| Object:After obesity,the increased release of adipose tissue derived miR-4431,miR-548ab/ag,miR-450a-5p can lead to Insulin Resistance（IR）in target organs and induce Type 2 Diabetes Mellitus（T2DM）.However,the molecular mechanism of obesity leading to increased release of miR-4431,miR-548ab/ag,miR-450a-5p from adipose tissue has not been reported in the literature.Based on the collecting of serum and abdominal adipose tissue samples from normal weight and obese individuals,the culturing of 3T3-L1mouse adipocytes in vitro,and the constructing of diet induced obese mice model,determine whether the increase of palmitic acid（PA）content after obesity can activate NF-κB inflammatory signal pathway induces endoplasmic reticulum stress,and then promotes the release of miR-4431,miR-548ab/ag,miR-450a-5p from exosomes which are secreted by adipose tissue/cell.It will provide experimental basis for further clarifying the molecular mechanism of obesity inducing T2DM and finding molecular targets for prevention and treatment of T2DM.Method:1.Human tissue sample experiment:（1）From September 2021 to June 2022,serum of normal weight（n=24）,obesity（n=24）and T2DM individuals（n=12）and abdominal adipose tissue samples（normal weight individuals n=6,obese individuals n=6）were collected in the First Affiliated Hospital of Shihezi University School of Medicine.（2）Detected the concentration of PA in serum of individuals by Elisa;detected the expression level of p-p65,the key factors of NF-κB inflammatory signaling pathway,inflammatory factors（IL-6,MCP-1,TNF-α）,and endoplasmic reticulum stress markers（GRP78,ATF6,IRE1α,PERK）in adipose tissue by q RT-PCR and Western Blot,and detected the content of miR-4431,miR-548ab/ag,miR-450a-5p in adipose tissue and serum.To analyze whether the increase of PA content after obesity will affect the activation of NF-κB,ER stress pathway and the increase of miR-4431,miR-548ab/ag,miR-450a-5p.2.Cell experiment in vitro:（1）3T3-L1,mouse embryonic fibroblast,was cultured and then induced into mature adipocytes in vitro,next,divided them into NC group,200μM PA group,500μM PA group;NC control group,200μM PA group,200μM PA treatment of cells with 5μM Bay11-7082 block p-p65 group（PA+Bay11-7082）;NC control group,200μM PA group,200μM PA treatment of cells with 200μM TUDCA block endoplasmic reticulum stress group（PA+TUDCA）,after 24h,collected samples for the next experimental operation.（2）Detected the expression level of the key factors of NF-κB inflammatory signaling pathway（p-p65,p65）,inflammatory factors（IL-6,MCP-1,TNF-α）,and endoplasmic reticulum stress markers（GRP78,ATF6,IRE1α,PERK）by q RT-PCR and Western Blot.Collected cell culture supernatant,q RT-PCR was used to detect of miR-4431,miR-548ab/ag,miR-450a-5p content in cell supernatant and cell precipitation.（3）Collected the supernatant of cell culture and extract the exosomes by VEXTMExosome Isolation Reagent kit,transmission electron microscope was used to observe the morphology of exocrine body,detection of exocrine particle size by NTA nanoparticle tracking analysis technology,detected the protein expression level of exosomes marker HSP70 and TSG101 by Western blot,q RT-PCR was used to detect of miR-4431,miR-548ab/ag,miR-450a-5p content in exosomes.3.Animal experiment in vivo:（1）12 male C57BL/6 mice aged 4 weeks were fed,after 1 week of adaptive feeding,they were fed with normal diet（Normal Chow Diet,NCD,n=6）and high-fat diet（High FAT Diet,HFD,n=6）respectively,dynamic monitoring of body weight,continue feeding until the 11th week.（2）In order to successfully build an obese mouse model,provide male C57BL/6 mice,which are 4weeks old with high-fat diet for 7 weeks.Then,they were segmented into simple high-fat diet group（HFD,n=6）,high-fat diet group with intraperitoneal injection of p-p65 blocker（HFD+Bay11-7082,n=6,2.5mg/kg body weight,once a week）and high-fat diet group with intraperitoneal injection of endoplasmic reticulum stress blocker（HFD+TUDCA,n=6,250mg/kg body weight,four times a week）,continuous injection for 4weeks,dynamic monitoring of body weight in mice.（3）In order to evaluate glucose tolerance and insulin sensitivity of mice,we adopted Intraperitoneal Glucose Tolerance Trial（IPGTT）and Insulin Tolerance Test（ITT）.Collected the serum of mice by the method of taking blood from the inner canthus,detection of blood glucose and PA content in mice.After sacrificing mice,collected adipose tissue and liver tissue of various parts of mice,detection of miR-4431,miR-548ab/ag,miR-450a-5p content and the expression level of p-p65/IL-6/MCP-1/TNF-α/GRP78/ATF6/IRE1α/PERK by q RT-PCR and Western Blot techniques.To determine whether the increase of PA content after obesity induces ER stress by activating NF-κB signal pathway,resulting in the increase of miRNAs.4.Statistical methods:Use SPSS 26.0 statistical analysis software to process and analyze the experimental data.The data conforms to normal distribution and is analyzed with t-test,if the experimental data do not conform to the normal distribution,we used nonparametric rank sum test for analysis.P<0.05,the difference is statistically significant.Results:1.NF-κB inflammatory signal pathway and endoplasmic reticulum stress in abdominal adipose tissue of obese individuals are activated,and miR-4431,miR-548ab/ag,miR-450a-5p content in serum and abdominal adipose tissue is increased.（1）Compared with normal weight subjects,the weight,Body mass index（BMI）,waist circumference,fasting blood glucose,triglycerides,total cholesterol and low-density lipoprotein of obese subjects increased significantly,the difference is statistically significant,P<0.05.（2）Compared with normal weight subjects,the expression level of p-p65,inflammatory factors（IL-6,MCP-1,TNF-α）and endoplasmic reticulum stress markers（GRP78,ATF6,PERK,IRE1α）in abdominal adipose tissue of obese subjects increased significantly,the difference is statistically significant,P<0.05.（3）Compared with normal weight subjects,the content of miR-4431,miR-548ab/ag,miR-450a-5p in abdominal adipose tissue and serum of obese subjects were noteworthy increased,and the difference is statistically significant,P<0.05.2.High concentration PA can activate NF-κB/endoplasmic reticulum stress pathway,promoting the release of miR-4431,miR-548ab/ag,miR-450a-5p from adipocyte exosomes.（1）Compared with untreated the control group,after 24h of 200μM and 500μM PA treatment,the m RNA and protein expression levels of p-p65,the key protein of NF-κB signal pathway,inflammatory factors（IL-6,MCP-1,TNF-α）,and endoplasmic reticulum stress markers（GRP78,ATF6,PERK,IRE1α）in3T3-L1 adipocytes increased significantly,and the content of miR-4431,miR-548ab/ag,miR-450a-5p in cell precipitation and cell culture supernatant increased significantly.The difference is statistically significant,P<0.05.（2）Compared with the untreated control group,after 24h of 200μM and 500μM PA treatment,the protein expression level of the exosomes marker Hsp70 and TSG101 increased obviously,in addition,miR-4431,miR-548ab/ag,miR-450a-5p content in exosomes increased clearly.The difference is statistically significant,P<0.05.3.Blocking NF-κB inflammatory signal pathway can significantly reverse the promotion of PA on the endoplasmic reticulum stress of adipocytes and the release of miR-4431,miR-548ab/ag,miR-450a-5p from the exosomes.（1）Compared with the PA treatment group,after 24h of adding p-p65 blocker（Bay 11-7082）at the same time of 200μM PA treatment,the m RNA and protein expression levels of p-p65,the key protein of NF-κB signal pathway,inflammatory factors（IL-6,MCP-1,TNF-α）,and endoplasmic reticulum stress markers（GRP78,ATF6,PERK,IRE1α）in 3T3-L1 adipocytes were significantly reduced,and miR-4431,miR-548ab/ag,miR-450a-5p in cell precipitation and cell culture supernatant diminished obviously.The difference is statistically significant,P<0.05.（2）Compared with the PA treatment group,after 24h of adding p-p65 blocker（Bay 11-7082）at the same time of 200μM PA treatment,the protein expression level of the exosomes markers Hsp70 and TSG101 decreased obviously;miR-4431,miR-548ab/ag,miR-450a-5p content in exosomes decreased clearly.The difference is statistically significant,P<0.05.4.Blocking endoplasmic reticulum stress can significantly reverse the promotion of PA on the release of miR-4431,miR-548ab/ag,miR-450a-5p from adipocyte exosomes.（1）Compared with the PA treatment group,after 24h of after adding endoplasmic reticulum stress blocker（TUDCA）at the same time of 200μM PA treatment,the m RNA and protein expression levels of endoplasmic reticulum stress markers（GRP78,ATF6,PERK,IRE1α）in 3T3-L1 adipocytes were significantly reduced,and miR-4431,miR-548ab/ag,miR-450a-5p in cell precipitation and cell culture supernatant diminished obviously.The difference is statistically significant,P<0.05.（2）Compared with the PA treatment group,after 24h of after adding endoplasmic reticulum stress blocker（TUDCA）at the same time of 200μM PA treatment,the protein expression level of the exosomes markers Hsp70 and TSG101 decreased obviously;miR-4431,miR-548ab/ag,miR-450a-5p content in exosomes decreased clearly.The difference is statistically significant,P<0.05.5.p-p65 blocker can significantly inhibit the inflammatory reaction and endoplasmic reticulum stress of epididymal adipose tissue in obese mice,reducing the content of miR-4431,miR-548ab/ag,miR-450a-5p in epididymal adipose tissue and serum,and improving glucose tolerance and insulin sensitivity in mice.（1）After 11 weeks of feeding healthy 4-week-old male C57BL/6 mice with high-fat diet,the weight,liver,Visceral White Adipose Tissue（Vis WAT）,Perirenal White Adipose Tissue（Per WAT）,Epididymal White Adipose Tissue（Epi WAT）and Subcutaneous White Adipose Tissue（Sub WAT）of HFD group mice were obviously bigger than those of NCD group,blood glucose level and PA content in serum were obviously higher than NCD group.The difference is statistically significant,P<0.05,it is showed that the diet induced obese mouse model has been successfully constructed.（2）Compared with the control group,the expression level of p-p65,the key protein of NF-κB signal pathway,inflammatory factors（IL-6,MCP-1,TNF-α）and endoplasmic reticulum stress markers（GRP78,ATF6,PERK,IRE1α）in adipose tissue of epididymis of obese mice increased significantly;the content of miR-4431,miR-548ab/ag,miR-450a-5p in epididymal adipose tissue and serum of obese mice was significantly increased.In the meantime,the glucose tolerance and insulin sensitivity of obese mice were damaged significantly.The difference is statistically significant,P<0.05.（3）Compared with the control group,p-p65 blocker（Bay 11-7082）can significantly inhibit the expression level of p-p65,the key protein of NF-κB signal pathway,inflammatory factors（IL-6,MCP-1,TNF-α）and endoplasmic reticulum stress markers（GRP78,ATF6,PERK,IRE1α）in epididymal adipose tissue of obese mice;p-p65 blocker（Bay 11-7082）can significantly reduce the content of miR-4431,miR-548ab/ag,miR-450a-5p in epididymal adipose tissue and serum of obese mice.Simultaneously,p-p65blocker（Bay 11-7082）can sharply restore the impaired glucose tolerance and insulin sensitivity of obese mice.The difference is statistically significant,P<0.05.6.Endoplasmic reticulum stress blocker can significantly reduce the content of miR-4431,miR-548ab/ag,miR-450a-5p in epididymal adipose tissue and serum of mice,and restore the impaired glucose tolerance and insulin sensitivity of obese mice.Compared with the control group,endoplasmic reticulum stress blocker（TUDCA）can sharply inhibit the expression of endoplasmic reticulum stress markers（GRP78,ATF6,PERK,IRE1α）in epididymal adipose tissue of obese mice;endoplasmic reticulum stress blocker（TUDCA）can significantly reduce the content of miR-4431,miR-548ab/ag,miR-450a-5p in epididymal adipose tissue and serum of obese mice.Meanwhile,endoplasmic reticulum stress blocker（TUDCA）can apparently restore the impaired glucose tolerance and insulin sensitivity of obese mice.The difference is statistically significant,P<0.05.Conclusion:The increase of palmitic acid content after obesity can induce endoplasmic reticulum stress by activating NF-κB inflammatory signal pathway and promote the release of miR-4431,miR-548ab/ag,miR-450a-5p from adipocyte exosomes. |
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