| ObjectivesObesity is a pathological state that can affect human health,which is due to the imbalance of body energy between intake and consumption.Digestion and absorption of nutrients are carried out and completed in the gastrointestinal tract.The function of gastrointestinal motility directly or indirectly affects the balance of energy metabolism in the body.The intestinal inflammation is an important factor that leads to gastrointestinal motility abnormalities.Active intervention to intestinal inflammation is the key to improve gastrointestinal motility function and treat obesity with insulin resistant.Therefore,based on previous studies,this experiment which take obese rats with insulin resistance as a model,breaking in from the nuclear factor kappa B(NF-κB)mediated the anti-inflammatory pathway regulated by silent information regulation factor 1(SIRT1),is to investigate the molecular mechanism of electroacupuncture(EA)in improving gastrointestinal motility of obesity with insulin resistance by controlling inflammation of the small intestine.MethodsOne hundred and twenty male SPF Wistar rats were 8 weeks old.Fifteen rats were fed with normal diet,while the remaining 105 rats were fed with high-fat diet for modeling.After 8 weeks,13 rats which fed with normal diet were randomly selected from the normal group,while 65 rats which reached the obesity standard were divided into model group(MOD),EA group,EA combined inhibitor group(EI),inhibitor(INH)group and resveratrol(RSV)group,with 13 rats in each group.Three rats from each group were randomly selected for hyperinsulinemia-euglycemic clamp to detect the success of the modeling.Each group was then given corresponding intervention.(1)NOR group: ordinary diet without other intervention.(2)MOD group: fed with high-fat diet without other intervention.(3)EA group: Zusanli(ST36),Guanyuan(CV4),Zhongwan(CV12)and Fenglong(ST40)with a EA frequency at 2Hz,1mA,continuous wave connected with Korean EA therapeutic apparatus.10 minutes treated by EA once time,three times a week for 8 weeks.(4)EI group: EA treatment plan was the same as EA group.In addition,Sirtinol inhibitor solution was injected into the tail vein per 1mg/kg according to the weight of the rat.Three times a week for eight weeks.(5)(INH)group: inhibitor solution was injected into the tail vein per 1mg/kg according to the weight of the rat.Three times a week for eight weeks.(6)RSV group: resveratrol solution was given to the rats by gavage per 200mg/kg according to the weight of the rat.Three times a week for eight weeks.During the intervention,the rats’ body weight,food intake and anal to nasal length were measured at week 0,2,4,6 and 8 of the intervention,and Lee’s index was calculated.Intraperitoneal insulin tolerance(IPITT)and intraperitoneal glucose tolerance(IPGTT)were measured at week 6 of the intervention.After the intervention,a second clamp was performed to measure systemic insulin sensitivity.Before sampling,1 rat in each group was selected to detect the bioelectric slow wave of gastric and intestinal muscles,and 2 rats in each group were selected to detect gastric emptying and intestinal propulsion rate.Serum was taken to measure the content of insulin and CRP.Protein expression levels of SIRT1,TNF-α,il-6 and acetylated NF-κ B(Ac-NF-κ B)were detected by western blotting.Gene expression levels of SIRT1,TNF-αand il-6 were detected by RT-PCR.Results 1.Effects of EA on body weight,food intake,insulin sensitivity and serum CRP in insulin-resistant obese rats.(1)The body weight results: except the agonist group and the EA group,the body weight of the rats in the other groups showed an obvious increase during the intervention.Before intervention(at the 0 week),compared with the NOR group,the weight of rats in each group fed with high-fat diet increased significantly(P<0.01),which was more than 20% higher than that in the NOR group.After the treatment,compared with the MOD group,the RSV group began to lose weight from the 4th week(P<0.05).From the 6th week,the weight of the EA group and the RSV group decreased significantly(P<0.01).There was no significant difference in body weight between the EI group or the INH group and the MOD group(P>0.05).From the 6th week,the weight of rats in the EA group and the RSV group began to drop,with a significant difference compared with the MOD group.(2)the Lee’s index results: before intervention(at the 0 week),Lee’s index of rats in each group fed with high-fat diet was significantly higher than that in the NOR group(P<0.01).From the 6th week,the Lee’s index of the rats in the EA group and the RSV group began to decrease,and there was a statistical difference compared with the MOD group(P<0.05,P<0.01).There was no significant difference in Lee’s index between the INH group or the EI group compared with the MOD group(P>0.05),but the INH group was significantly higher than the EA group at the 8th week(P<0.05,P <0.01).From the 6th week,the Lee’s index of rats in the EA group and the RSV group began to decline,and there was a significant difference compared with the MOD group(P<0.01).(3)food intake results: the food intake of the MOD rats fed with high-fat diet was significantly higher than that of the NOR group throughout the whole intervention process,with a statistical difference(P<0.01).Before intervention(at the 0 week),the feeding amount of rats in each group fed with high-fat diet was significantly higher than that in the NOR group(P<0.01).From the 4th week,the food intake of rats in the EA group and the RSV group began to decline,and there was a significant difference compared with the MOD group(P<0.01).EA and SIRT1 activator could reduce the food intake of obese rats.During the whole intervention process,there was no significant difference between the intake of rats in each INH group or EI group and the MOD group(P>0.05),but the intake of rats in the EI group was significantly higher than that in the EA group from the second week(P<0.05,P<0.01).(4)serum insulin results: the serum insulin level of the MOD rats fed with high-fat diet were significantly higher than that of the NOR group(P<0.01).After the intervention of EA and SIRT1 activator,the serum insulin level of the two groups of obese rats was significantly lower than that of the MOD group(P<0.01).The serum insulin level of the rats after SIRT1 inhibitor intervention was slightly higher than that of the MOD group,and the serum insulin level of the rats after EA combined with inhibitor intervention was lower than that of the model group,but the difference was not statistically significant(P>0.05),and the serum insulin level of the inhibitor group was significantly higher than that of the EA group P<0.01).(5)IPGTT test results: there was no significant difference in fasting blood glucose between the groups.After 15 minutes of intraperitoneal glucose injection,the blood glucose of all rats rose rapidly,peaked at 30 minutes,and then decreased gradually.Compared with the NOR group,from 30 minutes to 120 minutes,the blood glucose of the rats fed with high-fat diet was always higher than that of the NOR group(P<0.05,P<0.01).Compared with the MOD group,the blood glucose of the RSV group was lower from the 15 th minute and the EA group from the 30 th minute to the 120 th minute(P<0.05).The blood glucose index of the rats in the EI group was lower than that in the MOD group from the 15 th minute to the 120 th minute,but it was statistically significant at the 120 th minute.The glucose level of rats in the INH group was higher than that in the MOD group from the beginning of 30 min to the end of 120 min,which was statistically significant.Compared with the EA group,the blood glucose of the RSV group was lower from the 15 th minute to the end,but there was no statistical significance.The blood glucose of the rats in the INH group was always higher from the 30 th minute(P<0.01).Starting from the 15 th minute,the blood glucose of the EI group was higher than that of the EA group and the RSV group,but lower than that of the model group and the INH group.(6)IPITT experiment results: there was no significant difference in basal blood glucose between the groups under fasting condition.After 15 minutes of intraperitoneal insulin injection,the blood glucose of all rats dropped rapidly,dropped to the lowest level after 60 minutes,and then rose gradually.Among them,the agonist group,EA group and NOR group showed the fastest decline,while the MOD group,INH group and EI group showed a slower decline in blood glucose.Compared with the NOR group,from 15 minutes to 120 minutes,the blood glucose of the rats fed with high-fat diet was higher than that of the NOR group(P<0.05,P<0.01).Compared with the MOD group,the blood glucose of the rats in the RSV group and the EA group was always lower from the 15 th minute(P<0.05).Compared with the MOD group,there was no significant difference between the EI group and the INH group(P>0.05),but since the 15 th minute,the blood glucose value of the INH group was higher than that of the EA group and lower than that of the MOD group,and the blood glucose value of the INH group was slightly higher than that of the MOD group.Compared with the EA group,the blood glucose of the RSV group was slightly lower from the 30 th minute(P<0.05).The glucose level of the INH group was significantly higher than that of the EA group(P<0.01),and that of the EI group was significantly higher than that of the EA group from the 60 th minute(P<0.01).(7)GIR results: before intervention(at the 0 week),GIR was significantly lower in rats fed with high-fat diet than the rats fed with normal diet(P<0.01),and was lower than 20% of the mean value of the normal group.After 8 weeks of intervention,GIR in the EA group and SIRT1 activator group was significantly higher than that before intervention(P<0.01).There was no significant change in GIR in NOR group compared with that before intervention(P>0.05).After 8 weeks of intervention,GIR in the MOD group was significantly decreased compared with the NOR group(P<0.01).Compared with the MOD group,GIR was significantly increased in the EA group and RSV group(P<0.05,P<0.01).The GIR in EI group was higher than that of MOD group,but lower than that of EA group.(8)serum CRP results: compared with the NOR group,serum CRP in the MOD group was significantly increased(P<0.01).Compared with the MOD group,the serum CRP level of EA,SIRT1 agonist and EI group significantly decreased(P<0.01).Compared with the EA group,the serum CRP level of the INH group was significantly higher(P<0.01).The serum CRP level of the EI group was between the EA group and the MOD group.2.Effects of EA on bioelectric waves,gastric emptying and intestinal propulsion rate of obese rats in the stomach and small intestine.(1)the stomach muscle biological waves: compared with NOR group,the stomach muscle power slow wave amplitude and frequency are increased in IR obese rats fed with high-fat diet(P<0.05,P<0.01).Compared with the MOD group,the amplitude and frequency of gastric emg in the EA group and the RSV group decreased(P<0.05,P<0.01).The amplitude and frequency of gastric emg in the EI group or INH group was no statistical significance(P>0.05)Compared with the MOD group,which suggests that SIRT1 inhibitors reverse the effect of electroacupuncture,indicating that the effect of electroacupuncture on the stomach is exerted by activating SIRT1.(2)the intestinal muscle biological waves: compared with NOR group,intestinal muscle power slow wave amplitude increases,the frequency drop in MOD group,and the difference was statistically significant(P<0.05,P<0.01).Compared with MOD group,the EA group and RSV group rat small intestine muscle power slow wave amplitude decreased,but faster frequency(P<0.05,P<0.01).The EI group and INH group small intestine muscle slow wave amplitude and frequency with the MOD group no statistical difference(P>0.05).Compared with the EA group,the small intestinal electromyographic slow wave amplitude of the INH group was higher and the frequency was smaller,and the difference was statistically significant(P<0.05,P<0.01).(3)gastric emptying rate and intestinal propulsion rate: compared with the NOR group,the gastric empting rate of the IR-obese rats fed with high-fat diet increased,while the intestinal propulsion rate decreased.The differences among the MOD group,the EI group and the INH group were statistically significant(P<0.05,P<0.01).Compared with the MOD group,the gastric emptying rate of rats in the EA group and the RSV group decreased,while the intestinal propulsion rate increased(P<0.05).There was no significant difference between the EI group or the INH group and MOD group(P>0.05),but the changes of gastric emptying rate and intestinal propulsion rate in the EI group were between the EA group and the MOD group.Compared with the EA group,the gastric emptying rate of rats in the EI group and the INH group was significantly accelerated,and the intestinal propulsion rate was slowed down with statistical differences(P<0.05,P<0.01),while the changes in the RSV group were not significantly difference(P> 0.05).3.Effects on the expression of Ac-NF-κ B and its downstream inflammatory cytokines TNF-α and IL-6 in the small intestine of IR-obese rats.(1)expression of TNF-αand IL-6 proteins: compared with the NOR group,expression of TNF-αand IL-6 proteins in the small intestine of the MOD group was significantly increased(P<0.01).Compared with the MOD group,the expression of TNF-αand IL-6 decreased significantly both in EA group and RSV group(P<0.01,P<0.05),while there was no significant difference between the INH group and the MOD group(P>0.05).Compared with the MOD group,the expression levels of TNF-αand IL-6 in EI group were decreased,but the expression levels of TNF-α was decreased with statistical significance(P<0.05),while the changes of IL-6 were not statistically significant(P>0.05).Compared with the EA group,the protein expressions of TNF-αand IL-6 in the small intestine of rats in the INH group were significantly higher(P<0.01,P<0.05),while that in the EI group were higher(P>0.05),but lower than those in the INH group.The expression levels of TNF-αand IL-6 in the small intestine of rats in the RSV group were not different from those in the EA group(P>0.05).(2)protein expression of Ac-NF-κB: compared with the NOR group,the expression level of Ac-NF-κB in the MOD group was significantly increased(P<0.05).Compared with the MOD group,the expression level of Ac-NF-κB in the EA group and RSV group was significantly decreased(P<0.05).The protein expression level of Ac-NF-κB in the small intestine tissues of the INH group and the EI group were not different from that of the MOD group(P>0.05).Compared with the EA group,the expression level of Ac-NF-κ B protein in the small intestine of the EI group was increased,but there was no significant difference(P>0.05).The expression level of Ac-NF-κ B protein in the small intestine of the INH group was significantly increased(P<0.05).The expression level of Ac-NF κB protein in the small intestine of rats in the RSV group was not significantly different(P>0.05).(3)TNF-α and IL-6 gene expression: compared with the NOR group,the relative mRNA expression levels of TNF-α and IL-6 in the MOD group were significantly increased(P< 0.05,P<0.01).Compared with the MOD group,the expression of TNF-αand IL-6 mRNA in the EA group and RSV group decreased significantly(P<0.05).There was no significant difference between the INH group or the EI group and the MOD group(P>0.05).Compared with the EA group,the relative expression levels of TNF-α and IL-6 mRNA in the small intestine tissues of the EI group and the INH group were both increased,and there was no statistical difference in the changes of TNF-α mRNA in the EI group,while there were statistical differences in the rest groups(P< 0.05,P <0.01).There was no significant difference in the expression of TNF-α and IL-6 mRNA in the small intestine of the RSV group(P>0.05).4.Effects of EA on SIRT1 expression in small intestine and co-expression of SIRT1/Ac-NF-κ B in the nucleus of insulin resistant obese rats.(1)SIRT1 protein expression: Compared with the NOR group,the relative expression level of SIRT1 protein in the small intestine of the MOD group decreased significantly(P<0.01).Compared with the MOD group,the expression of SIRT1 protein in the small intestine of obese rats increased significantly in the EA group and the RSV group(P< 0.05).The expression level of SIRT1 protein in the EI group was no significant difference compared with the MOD group(P>0.05).The expression of SIRT1 protein in the small intestine of the INH group was not significantly different from that of the MOD group(P>0.05),but significantly lower than that of the EA group and the NOR group(P <0.01).Compared with the EA group,the expression of SIRT1 protein in the small intestine of the RSV group was not significantly different(P>0.05).(2)SIRT1 gene expression: compared with the NOR group,the relative expression of SIRT1 mRNA in the small intestine of the MOD group was significantly decreased(P<0.01).Compared with the MOD group,the expression of SIRT1 mRNA in the small intestine of obese rats increased significantly both in EA group and RSV group(P<0.01).SIRT1 in the EI group was higher than that in the MOD group(P<0.05),but lower than that in the EA group.Expression of SIRT1 mRNA in the small intestine tissues of the INH group showed no significant change compared with that of the MOD group,but was significantly lower than that of the EA group and the NOR group(P<0.01).(3)co-expression of SIRT1/Ac-NF-κB: the co-expression of SIRT1 and Ac-NF κ B was clearly seen at 400 X magnification in each group.As can be seen from the SIRT1/Ac-NF-κB ratio(hereinafter referred to as the ratio),compared with the NOR group,the ratio in the MOD group was significantly decreased(P <0.01).Compared with the MOD group,the ratio both in the EA group and RSV group were significantly increased(P<0.05).The ratio of the INH group was higher than that of the MOD group(P<0.05),but lower than that of the EA group.Compared with the EA group,the ratio of the RSV group was not significantly changed(P>0.05).Conclusions 1.EA can reduce the weight of insulin-resistant obese rats,reduce their food intake and improve insulin sensitivity.This effect of EA is related to the up-regulated expression of SIRT1 protein.2.EA can reduce serum CRP level of insulin-resistant obese rats,suggesting that EA has the effect of improving systemetic inflammation.3.EA has a two-way regulating effect on gastrointestinal motility in insulin-resistant rats,thus to improve gastrointestinal motility disorders.That is,EA can inhibit the stomach movement,slow down the stomach emptying,and reduce the peristalsis of the small intestine,but accelerate the rate of intestinal propulsion.4.EA can up-regulate the expression of SIRT1 in the small intestine of insulin-resistant rats.SIRT1 deacetylates the NF-κB,then inhibits its transcriptional activity,down-regulates the expression of inflammatory factor genes in the downstream of its pathway,and reduces the expression levels of inflammatory factors TNF-α and IL-6 in the small intestine.5.Both EA and SIRT1 agonists have the similar biological effects,which can be blocked by SIRT1 specific inhibitors,indicating that the target of EA regulation is SIRT1.Above all,EA can specificity increase the expression of SIRT1 protein in the small intestine tissues of insulin-resistant obese rats.SIRT1 deacetylates the NF-κ B,inhibits its mediated inflammatory signaling pathways,reduce the expression of the inflammation factor,such as TNF-α and IL-6,alleviate the intestinal inflammation,and thus improve the gastrointestinal motility.This provides a theoretical basis for EA to improve abnormal gastrointestinal dynamics,and then to prevent obesity and insulin resistance-related diseases. |
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