| ObjectivesInsulin resistance(IR) is not only a precursor condition of type 2 diabetes mellitus(T2DM),but also an independent risk factor for hypertention.IR is closely associated with metabolic syndrome and cardiovascular diseases. To intervene with IR at early time is the key to treat T2 DM, and it means a lot for prevention of hypertention, metabolic syndrome and related cardiovascular diseases. At present, western medicine is kind of strategy for treatment of IR, but it always needs a long-term and even lifetime medication. Moreover, it always accompanied with side effects. Acupuncture has the effect of regulation on insulin secretion, nerve, hormone and cells. It improves disorder of glucolipide metabolism, and insulin sensitivity of insulin-target organs. Acupuncture is characterized as safe, convenient, effective and with no adverse effect. For the above reasons, acupuncture is widely used in prevention and treatment for IR and its related diseases.However, present research in this field is too much focused on metabolic changes in peripheral target organs, such as liver, adipose tissue and skeletal muscle. There are less consideration on the relationship between central nervous system, especially central inflammation, and insulin resistance. Based on previous stuy, given consideration on close correlation between IR and chronic low-grade inflammation, we conduct this study. In this study, genetic insulin resistant rats –zucker diabetic fatty(ZDF) rats- were used. Mechanisms of the effect of EA on IR were studied, via its regulation on hypothalamic SIRT1/NF-κB pathway and its downstream inflammatory cytokines. It was expected to provide experimental evidence for application of EA in treatment of IR and its related diseases. MethodsTwelve male seven-week old ZDF rats were randomly divided into three groups: model control group, non-electric stimulation group(NES) and EA group, four rats in each group. Four zucker lean rats were adopted for background control, assigned to blank control group. In NES and EA groups, acupoints of Zusanli(ST36), Guanyuan(CV4), Zhongwan(CV12) and Fenglong(ST40) were selected. A standard for 0.30×13mm stainless steel needls were used for an insert depth of 3mm to 5mm. Needles at ST36 and ST40 were inserted perpendicularly, while at CV4 was obliquely to xiphoid and to symphysis pubis at CV 12. For EA group, electric acupuncture apparatus was connected with the needles, with intensity of 1m A and frequency of 2Hz, 10 minutes each time. For NES group, electric acupuncture apparatus was also connected with the needles, but with no power. EA and NEA interventions will be conducted three times a week and last for thre weeks. Rats in the other two groups will be grabbed and fixed at the same time with EA and NES groups.In these three weeks,their food intake, weight gain and blood glucose levels will be recorded each week. At the end of third week, their serum will be collected to detect level of hs-CRP. Their hypothalamus will be stored for following detections. Western blotting was used to measure the protein levels of SIRT1, Ac-NF-κB p65 and its downstream inflammatory cytokines, including TNF-α and IL-6. Double labelingimmunofluorescence technique was used to detect the expressions of SIRT1 and Ac-NF-κB p65. Real time PCR was used for detection of m RNA levels of SIRT1、TNF-α and IL-6. All data will be analyzed statistically. Results 7. Effects of EA on food intake, body weight, blood glucose levels and serumhs-CRP of ZDF rats.At the beginning, food intakes in model control group, NES group and EA group were of no difference(P>0.05), but in blank control group, it was significantly less than in the other three groups(P<0.01). At the end of first week, food intakes in NES and EA groups were less than in model control group, but the difference was lack of significance(P>0.05). At the end of second week, food intakes in EA group was significantly less than in model control group(P<0.01), while it in NES group was also reduced(P<0.05). At the end of third week, this difference were more significant(P<0.01).At the beginning, body weight in model control group, NES group and EA group were of no difference(P>0.05), but in blank control group, it was significantly less than in the other three groups(P<0.01). At the end of first week, body weight in NES and EA groups were less than in model control group, but the difference was lack of significance(P>0.05). At the end of second week, body weight in EA group was significantly less than in model control group(P<0.05), while there was of no difference between NES group and model control group(P>0.05). At the end of third week, difference between EA group and model group was still exist(P<0.05), while reduction in NES group was of no significance(P>0.05).At the beginning, fasting blood glucose(FBG) levels in blank control group was significantly lower than in the other three groups(P<0.05), and the FBG levels in the other three groups were of no difference(P>0.05). At the end of first and second week, FBG levels were increased in each group, and it is still lower in blank control group(P<0.01)At the end of third week, FBG level in EA group was significantly lower than in model control group. It was also decreased in NES group, but with no significance(P>0.05).At the beginning, postprandial blood glucose(PBG) levels in blank control group was significantly lower than in the other three groups(P<0.05), and the PBG levels in the other three groups were of no difference(P>0.05). At the end of first week, PBG levels in EA and NES groups were lower than in model control group, but with no significance(P>0.05). At the end of second and third week, PBG levels in EA and NES groups were significantly lower than in model control group(P<0.05)。At the end of third week, compared with in control group, serum hs-CRP in model control group was much higher(P<0.05), and no differences were observed in EA and NES groups. Compared with model control group, serum hs-CRP in EA group was significantly decreased(P<0.05). 8. Effects of EA on protein expression levels of hypothalamic SIRT1 andAc-NF-κB p65 in ZDF ratsCompared with control group, hypothalamic SIRT1 protein expression levels were much lower(P<0.01), but no difference was observed in EA group(P>0.05).Compared with model control group, Ac-NF-κB p65 protein expression levels were lowere in the other three groups. Compared with blank control group, it was higher in NES group, but no difference was observed in EA group. 9. Effects of EA on expression sites and relationship of hypothalamic SIRT1and Ac-NF-κB p65 in ZDF rats.In terms of cell calculation, SIRT1 in blank control and EA groups was more than Ac-NF-κB p65,but the difference was reversed in model control and NES groups. From the value of SIRT1/Acetyl NF-κB p65, it was bigger than 1, and the difference in blank control group was more obvious. The value was smaller than 1 in model control and NES groups, especially in model control group. In pictures of high power lens, it was observed that the expressions of SIRT1 and Ac-NF-κB p65 in hypothalamus were abundant,and most of them were nuclear expressions. In some cells, a phenomenon of colocalization was observed. 10. Effect of EA on the m RNA expression of hypothalamic SIRT1 in ZDF ratsCompared with blank control group, SIRT1 m RNA expressions were much lower in the other three groups(P<0.01). In comparison with model control group, it was obviously higher in EA group( P<0.01). 11. Effects of EA on protein expression levels of hypothalamic TNF-α andIL-6 in ZDF ratsCompared with blank control group, protein expression levels of TNF-αand IL-6 were much higher(P<0.01). In comparison with model controlgroup, their expression levels were significantly decresed in EA and NESgroups(P<0.01)。 12. Effects of EA on m RNA expression levels of hypothalamic TNF-α andIL-6 in ZDF ratsCompared with blank control group, m RNA expressions of TNF-α and IL-6 were much higher in the other three groups(P<0.01). In comparison with model control group, it was decreased in EA and NES groups,but with no statistical significance(P>0.05). Conclusions6. EA has the effects of controlling food intake, reducing body weight, decreasing FBG and PBG levels, and no obvious difference is observed between EA and NES method, which implys their reliable ability to ameliorate IR.7. Circulating hs-CRP level in ZDF rats is reduced by EA, implys EA has the effect of improving systemetic inflammation.8. In ZDF rats, EA is supposed to upregulate hypothalamic SIRT1 expression, and deacetylate NF-κB, which act a role in regulation of its downstream related genes.9. EA has the effect of ameliorate central inflammation, via regulation of hypothalamic SIRT1/NF-κB pathway, and reduction levels of downstream inflammatory cytokines TNF-α and IL-6.10. From the view of their synthetical effects, EA has a better effect in improving IR, maybe associated with the activation of nervous fiber and receptors, and the releasing of central active substances by electric stimulation. |
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