| Objectives : Non-alcoholic fatty liver disease(NAFLD)has become the largest chronic liver disease in China and is closely related to insulin resistance(IR)and metabolic inflammation.Electroacupuncture(EA)is widely used in obesity and IR-related diseases,but its mechanism of action on NAFLD is still unclear.Inflammatory factors released in the process of cell scorching aggravate metabolic inflammation and promote the progress of NAFLD.Therefore,in this study,NAFLD mice were used as a model to observe the effect of EA on inflammatory factors and insulin sensitivity in liver tissue of NAFLD mice and pyroptosis induced by NLRP3,and to explore the molecular mechanism of EA in improving NAFLD,so as to provide theoretical and experimental basis for clinical application of EA in the prevention and treatment of NAFLD.Methods:Sixty eight-week-old SPF C57BL/6J mice were randomly selected and 15 mice were fed with common diet(CD)as the normal control group,and the rest mice were fed with high-fat diet(HFD)as the model group for NAFLD.At the end of the 12 th week,five mice were randomly selected from the control group and the model group and killed.The body weight and Lee’s index were used as the evaluation criteria for obesity to determine whether the experimental obesity mouse model was constructed.Ten mice from 40 model group were randomly selected as preventive treatment of disease(PTDG),and from the 13 th week,the mice in the preventive group were treated with EA three times a week;the remaining 30 model mice continued to be fed with HFD without any treatment.At the end of the 24 th week,five mice were randomly selected from the control group,the EA group(Zusanli and Sanyinjiao)and the model group to kill,to verify whether the NAFLD model was successfully constructed,and to evaluate the efficacy of the treatment group.The remaining 25 mice in the model group were randomly assigned to model group,EA group,non-meridian non-acupoint group,agonist group,EA+inhibition group,5 mice in each group,and continued to be fed with HFD.The treatment group continued to be fed with HFD;the control group continued to receive ordinary feed.At the 25 th week,the mice in each group were given EA and drug intervention: the control group and model group were not given any treatment;EA group and non-acupoint group were treated with EA;In the agonist group,only SIRT1 agonist(SRT1720)was injected intraperitoneally without EA;The EA+inhibition group was also given intraperitoneal injection of SIRT1 inhibitor(EX-527)while undergoing EA treatment.The treatment group was treated 3 times a week for 8 weeks;at the end of the 32 nd week,the mice were killed and samples were taken for testing.Regularly observe the general situation,food consumption and body weight of mice,measure the length of anus and nose,and calculate Lee’s index.The liver index was calculated after the mice were killed.Blood was taken from the apex of the heart to retain the serum,and the contents of biochemical indexes(AST,ALT,TG and TC)of mice in each group were measured.Detection of serum CRP,TNF-α,IL-1β,IL-10,IL-18 and insulin content by enzyme-linked immunosorbent assay(ELISA).The liver was preserved and embedded in paraffin,and HE staining was performed to evaluate the fatty change and inflammation of the live.The infiltration of hepatic macrophages was analyzed by immunohistochemistry.Detection of SIRT1,NLRP3,GSDMD,IL-1βand IL-18 m RNA expression level in mouse liver by q RT-PCR;Western blotting method was used to detect the protein expression level of SIRT1,NLRP3 and GSDMD in mouse liver tissue.The above data were statistically analyzed.Results:1.EA can reduce the weight of NAFLD mice and reduce the serum transaminase and blood lipid levels of mice(1)The results of body weight showed that the weight of mice in the model group increased gradually after feeding with HFD.At the end of the 12 th week,compared with the control group,the weight of the model group increased significantly.Compared with the normal group and before the model,the weight of mice in the model group increased significantly,and exceeded the average weight of the normal group by more than 20%,which was judged as an experimental obesity mouse model.Compared with the model group,the body weight of mice in the treated group did not increase significantly after EA treatment,and the body weight of the treated group began to decrease gradually from the 16 th week.At the end of the 24 th week,compared with the model group,the weight of mice in the control group and the treatment group was statistically different;Compared with the normal control group,there was no statistical difference in the weight of the mice in the treatment group,while the weight of the mice in the model group increased significantly with statistical significance.From the 25 th week,the weight of mice in the treatment group remained stable.From the 28 th week,the weight of mice in the EA group and the agonist group gradually decreased significantly,which was statistically significant compared with the model group;The weight of mice in the non-acupoint group also decreased slightly,but there was no significant difference between the non-acupoint group and the model group.There was no statistical difference between the EA+inhibitor group and the model group.By the end of the 32 nd week,compared with the control group,the weight of the model group mice increased significantly.Compared with the model group,the weight of mice in the treatment group,the EA group and the agonist group decreased significantly;However,the weight loss of mice in non-meridian non-acupoint group and EA + inhibitor group was not significant,and the difference was not statistically significant.Compared with the EA group,the body weight of mice in the non-meridian non-acupoint group and EA + inhibitor group was statistically significant;the body weight of mice in the agonist group was not statistically significant.At the end of the 12 th week,compared with the normal group,the weight of the model group increased significantly,and the Lee’s index increased significantly.From the 16 th week,compared with the model group,the Lee’s index of mice in the treated group decreased significantly.At the end of the 24 th week,compared with the control group,there was no statistical difference in the Lee’s index in the treatment group;Compared with the model group,the Lee’s index of mice in the normal group and the treated group had statistical differences.From the 25 th week,the Lee’s index of mice in the treated group remained stable.From the 28 th week of intervention,compared with the model group,the Lee’s index of the EA group and the agonist group both decreased significantly.The Lee’s index of mice in the non-acupoint group decreased,but there was no significant difference between the non-acupoint group and the model group.There was no statistical difference between the EA plus inhibition group and the model group.By the end of the 32 nd week,compared with the control group,the Lee’s index of mice in the model group increased significantly.Compared with the model group,the Lee’s index of mice in the preventive treatment group,the EA group and the agonist group decreased with statistical significance.However,the Lee’s index of mice in the non-meridian non-acupoint group and the EA+inhibitor group did not decrease significantly,and the difference was not statistically significant.Compared with the EA group,the Lee’s index of mice in the non-meridian non-acupoint group and EA+inhibitor group was statistically significant;The Lee’s index of mice in the agonist group was not statistically significant.(2)The results of serum AST,ALT,TG and TC showed that compared with the control group,the levels of serum ALT,AST,TG and TC in the model group were significantly higher;Compared with the model group,the serum AST,ALT,TG and TC contents of the mice in the treatment group,the EA group and the agonist group decreased significantly;However,the serum AST,ALT,TG and TC contents of mice in non-meridian non-acupoint group and EA+inhibitor group did not significantly decrease.Compared with the EA group,the contents of AST,ALT,TG and TC in serum of mice in the non-meridian non-acupoint group and EA+inhibitor group were significantly increased,with statistical significance.The contents of AST,ALT,TG and TC in serum of mice in the agonist group were basically the same,and there was no significant difference.2.EA reduces inflammation in NAFLD mice and improves insulin sensitivity(1)The results of serum CRP,TNF-α,IL-1 and IL-18 showed that compared with the control group,the serum CRP,TNF-α,IL-1β and IL-18 in the model group were significantly higher.After EA and drug intervention,compared with the model group,the content of serum CRP,TNF-α,IL-1βand IL-18 in the treated group,EA group and agonist group decreased significantly;There was no significant difference in the contents of serum CRP,TNF-α,IL-1βand IL-18 between the non-meridian non-acupoint group and the EA+inhibitor group.Compared with the EA group,the levels of inflammatory factors CRP,TNF-α,IL-1βand IL-18 in the serum of mice in the treatment group and the non-acupoint group were lower,with significant statistical differences;There was no significant difference in serum inflammatory factors in the agonist group;The content of serum CRP,TNF-α,IL-1βand IL-18 in the EA+inhibitor group was significantly different.(2)The results of serum IL-10 showed that compared with the control group,the level of serum IL-10 in the model group was significantly lower.Compared with the model group,the serum IL-10 of mice in the treatment group,the EA group and the agonist group increased significantly.There was no significant difference in serum IL-10 between the non-meridian non-acupoint group and the EA+inhibitor group compared with the model group.Compared with the EA group,the level of inflammatory factor IL-10 in the blood of the mice in the treatment group increased,and there was no significantly difference;the level of serum IL-10 in the agonist group was not statistically significant.(3)The results of insulin sensitivity showed that the values of fasting blood glucose(FBG),fasting insulin(FINS)and insulin resistance index(HOMA-IR)in the model group were significantly higher than those in the control group.Compared with the model group,the FBG,FINS and HOMA-IR values of mice in the treatment group,the EA group and the agonist group decreased.Compared with the EA group,the FBG,FINS and HOMA-IR of mice in the treatment group decreased,but there was no statistical difference.There was no significant difference between FBG,FINS and HOMA-IR in the agonist group.However,the increase of FBG,FINS and HOMA-IR values in non-acupoint group and EA plus inhibitor group was statistically significant.3.EA can improve the liver histopathology of NAFLD mice and inhibit the infiltration of liver macrophages(1)HE staining results of liver tissue showed that NAFLD mouse model was successfully constructed.The liver tissue structure of mice in the control group has no obvious fat lesion and inflammatory cell infiltration,the liver cells are arranged in order,without damage,the nucleus has no fat drop vacuole in the central cytoplasm of the cell,no inflammation and liver lesion,and small fat drops are occasionally seen.HE staining of mice in model group,EA+inhibitor group and non-acupoint group showed different degrees of lipid accumulation and inflammatory infiltration in hepatic lobule and portal area;Circular lipid droplets of different sizes can be seen in the cytoplasm,bullous and vesicular lipid droplets alternate,the nucleus is inclined to the edge,and balloon-like degeneration can be seen.The lipid accumulation in the liver of mice in the preventive treatment group,the EA group and the agonist group was significantly reduced,mainly in the form of vesicular lipid droplets,and the inflammatory infiltration was inhibited;Among them,the lipid accumulation and inflammatory infiltration in the treatment group were better than those in the EA group and the agonist group.The pathological changes in the EA group were basically similar to those in the agonist group.(2)The results of liver macrophage F4/80 and CD68 infiltration showed that compared with the control group,the number of liver F4/80 and CD68 staining positive cells in the model group was significantly increased;Compared with the model group,the number of liver F4/80 and CD68 positive cells in the treated group,the EA group and the agonist group decreased significantly;The number of F4/80 and CD68 staining positive cells in the liver of mice in the non-acupoint group was basically the same as that in the model group.4.EA increases the expression level of SIRT1 mRNA in liver tissue of NAFLD mice,inhibits the expression of NLRP3 m RNA,and reduces the expression level of GSDMD,IL-1β and IL-18 m RNA(1)The results of SIRT1 m RNA showed that the expression level of SIRT1 m RNA in liver tissue of model group was significantly lower than that of normal group.Compared with the model group,the expression level of SIRT1 m RNA in the liver of mice in the treatment group,the EA group and the agonist group increased significantly.Compared with the model group,the expression level of SIRT1 m RNA in the liver tissue of mice in the agonist group was significantly higher than that in the model group.There was no significant difference in the expression level of SIRT1 m RNA in the liver between the non-meridian non-acupoint group and the model group.There was no significant difference in the expression level of SIRT1 m RNA in the liver between the EA group and the agonist group.There was no significant difference in the expression level of SIRT1 m RNA between the EA+inhibitor group and the model group.(2)The results of NLRP3 and GSDMD m RNA showed that the expression level of NLRP3 and GSDMD m RNA in the liver tissue of the model group was significantly higher than that of the normal group.Compared with the model group,the expression level of NLRP3 and GSDMD m RNA in the liver tissue of mice in the treatment group,the EA group and the agonist group decreased significantly.There was no significant difference in the expression level of NLRP3 and GSDMD m RNA in the liver between the EA group and the agonist group;there was no significant difference between the non-meridian non-acupoint group and the model group.The expression level of NLRP3 and GSDMD m RNA gene in the agonist group was significantly lower than that in the model group.Compared with the model group,the expression level of NLRP3 and GSDMD m RNA in liver tissue in the EA+inhibitor group decreased,but the expression level of NLRP3 and GSDMD m RNA was significantly higher than that in the EA group.(3)The results of IL-1β and IL-18 m RNA showed that the expression level of IL-1β and IL-18 m RNA in the liver tissue of the model group was significantly higher than that of the normal group.Compared with the model group,the expression levels of IL-1β and IL-18 m RNA in liver tissue of mice in the treatment group,EA group and agonist group decreased significantly.The expression level of IL-1β and IL-18 m RNA in the liver tissue of mice in the agonist group was significantly lower than that in the model group.There was no significant difference between the non-meridian non-acupoint group and the model group.Compared with the model group,the expression level of IL-1β and IL-18 m RNA in liver tissue of the EA+inhibitor group decreased,but the expression level of IL-1β and IL-18 m RNA increased significantly compared with the EA group.5.EA increases the expression of SIRT1 protein in liver tissue of NAFLD mice,inhibits the expression of NLRP3 protein,and reduces the expression of GSDMD protein(1)The results of SIRT1 protein expression showed that the expression of SIRT1 protein in the liver tissue of the model group was significantly higher than that of the normal group.Compared with the model group,the expression of SIRT1 protein in liver tissue of mice in the treatment group,EA group and agonist group increased significantly.There was no significant difference in the expression of SIRT1 protein in the liver tissue between the EA group and the agonist group.There was no significant difference in SIRT1 protein in liver tissue between non-meridian non-acupoint group and model group mice.The expression of SIRT1 protein in the liver tissue of mice in the agonist group was significantly lower than that in the model group.There was no significant difference in the expression of SIRT1 protein in liver tissue between the EA+inhibitor group and the model group.(2)The results of NLRP3 and GSDMD protein expression showed that the expression of NLRP3 and GSDMD protein in the liver tissue of the model group was significantly higher than that of the normal group;Compared with the model group,the protein expression of NLRP3 and GSDMD in liver of mice in the treatment group,EA group and agonist group decreased significantly.There was no significant difference in the expression of SIRT1 protein in liver tissue between the EA group and the agonist group;the expression of NLRP3 and GSDMD protein in liver tissue of mice in agonist group was significantly lower than that in model group.There was no significant difference between the non-meridian non-acupoint group and the model group.Compared with the model group,the expression of NLRP3 and GSDMD protein in the liver tissue of the EA+inhibitor group decreased,but there was no statistical significance.Compared with the EA group,the expression of NLRP3 and GSDMD protein in the liver tissue of the mice was significantly increased.Conclusion: 1.EA(Zusanli and Sanyinjiao points)can effectively reduce the body weight and Lee’s index of NAFLD mice,and reduce liver steatosis and inflammatory reaction of mice;However,the non-meridian non-acupoint group could not effectively reduce the body weight and Lee’s index of mice,indicating the specificity of Zusanli and Sanyinjiao treatment;EA+inhibitor cannot effectively reduce the body weight and Lee’s index of mice,indicating that SIRT1 inhibitor may partially inhibit the therapeutic effect of EA;Therefore,it is speculated that EA may also play a role in treating NAFLD through other ways.2.EA can reduce the infiltration of macrophages in liver tissue of NAFLD mice,and reduce the levels of CRP,TNF-α,IL-1β and IL-18 in peripheral blood;increase the level of serum IL-10;reduce blood glucose and insulin levels and improve IR status.Non-point therapy cannot inhibit the infiltration of macrophages and reduce the level of related inflammatory factors;SIRT1 inhibitor may partially block the anti-inflammatory effect of EA.3.EA can promote the expression level of SIRT1 mRNA in liver tissue of NAFLD mice,down-regulate the level of NLRP3 m RNA,inhibit the activation of inflammatory factors,and reduce the expression level of IL-1β and IL-18 m RNA in liver tissue.Non-point therapy and SIRT1 inhibitor cannot achieve the same effect,and the anti-inflammatory effect of EA may be partially blocked by SIRT1 inhibitor.4.EA can increase the expression of SIRT1 protein in liver tissue,down-regulate the level of NLRP3 protein,inhibit the activation of NLRP3 inflammatory corpuscles,reduce the protein level of NLRP3 and GSDMD,inhibit liver cell pyrosis,and improve NAFLD.SIRT1 inhibitor partially blocked the down-regulation of NLRP3 and GSDMD by EA.5."Acupuncture for preventive treatment of disease" can effectively prevent the occurrence and development of NAFLD mouse model.After the formation of obesity model in mice fed with HFD for 12 weeks,EA treatment was started at the 13 th week.The liver steatosis and inflammation,SIRT1,NLRP3,GSDMD-related protein and gene expression in mice in the treatment group were the lowest.Acupuncture without meridian and non-acupoint has no obvious effect on the inflammatory state and insulin sensitivity of NAFLD mice.SIRT1 inhibitor can partially block the effect of EA on activating SIRT1,reduce the anti-inflammatory effect of EA to a certain extent,and increase insulin sensitivity.To sum up,EA may be one of the molecular mechanisms for improving the NAFLD mouse model by regulating SIRT1/NLRP3 to inhibit hepatocyte pyroptosis. |
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