| ObjectiveAcupoint compatibility is the key factor to determine the clinical curative effect.Different acupoint selection directly leads to the difference of acupuncture effect.For example,electroacupuncture at ST36 can enhance gastric motility,while electroacupuncture at ST25 can cause gastric relaxation.The different effects may be due to the activation of gastrointestinal sympathetic nerve and inhibition of gastrointestinal movement when acupuncture stimulation acts on the abdomen of rats,and the activation of parasympathetic nerve and promotion of gastrointestinal movement when it acts on limbs.However,in contradiction with this,abdominal and limb acupoints are often combined in the clinical treatment of obesity.Acupoint compatibility can produce the clinical efficacy only by improving the synergistic effect.In the obese rat model,what are the effects of acupuncture at different parts on obesity model rats with insulin resistance?Can compatibility therapy enhance acupuncture effect?In order to further study whether the compatibility of simple abdominal acupoints and limbs acupoints has an effect on the regulation of obesity insulin resistance,and whether the compatibility of abdominal combined with limbs acupoints has a synergistic effect?In this project,it is proposed to intervene the obese rat model of insulin resistance by Electroacupuncture of "biaoben acupoint combination",using immunoblotting,immunohistochemistry,RT-PCR and other technologies,based on the changes of food intake,body weight,biochemical indexes related to glucose and lipid metabolism and peripheral insulin sensitivity,and TLR4/NF-k Taking the B signal pathway as the starting point,this paper discusses the specificity of the compatibility of acupoints in different parts on the regulation of fat metabolism,verifies whether there is a synergistic effect between the compatibility of acupoints in abdomen and limbs,and explains that the "biaoben acupoint combination" electroacupuncture may regulate TLR4/NF-kB improve the mechanism of insulin resistance and obesity.The completion of this project will open a new window for the study of acupoint compatibility,and provide a new scientific basis for the clinical use of acupuncture acupoint compatibility in the prevention and treatment of insulin resistance,obesity and related diseases.MethodThe first stage is to compare and study the different effects of different acupoint compatibility on obesity insulin resistance,100 8-week-old male without specific pathogen free（SPF）Wistar rats were used.15 rats were selected as the normal group by random number table method,and the remaining 85 rats were used as the model group.The rats in each group were marked by ear labeling method.The rats in the normal group were fed with ordinary feed,the rats in the model group were fed with high-fat feed,and the rats in each group were fed with corresponding feed for 8 weeks.After the completion of modeling,60 rats were randomly selected and divided into model group,abdominal electroacupuncture group,limb electroacupuncture group and "biaoben acupoint combination" electroacupuncture group,15 rats in each group and were fed with high-fat diet.（1）Normal group（n=15）:only ordinary food was given without other intervention;（2）Model group（n=15）:model rats,no other intervention;（3）Abdominal electroacupuncture group（n=15）:model rats,Guanyuan（CV4）,Zhongwan（CV 12）and bilateral Tianshu（ST25）were selected,and Han’s electroacupuncture instrument was used,2Hz,1mA,continuous wave for 15min.The intervention was conducted once every other day,three times a week for 8 weeks;（4）Limb electroacupuncture group（n=15）:model rats,bilateral Zusanli（ST36）and Fenglong（ST40）were selected,and Han’s electroacupuncture instrument was used,2Hz,1mA,continuous wave for 15min.The intervention was conducted once every other day,three times a week for 8 weeks;（5）"Biaoben acupoint combination" electroacupuncture group（n=15）:model rats were selected from Guanyuan（CV4）,Zhongwan（CV12）,bilateral Tianshu（ST25）,Zusanli（ST36）and Fenglong（ST40）.Han’s electroacupuncture parameters are set as:2Hz,1mA,continuous wave.The duration of single electroacupuncture was 15min,and was conducted once every other day,three times a week,last for 8 consecutive weeks.During the modeling period,the body mass of rats in each group was measured on the last day of the 8th week of high-fat diet modeling,and then 3 rats were selected from the ordinary diet group and 15 rats from the model group were selected for high insulin-n-glucose clamp experiment to detect the glucose infusion rate（GIR）.Whether the obese insulin resistance rats were successfully modeled was judged by body mass and GIR.Body weight,food intake,Lee’s index,fasting blood glucose of peripheral blood and postprandial blood glucose were measured before electroacupuncture（week 0）and at weeks 2,4,6 and 8 after intervention;After 6 weeks of intervention,the intraperitoneal insulin tolerance（IPITT）and intraperitoneal glucose tolerance（IPGTT）were measured in some rats.After electroacupuncture treatment,the fat content of epididymis,adipocyte volume,subcutaneous fat content,liver weight and fat content were measured.Insulin sensitivity was measured by hyperinsulinemic euglycemic clamp before the rats in each group were killed;The blood of cardiac apex was taken immediately after the rats were killed,and the serum IL-1 was detected by ELISA β、IL-6、TNF-α Content of.Finally,fresh liver tissues were taken to detect the expression levels of macrophage marker genes F4/80,CD68 and CD11c in rat liver tissues.The second stage is to study the mechanism of "Biaoben acupoint combination" electroacupuncture on TLR4/NF-κ B signal pathway in the liver of obese rats with insulin resistance.100 8-week-old male without specific pathogen free（SPF）Wistar rats were used.15 rats were selected as the normal group by random number table method,and the remaining 85 rats were used as the model group.The rats in each group were marked by ear labeling method.The rats in the normal group were fed with ordinary feed,the rats in the model group were fed with high-fat feed,and the rats in each group were fed with corresponding feed for 8 weeks.After the completion of modeling,60 rats were randomly selected and divided into model group,acupuncture group,silencing group and acupuncture+silencing group,with 15 rats in each group and fed with high-fat diet.（1）Normal group（n=15）:only ordinary food was given without other intervention;（2）Model group（n=15）:model rats,no other intervention;（3）Acupuncture group（n=15）:model rats were treated with Guanyuan（CV4）,Zhongwan（CV12）,bilateral Tianshu（ST25）,Zusanli（ST36）and Fenglong（ST40）.Han’s electroacupuncture instrument was used,2Hz,1mA,continuous wave for 15min,once every other day,three times a week for 8 weeks;（4）Silencing group（n=15）:model rats were injected with shRNA into caudal vein to silence the expression of TLR4;（5）Acupuncture+silencing group（n=15）:model rats,with the Biaoben acupoint combination" electroacupuncture on Guanyuan（CV4）,Zhongwan（CV 12）,bilateral Tianshu（ST25）,Zusanli（ST36）and Fenglong（ST40）,use Han’s electroacupuncture instrument,2Hz,1mA,continuous wave,time 15min,intervene once every other day,three times a week for 8 weeks,and inject shRNA into caudal vein synchronously to silence the expression of TLR4.The body mass of rats in each group was measured on the last day of the 8th week of high-fat diet modeling,and then 3 rats were selected from the general diet group and 15 rats from the model group were selected for high insulin-n-glucose clamp experiment to detect the glucose infusion rate（GIR）.Whether the obese insulin resistance rats were successfully modeled was judged by body mass and GIR.The liver tissues of rats were taken 24 hours after the end of the 8th week after electroacupuncture intervention,and the protein expression of TLR4,NF-κ B and AP-1 in the liver of rats in each group were detected by Western blot immunofluorescence method.Result1.Effects of Electroacupuncture on body weight,body fat content,glucose and lipid metabolism and insulin sensitivity（1）The results of body weight showed that the body weight of rats in each group was significantly higher than that in the normal group before intervention（P<0.01）,suggesting that high-fat diet can successfully prepare the obese rat model.Compared with the model group,after 8 weeks of intervention,the body weight of rats in abdominal electroacupuncture group,limb electroacupuncture group（P<0.05）and biaoben acupoint combination electroacupuncture group（P<0.01）increased slowly,suggesting that electroacupuncture stimulation of abdominal acupoints and limb acupoints can slow down the body weight growth of obese rats induced by high-fat diet.Compared with abdominal electroacupuncture group and limb electroacupuncture group,the effect of biaoben acupoint combination electroacupuncture group on slowing down the weight growth of rats is more obvious（P<0.05）,which further shows that the electroacupuncture effect of biaoben acupoint combination is better than that of simple local point group combination or distal point combination group.（2）The results of Lee’s index showed that before the intervention,compared with the normal group,the Lee’s index of rats in each group increased significantly（P<0.01）.Compared with the model group,after 8 weeks of intervention,the Lee’s index of rats in abdominal electroacupuncture group,limb electroacupuncture group and biaoben acupoint combination electroacupuncture group decreased significantly（P<0.05）,indicating that electroacupuncture can effectively improve the obesity degree of islet resistant obese rats.However,there was no significant difference in Lee’s index among abdominal electroacupuncture group,limb electroacupuncture group and specimen acupoint electroacupuncture group.（3）The change of visceral white fat weight showed that compared with the normal group,the weight of visceral white fat of four groups of rats fed with high-fat diet（model group,abdominal electroacupuncture group,limb electroacupuncture group and biaoben acupoint combination electroacupuncture group）increased significantly（P<0.01）,indicating that high-fat diet can induce the growth of white fat.Compared with the model group,the weight of white fat in abdominal electroacupuncture group,limb electroacupuncture group（P<0.05）and biaoben acupoint combination electroacupuncture group（P<0.01）was significantly reduced,indicating that electroacupuncture can inhibit the accumulation of white fat.Compared with abdominal electroacupuncture group and limb electroacupuncture group,the effect of biaoben acupoint combination electroacupuncture group on reducing the weight growth of visceral white fat is more obvious（P<0.05）,which further shows that the electroacupuncture effect of biaoben acupoint combination is better than that of simple local point combination or distal point combination,but there is no significant difference between abdominal electroacupuncture group and limbs.（4）The contents of serum TC,TG,FFA,LDL-C and HDL-C showed that compared with the normal group,the contents of serum TC,TG,FFA and LDL-C in other groups increased significantly（P<0.01）,and the content of HDL-C decreased significantly（P<0.01）;Compared with the model group,after 8 weeks of intervention,the contents of TC,TG,FFA and LDL-C in serum of the three groups of electroacupuncture intervention（abdominal electroacupuncture group,limb electroacupuncture group and biaoben acupoint combination electroacupuncture group）were significantly reduced,while the content of HDL-C was increased,and the difference was statistically significant.Compared with abdominal electroacupuncture group and limb electroacupuncture group,the biaoben acupoint combination electroacupuncture group had more obvious regulatory effects on TC,TG,FFA,LDL-C and HDL-C（P<0.05）,but there was no significant difference between abdominal electroacupuncture group and limb electroacupuncture group（P>0.05）.This shows that electroacupuncture can regulate lipid metabolism,and the effect of biaoben acupoint combination is better than simple local acupoint combination or remote acupoint combination.（5）The changes of blood glucose metabolism in rats showed that before intervention,compared with the normal group,the fasting blood glucose（FPG）and postprandial blood glucose（PBG）of rats in the four groups fed with highfat diet（model group,abdominal electroacupuncture group,limb electroacupuncture group and biaoben acupoint combination electroacupuncture group）increased,but only the difference of PBG was statistically significant,There was no significant difference in FPG data between groups.After 8 weeks of intervention,compared with the model group,FBG and PBG in the three groups of electroacupuncture intervention（abdominal electroacupuncture group,limb electroacupuncture group and biaoben acupoint combination electroacupuncture group）decreased,but only the difference of PBG was statistically significant,and the difference of FPG data between the groups was not statistically significant.Although there was a change trend among the three groups of electroacupuncture intervention,the difference was not statistically significant.（6）The changes of ipgtt in rats showed that:after the 6th week of intervention,the intraperitoneal glucose tolerance was detected by intraperitoneal injection of glucose.The overall change trend of ipgtt began to rise,reached the highest blood glucose concentration in 30min,and then decreased.From 30 min after injection,the blood glucose values of the four groups fed with high-fat diet（model group,abdominal electroacupuncture group,limb electroacupuncture group and biaoben acupoint combination electroacupuncture group）were significantly higher than those of the normal group（P<0.01）,reflecting the decline of abdominal glucose tolerance in obese insulin resistant rats;90 minutes after injection,the blood glucose values of abdominal electroacupuncture group,limb electroacupuncture group and biaoben acupoint combination electroacupuncture group were significantly lower than those of model group（P<0.05）,indicating that electroacupuncture is beneficial to improve the abdominal glucose tolerance of obese insulin resistant rats;In the three intervention groups,the effect of biaoben acupoint combination electroacupuncture group is more obvious,while there is no significant difference between abdominal electroacupuncture group and limb electroacupuncture group,indicating that the selection of biaoben acupoint combination is better than simple abdominal acupoints and limb acupoints in regulating abdominal glucose tolerance of obese insulin resistance rats.（7）Insulin tolerance of rats was measured by intraperitoneal injection at the 6th week.The overall change trend of ipitt is that the blood glucose concentration gradually decreases after injection,reaches the lowest at 60min,and then increases.From 30 min,the blood glucose values of the four groups fed with high-fat diet（model group,abdominal electroacupuncture group,limb electroacupuncture group and biaoben acupoint combination electroacupuncture group）were significantly higher than those of the normal group（P<0.01）,and the decrease range of blood glucose was low,reflecting the decrease of abdominal islet tolerance in obese insulin resistant rats.After 60 min after injection,the blood glucose values of abdominal electroacupuncture group,limb electroacupuncture group and biaoben acupoint combination electroacupuncture group were significantly lower than those of model group（P<0.05）,indicating that electroacupuncture is beneficial to improve the abdominal insulin tolerance of obese insulin resistant rats;In the three intervention groups,the effect of biaoben acupoint combination electroacupuncture group is more obvious,while there is no significant difference between abdominal electroacupuncture group and limb electroacupuncture group,indicating that the selection of biaoben acupoint combination is better than simple abdominal acupoints and limb acupoints in regulating abdominal insulin tolerance of obese insulin resistance rats.（8）The changes of HOMA-IR index and ISI index of rats showed that compared with the normal group,the HOMA-IR index of rats in the four groups fed with high-fat diet（model group,abdominal electroacupuncture group,limb electroacupuncture group and biaoben acupoint combination electroacupuncture group）was significantly higher（P<0.01）,while the ISI index was significantly lower（P<0.01）,reflecting the decline of insulin sensitivity and insulin resistance.After electroacupuncture intervention,HOMA-IR index of rats was significantly lower（P<0.05,P<0.01）,while ISI index was significantly higher（P<0.05,P<0.01）.Among the three intervention groups,the effect of biaoben acupoint combination electroacupuncture group was more obvious（P<0.05）,while there was no significant difference between abdominal electroacupuncture group and limb electroacupuncture group,which further showed that the effect of electroacupuncture combined with biaoben acupoint combination combination on regulating systemic insulin sensitivity was better than that of simple abdominal point and limb point.（9）The changes of gir level in rats showed that compared with the normal group,the gir of rats in the four groups fed with high-fat diet（model group,abdominal electroacupuncture group,limb electroacupuncture group and biaoben acupoint combinationelectroacupuncture group）was significantly lower（P<0.01）,and all were lower than 20%of the normal group,indicating that the insulin resistance rat model was successful.After 8 weeks of intervention,compared with the model group,the gir of the three groups after electroacupuncture treatment was higher than that of the model group（P<0.05,P<0.01）,indicating that electroacupuncture can regulate systemic insulin sensitivity.Among the three intervention groups,the effect of biaoben acupoint combination electroacupuncture group was more obvious（P<0.05）,while there was no significant difference between abdominal electroacupuncture group and limb electroacupuncture group,which further showed that the effect of electroacupuncture combined with biaoben acupoint combination combination on regulating systemic insulin sensitivity was better than that of simple abdominal point and limb point.2.Effect of electroacupuncture on IL-1 in serumβ、IL-6、TNF-α Impact of changeCompared with the normal group,IL-1 in serum of four groups of rats fed with high-fat diet（model group,abdominal electroacupuncture group,limb electroacupuncture group and biaoben acupoint combination electroacupuncture group）β、IL-6、TNF-α The content of insulin resistance increased significantly（P<0.01）,indicating that the inflammatory level of obese insulin resistant rats was higher.Compared with the model group,IL-1 in serum of rats in abdominal electroacupuncture group,limb electroacupuncture group（P<0.05）and biaoben acupoint combination electroacupuncture group（P<0.01）β、IL-6、TNF-α It shows that electroacupuncture can inhibit the release of peripheral inflammatory factors.Compared with abdominal electroacupuncture group and limb electroacupuncture group,the biaoben acupoint combination electroacupuncture group is better for IL-1 β、IL-6、TNF-α The regulation effect was more obvious（P<0.05）,which further showed that the efficacy of electroacupuncture with biaoben acupoint combination was better than that of simple local or distal points,but there was no significant difference between abdominal electroacupuncture group and limb electroacupuncture group.3.Effect of electroacupuncture on the expression of macrophage markers in liver tissueCompared with the normal group,the expressions of macrophage markers F4/80,CD68 and CD 11c mRNA in the liver of rats fed high-fat diet,i.e.insulin resistance obesity model,were significantly higher（P<0.01）.Compared with the model group,the expression of F4/80,CD68 and CD11c mRNA in liver tissue of the three groups of electroacupuncture decreased significantly（P<0.05,P<0.01）.Compared with the three groups of electroacupuncture,it was found that the expression of F4/80,CD68 and CD11c mRNA in the biaoben acupoint combination group was significantly lower than that in the abdominal point combination group and limb point combination group（P<0.05）,But the difference was not statistically significant.4.Effect of electroacupuncture on liver weight and liver fat contentCompared with the normal group,the weight of liver and liver fat in the four groups fed with high-fat diet increased significantly（P<0.01）.Compared with the model group,the liver weight and liver fat weight of the three groups of electroacupuncture were significantly reduced（P<0.05,P<0.01）.Compared with the three groups of electroacupuncture,it was found that the liver weight and liver fat weight of the biaoben acupoint combination group were significantly reduced（P<0.05）compared with the abdominal point combination group and the limbs point combination group,although there were differences between the abdominal point combination group and the limbs point combination group,But the difference was not statistically significant.5.Effect of electroacupuncture on TLR4 and NF in liver-κ Influence of B expressionCompared with the blank group,the model group TLR4,NF-k The expression of B increased（P<0.05）.Compared with the model group,TLR4 and NF in the liver of rats in the biaoben acupoint combination acupuncture group-kB.The content of AP-1 decreased significantly（P<0.05）,indicating that acupuncture at the biaoben acupoint combination may play a role in improving insulin resistance in obese rats by inhibiting TLR4 and inhibiting liver inflammation.6.Effects of electroacupuncture on liver TLR4 and NF-κ B.Effect of AP-1 protein expressionCompared with the blank group,the expression of TLR4 in the model group was increased（P<0.05）,which showed that the immunity of obesity induced insulin resistance rats was activated.NF-kB.The content of AP-1 was also higher than that in the normal group（P<0.05）,indicating that the inflammatory pathway was activated and the inflammatory reaction of liver appeared.Compared with the model group,TLR4 and NF in the liver of rats in the biaoben acupoint combination acupuncture group-kB.The content of AP1 decreased significantly（P<0.05）,indicating that acupuncture at the biaoben acupoint combination can inhibit the immune response of rat liver and reduce inflammation.TLR4 and NF in acupuncture group,silencing group and acupuncture+silencing group-kB.There was no significant difference in the expression of AP-1,indicating that the therapeutic effect of acupuncture with acupoints was weakened by TLR4 silencing,and the inhibition of inflammation by acupuncture may play a role through TLR4.Conclusion1.After electroacupuncture treatment,the contents of TC,TG,FFA and LDLC decreased significantly,and the content of visceral white fat decreased,suggesting that electroacupuncture can improve the level of lipid metabolism.In addition,electroacupuncture can also improve the disorder of glucose metabolism.Although the postprandial blood glucose concentration of obese insulin resistant rats increased,the postprandial blood glucose concentration of the three groups of rats decreased after electroacupuncture treatment.In the three groups of electroacupuncture,it was further found that the effect of biaoben acupoint combination group was better than that of simple abdominal point combination and limb point combination,Thus,it also reflects the advantages of combination points with specimens,which can effectively improve the treatment efficiency and bring guiding significance to the clinic.2.After electroacupuncture,it was found that the gir level of abdominal electroacupuncture group,limb electroacupuncture group and biaoben acupoint combination group increased,and the blood glucose concentration measured by ipgtt and ipitt after 90 min was significantly lower than that of the model group,which further showed that electroacupuncture could effectively improve insulin resistance and systemic insulin sensitivity.In the process of comparison among the three electroacupuncture groups,it was found that the effect of biaoben acupoint combination was better than that of simple abdominal acupoint combination and limb acupoint combination,which effectively improved the peripheral insulin sensitivity.3.Electroacupuncturecan effectively reduce IL-1,IL-6 and TNF in peripheral blood-a Concentration,thereby reducing the systemic inflammatory response.For comparing the effects of different acupoint compatibility on systemic inflammatory response,it is found that the effect of biaoben acupoint combination is more prominent than that of simple abdominal acupoint combination and limb acupoint combination,which further shows that the curative effects of different acupoint compatibility are also different.4.The expression of F4/80,CD68 and CD 11c mRNA in the liver tissue of the three groups treated with electroacupuncture decreased significantly,indicating that electroacupuncture can inhibit the infiltration of macrophages and reduce the inflammatory response.Among the three groups of electroacupuncture,the effect of biaoben acupoint combination group was significantly better than that of the other two groups.5.After acupuncture treatment,the expression of TLR4 in liver decreased significantly,indicating that acupuncture can inhibit TLR-4/NF-kB pathway improves insulin resistance in obese rats.After TLR4 was silenced by virus,the indexes of insulin resistance improved similar to that of acupuncture,and no better effect was found in rats receiving TLR silencing and acupuncture treatment at the same time,which proved that acupuncture treatment of insulin resistance in obese rats may be achieved by inhibiting TLR4.Similarly,we found that NF in the model group-k The expression levels of B and AP-1 increased,which indicates that the inflammatory level of insulin resistant liver increased.After acupuncture intervention,these indexes also decreased significantly,and the trend is consistent with TLR4,which further proves that acupuncture at the biaoben acupoint combination can improve liver inflammation and inhibit insulin resistance in obese rats by inhibiting TLR4.To sum up,theoretical and experimental studies have confirmed that the combination of ST36,ST40,CV12,CV4 and ST 25 points.CV4 has the function of strengthening and protecting innate vitality,ST36 has the function of solidifying and protecting the stomach qi,CV12 can promote the movement of Zhongjiao,ST40 can dispel dampness and phlegm,and ST25 can raise the clear and lower the turbid symptoms.Therefore,the combination of these acupoints has laid a good foundation for the treatment of insulin resistance. |
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