| Objective:Obesity is a metabolic disorder characterized by adipose tissue deposition and chronic low-grade inflammation.Increased intestinal permeability may play an important role in the development of obesity.By observing the changes of obesity-related indexes,intestinal permeability and TLR4-MyD88-NF-κB inflammation signaling pathway in obese model mice induced by high-fat diet before and after electro-acupuncture intervention,we explore the molecular mechanism of electro-acupuncture in the treatment of obesity,and to provide theoretical and experimental basis for acupuncture treatment of obesity and its related complications.Methods:There were 130 C57BL/6 mice.Twenty of them were randomly selected as normal control group,which were feeded with non-high fat diet for 11 weeks,and then randomly divided into two group(N1 and N2),with 10 in each group.The remaining 110 mice were fed with high fat diet to establish obese mice model.After 11 weeks,mice with a body weight greater than 15% of the average body weight of the control group at the same time point were screened out as obese mice,which were randomly divided into six groups: model group(M1 and M2),electro-acupuncture treatment group(A1,A2,A3 and A4).10 mice were randomly selected near the mean body weight of the mice that were failed to establish obesity models,to form the obesity resistance group(R).Mice in N1 and M1 groups were drawn materials at the end of modeling.The mice in A1,A2,A3 and A4 groups were treated with electro-acupuncture after modeling,and the corresponding treatment cycles were 1w,2w,3w and 4w,respectively.The mice in N2,M2 and R groups were only tied up without needles.were sampled at the end of the treatment cycle.The mice in all groups were drawn materials at the end of the treatment cycle.The body weight growth value,Lee’s index and fat coefficient of mice in each group were calculated.The serum levels of CHO,TG,HDL-C and LDL-C of mice in each group were detected by automatic biochemical analyzer.The levels of IL-1beta,TNF-alpha,IL-4,IL-10,LP,ADPN in serum,LPS,PGN in plasma and IL-1beta,IL-10 in colon of mice in each group were detected by ELISA.HE staining was used to observe the morphological size of adipocytes,pathological structure and chronic inflammation of colon.IHC staining was used to detect the protein distribution density of intestinal tight junction protein ZO-1 and Claudin-2.The mRNA expression of TLR4,MyD88 and NF-kappa B in colon tissue was detected by q-PCR.The obese mice model induced by high fat diet was evaluated through the comparison between M1 and N1 group.By comparing the indices of mice in N2,M2 and R group,the effects of high fat diet on inducing obesity in different phenotypes of mice(obesity-prone and obesity-resistant)were explored.Through the comparison between model group and electro-acupuncture treatment group,the effect of electro-acupuncture intervention on obesity was determined.By comparing A2,A3,A4 with A1,we determined whether the effect of electro-acupuncture intervention on improving obesity index was enhanced by prolonging the treatment cycle.Results:1.After 11 weeks of high fat feeding,59 mice reached the obesity standard,and the success rate of modeling was about 55.66%(4 accidental deaths).After 11 weeks of feeding,the weight of mice in N1 and M1 groups increased.The body weight gain,Lee’s index,fat coefficient,the serum levels of CHO,TG,IL-1beta,TNF-alpha(P < 0.01),LDL-C(P < 0.05)in M1 group were significantly higher than those in N1 group,and HDL-C levels were significantly lower than that in N1 group(P < 0.01).Under light microscope(HE × 400),the adipocytes of mice in group M1 were larger,and of different shapes and sizes,while those in group N1 were smaller,different in shape and similar in size.Compared with group N1,in a single field of vision,the number of adipocytes decreased,and the cell length and short diameter increased in M1 group.2.Comparisons among groups not receiving electro-acupuncture treatment: At the end of the treatment period,the body weight,Lee’s index,fat coefficient,CHO,TG,LDL-C,LP,IL-1 beta,TNF-alpha in M2 group were significantly higher than those in N2 group(P < 0.01)and R group(P < 0.01),while HDL-C,ADPN,IL-4,IL-10 levels were lower than those in N2 group(P < 0.01)and R group(P < 0.01).Compared with N2 group,there were no differences in body weight,fat coefficient,CHO,LP,ADPN,IL-4,IL-10 in R group,but Lee’s index,TG,LDL-C,IL-1beta and TNF-alpha were higher than N2 group(P < 0.05),and HDL-C was lower than that in N2 group(P < 0.01).Comparisons between electro-acupuncture treatment groups and model groups:Compared with M2 group,mice in group A1,the body weight,TNF-alpha(P < 0.01),TG,LP(P < 0.05)levels decreased,while IL-10 levels increased(P < 0.05),and the other indicators showed no difference.Compared with M2 group,the body weight,fat coefficient,CHO,TG,LDL-C,LP and TNF-alpha levels in A2 group decreased(P < 0.01),while the levels of IL-10,IL-4(P < 0.01)and ADPN(P < 0.05)increased.Compared with M2 group,mice in group A3,the body weight,Lee’s index,fat coefficient,CHO,TG,LDL-C,LP,IL-1beta,TNF-alpha levels decreased(P < 0.01),while the contents of HDL-C,ADPN,IL-4,and IL-10 increased(P < 0.01).Compared with the M2 group,the results of the A4 group were consistent with those of the A3 group.Comparisons between electro-acupuncture treatment groups:Compared with group A1,the fat coefficient,TG,LP(P < 0.01),CHO,LDL-C,TNF-alpha(P < 0.05)levels in group A2 decreased,while the levels of IL-4 and IL-10 increased(P < 0.01),and the other indicators showed no difference.Compared with group A1,except for the same change trend as group A2,the body weight and the level of IL-1beta decreased(P < 0.01),while the levels of HDL-C(P < 0.01)and ADPN(P < 0.05)increased.Compared with A1 group,A4 group showed the same trend as A3 group.When the treatment cycle was prolonged,the effect of electro-acupuncture on obesity was enhanced.3.Comparisons among groups not receiving electro-acupuncture treatment: At the end of the electro-acupuncture treatment period,mice in the M2 group,the levels of IL-1β in colon,LPS and PGN in plasma were higher than those in the N2 group(P < 0.01)and the R group(P < 0.01 or P < 0.05),while the levels of IL-10,ZO-1 and Claudin-2 were lower than those in the N2 group(P < 0.01)and the R group(P < 0.01).Compared with N2 group,the contents of IL-1beta,LPS and PGN increased(P < 0.05),while IL-10 and ZO-1 decreased(P < 0.05)in R group,and there was no difference in Claudin-2.Comparisons between electro-acupuncture treatment groups and model groups:Compared with the M2 group,only the contents of IL-1β was decreased(P < 0.05)and the contents of IL-10 was increased(P < 0.01)in the A1 group,while the other indicators were not different.Compared with M2 group,the change trend of group A2 was the same as that of group A1.Compared with the M2 group,in addition to the decrease of IL-1β levels(P < 0.01)and the increase of IL-10 levels(P < 0.01),the A3 group had ZO-1(P < 0.05),Claudin-2(P < 0.01)expression increased.Compared with M2 group,mice in A4 groups,the levels of IL-1β(P < 0.01),LPS(P < 0.05)decreased,while the levels of IL-10,Claudin-2(P < 0.01),ZO-1(P < 0.05)increased,and the content of PGN did not change.Comparisons between electro-acupuncture treatment groups:Compared with the A1 group,the A2 and A3 groups only had IL-10 increased(P < 0.01),and the other indicators showed no difference.Compared with the A1 group,in addition to the increase in IL-10(P < 0.01),Claudin-2 expression also increased(P < 0.05)in the A4 group.Under light microscope(HE × 400),the colonic morphology structure of N2 group had no obvious pathological changes,with intact structure and no infiltration of inflammatory cells.Compared with the N2 group,the M2,R and electro-acupuncture treatment groups all had inflammatory cell infiltration,intestinal gland vacuolar degeneration pathological damage in varying degrees,and the model group had the most obvious pathological damage.Compared with the model group M2,the A4 group had some improvement in inflammatory cell infiltration,myocyte degeneration and necrosis.4.Comparisons among groups not receiving electro-acupuncture treatment: The gene expression of TLR4,MyD88,NF-kappa B in M2 group was significantly higher than that in N2 group(P < 0.01)and R group(P < 0.01).Compared with N2 group,the gene expression of TLR4,MyD88 increased in R group(P < 0.01),but the gene expression of NF-kappa B had no difference.Compared with M2 group,the gene expression of TLR4,MyD88,NF-kappa B in A1,A2,A3 and A4 groups decreased(P < 0.01).Compared with group A1,the expression of TLR4,MyD88,NF-kappa B gene in group A3 and A4 were significantly decreased(P < 0.01),but the gene expression of NF-kappa B in group A2 was higher than that in group A1(P < 0.01).Conclusion:High-fat diet can induce obesity with fat accumulation and chronic systemic inflammation,and increase intestinal permeability.Electroacupuncture can improve obesity induced by high-fat diet by inhibiting TLR4-MyD88-NF-kappa B inflammatory signaling pathway and reducing intestinal permeability. |
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