin sensitivity,and IL-37d&IL-33ko gene mice have worse insulin sensitivity than IL-37d transgenic mice,which proves that IL-33 deletion partially reverses the inhibitory effect of IL-37d on insulin resistance.3.Loss of IL-33 reverses the inhibition of fatty liver by IL-37dThe livers of IL-37d transgenic mice,wild-type mice and IL-37d&IL-33ko gene mice after dissection were taken from normal diet or high-fat diet,and the livers were frozen sectioned and stained with Oil Red O to detect the degree of fatty liver.Feeding with high-fat diet increased the accumulation of fat in the liver of wild-type mice,while IL-37d transgenic mice had less fat accumulation than wild-type mice,and IL-37d&IL-33ko gene mice had more liver fat accumulation than IL-37d transgenic mice.Mouse;After homogenizing the liver tissue,the supernatant was taken to detect the triglyceride(TG)content.It was found that the TG content of the IL-37d transgenic mice after high fat was significantly lower than that of the wild-type mice,while the IL-37d&IL-33ko genes were small The TG content of mice is higher than that of IL-37d transgenic mice.The above results prove that IL-33 deletion reverses the inhibition of IL-37d on fatty liver.4.IL-33 knockout reverses IL-37d’s promotion of type 2 immune response in adipose tissueTake the dissected normal diet or high-fat diet IL-37d transgenic mice,wild-type mice and IL-37d&IL-33ko gene mice subcutaneous fat and epididymal fat,culture the fat tissue in vitro for 24 hours,and detect it by ELISA The content of type Ⅱ cytokines IL-4,IL-5,and IL-13 in the supernatant IL-37d was significantly higher than that of wild-type mice,while IL-37d&IL-33ko gene mice had low type Ⅱ cytokines transgenic to IL-37d In mice,this result proves that IL-33 knockout reverses IL-37d’s promotion of type 2 immune response to adipose tissue.5.IL-33 knockout reverses IL-37d’s inhibition of adipose tissue inflammationObtain the epididymal fat of normal diet or high-fat diet IL-37d transgenic mice,wild-type mice and IL-37d&IL-33ko gene mice after dissection,the tissues were dehydrated and embedded,then paraffin sectioned,and stained with immunohistochemistry Methods The expression level of F4/80 was detected,and it was found that the wild-type mice after high fat showed a large number of crown-like structures,which were significantly more than IL-37d transgenic mice.At the same time,IL-37d&IL-33ko gene mice also had crown-like structures.More than IL-37d transgenic mice,it was proved that IL-33 knockout reversed IL-37d’s inhibition of fat tissue inflammation.II.The mechanism of IL-37d regulating IL-33/ST21.IL-37d regulates IL-33/ST2 not by affecting IL-33Take the subcutaneous fat and epididymal fat tissue of high-fat-induced WT or IL-37d transgenic mice to extract adipose tissue RNA with Trizol reagent,reverse transcription of RNA into DNA with a reverse transcription kit,and detect IL-33 by real-time fluorescent quantitative PCR There is no significant difference between IL-37d transgenic mice and wild-type mice.A549 cells transfected with IL-37d or mock were stimulated with apoptotic cells,and total cellular RNA was extracted After reverse transcription,the mRNA level of IL-33 was detected by real-time fluorescent quantitative PCR.There was also no significant difference.The above results prove that IL-37d is against IL-33/ST2 regulation does not affect IL-33.2.IL-37d regulates IL-33/ST2 not by affecting ST2Take the subcutaneous fat and epididymal fat tissue of high-fat-induced WT or IL-37d transgenic mice to extract adipose tissue RNA with Trizol reagent,reverse transcription of RNA into DNA with a reverse transcription kit,and detect ST2 mRNA by real-time fluorescent quantitative PCR Level,it is found that IL-37d transgenic mice and wild-type mice are not significantly different,which proves that IL-37d’s regulation of IL-33/ST2 does not affect ST23.IL-37d regulates IL-33/ST2 by inhibiting soluble ST2 induced by high fatTake the subcutaneous fat and epididymal fat tissue of high-fat-induced WT or IL-37d transgenic mice,extract adipose tissue RNA with Trizol reagent,reverse transcription of RNA into DNA with a reverse transcription kit,and detect sST2 by real-time fluorescent quantitative PCR The mRNA level of sST2 in the adipose tissue of mice after high fat was found to be greatly increased,while the mRNA level of sST2 in IL-37d transgenic mice was significantly lower than that of wild-type mice.Subcutaneous fat and epididymal fat tissue were cultured in vitro 24 The culture supernatant was taken at an hour to detect the content of sST2 in the supernatant by ELISA,and the same results were obtained,proving that IL-37d can inhibit sST2 induced by high fat.4.IL-37d inhibits soluble ST2 induced by TNF-α and regulates IL-33/ST2Take subcutaneous fat derived from WT or IL-37d transgenic mice,extract mesenchymal stem cells(S-ADSC)derived from subcutaneous fat tissue,and stimulate with TNF-α(10ng/ml)or PBS control for 24 hours to extract total cell RNA Reverse transcription of RNA into DNA with a reverse transcription kit,and use real-time fluorescent quantitative PCR to detect the level of sST2 mRNA;take cell culture supernatant and use ELISA to detect the content of sST2 in the supernatant TNF-α can stimulate sST2 mRNA The level and the content in the supernatant were significantly increased,while ADSCs derived from IL-37d transgenic mice expressed lower levels of sST2,proving that IL-37d can inhibit sST2 induced by TNF-α.5.IL-37d inhibits soluble ST2 by inhibiting the NF-κB pathwayTake the subcutaneous fat and epididymal fat tissue of high-fat-induced WT or IL-37d transgenic mice,extract adipose tissue RNA with Trizol reagent,reverse transcription of RNA into DNA with a reverse transcription kit,and detect NF-by real-time fluorescent quantitative PCR method The mRNA levels of IL-6,IL-1β and TNF-α regulated by the κB pathway,it was found that IL-37d transgenic mice expressed lower levels of IL-6,IL-1β and TNF-α;the subcutaneous fat was combined with RIPA lysate The epididymal adipose tissue was lysed,and the protein was extracted after high-speed centrifugation.The phosphorylation level of P65 was detected by western blot.It was found that high fat significantly increased the phosphorylation level of P65,while the phosphorylation level of adipose tissue of IL-37d transgenic mice was lower than that of wild-type mice.The above results prove that IL-37d can inhibit the NF-κB pathway,thereby inhibiting sST2.6.IL-37d pair inhibits the NF-κB pathway by binding to the receptor IL-1R8Take the subcutaneous fat of IL-37d transgenic mice,extract the mesenchymal stem cells(S-ADSC)derived from subcutaneous adipose tissue,use small interfering RNA to interfere with IL-1R8,use TNF-α(10ng/ml)or PBS control to stimulate 24 After hours,extract total cell RNA,reverse transcription of RNA into DNA with a reverse transcription kit,and detect the mRNA level of sST2 by real-time fluorescent quantitative PCR.It was found that interfering with IL-1R8 can reverse the inhibition of sST2 by IL-37d;use RIPA lysate the subcutaneous fat and epididymal fat tissue were lysed,and the protein was extracted after high-speed centrifugation.The P65 phosphorylation level was detected by western blot.It was found that interference with IL-1R8 also reversed the inhibition of IL-37d on the P65 phosphorylation level The above results prove that IL-37d is effective It inhibits the NF-κB pathway by binding to the receptor IL-1R8,thereby inhibiting sST2Conclusion1.Loss of IL-33 reverses the inhibition of IL-37d on obesity and insulin resistance2.IL-37d can inhibit obesity and insulin resistance by regulating IL-33/ST2,but does not directly affect IL-33 and ST2.3.IL-37d indirectly regulates IL-33/ST2 by inhibiting sST2.4.IL-37d inhibits NF-Kb through the receptor pathway,thereby inhibiting sST2.Innovation and significance1.It is proposed for the first time that IL-37d can inhibit obesity and insulin resistance by regulating IL-33/ST22.It is proposed for the first time that IL-37d indirectly regulates IL-33/ST2 by inhibiting sST2.3.It is proposed that the mechanism of IL-37d inhibiting sST2 is to inhibit the activation of NF-Kb through the receptor pathway.Limitation1.The mechanism of IL-37d to lose weight is not single,and there are other mechanisms to be studied besides inhibiting sST22.The specific mechanism of IL-37d inhibiting NF-κB has not yet been fully explored... |
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