电针联合壮医药线点灸对糖尿病胃轻瘫大鼠胃窦组织氧化应激反应的影响

Effect of electroacupuncture combined with Zhuang-medicine-thread moxibustion on oxidative stress in gastric antrum of diabetic gastroparesis rats

目的:观察电针联合壮医药线点灸对糖尿病胃轻瘫(DGP)大鼠氧化应激相关指标的影响,探讨电针联合壮医药线点灸治疗DGP的机制。方法:雄性SD大鼠随机分为正常组、模型组、西药组、电针组、点灸组、联合组,每组15只。采用腹腔注射链脲佐菌素(STZ)制备DGP大鼠模型。西药组予0.15 mg/mL枸橼酸莫沙必利混悬液灌胃给药;选取大鼠“中脘”“内关”“三阴交”穴,电针组电针20 min(10 Hz/50 Hz,2 mA),点灸组每穴点灸3壮,联合组为电针联合点灸治疗。均每日1次,连续3周。检测各组大鼠体质量、血糖、胃排空率、小肠推进率;采用硫代巴比妥酸法测定血清丙二醛(MDA)含量,黄嘌呤氧化酶法测定血清超氧化物歧化酶(SOD)活性,ELISA法检测血清活性氧(ROS)活性;HE染色观察胃窦组织的病理学改变;Western blot、实时荧光定量PCR检测胃窦组织血红素氧合酶1(HO-1)、核因子E2相关因子2(Nrf 2)、还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)、过氧化物酶体增殖物激活受体γ辅助活化因子1α(PGC-1α)蛋白及mRNA表达水平。结果:与正常组比较,模型组血糖升高(P<0.01),体质量、胃排空率、小肠推进率、血清SOD活性和胃窦组织HO-1、PGC-1α、总Nrf2蛋白及mRNA表达水平及Nrf2核转移均降低(P<0.01),血清MDA含量、ROS活性和胃窦组织NOX4蛋白及mRNA表达水平均升高(P<0.01)。模型组可见胃窦组织固有层与黏膜下层大量炎性细胞浸润,固有层胃腺显著扩张,黏膜下层重度水肿。与模型组比较,电针组、点灸组、联合组血糖均降低(P<0.01);西药组、电针组、点灸组、联合组体质量、胃排空率、小肠推进率和胃窦组织HO-1、PGC-1αmRNA表达均升高(P<0.01,P<0.05),血清MDA含量、ROS活性和胃窦组织NOX4蛋白及mRNA表达均降低(P<0.01);西药组、点灸组、联合组血清SOD活性、胃窦组织总Nrf2蛋白表达均升高(P<0.01,P<0.05);西药组、联合组胃窦组织HO-1、PGC-1α蛋白表达及Nrf2核转移均升高(P<0.05,P<0.01);西药组、电针组、联合组胃窦组织Nrf2 mRNA表达均升高(P<0.01,P<0.05)。西药组、电针组、点灸组、联合组均可见胃窦组织固有层与黏膜下层炎性细胞浸润减少,胃腺扩张和黏膜水肿不同程度改善。与联合组比较,西药组、电针组、点灸组血糖升高(P<0.01),体质量、胃排空率、小肠推进率均显著降低(P<0.01,P<0.05);电针组、点灸组血清MDA含量及ROS活性升高(P<0.01),血清SOD活性降低(P<0.01),胃窦组织总Nrf2蛋白、PGC-1α蛋白及mRNA、HO-1 mRNA表达降低(P<0.01,P<0.05),NOX4蛋白及mRNA表达升高(P<0.05);点灸组胃窦组织Nrf2 mRNA表达降低(P<0.05)。结论:电针联合壮医药线点灸能提高抗氧化酶活性,减少脂质过氧化物产生,良性调控抗氧化相关蛋白及mRNA的表达,这可能是其治疗DGP的作用机制之一。

Objective To observe the effect of electroacupuncture(EA)combined with Zhuang-medicine-thread moxibustion on oxidative stress-related indicators in diabetic gastroparesis(DGP)rats,so as to explore its mechanism underlying improvement of DGP.Methods Male SD rats were randomly divided into normal,model,medication,EA,Zhuang-medicine-thread moxibustion(moxibustion)and EA+Zhuang-medicine-thread moxibustion(combination)groups(15 rats in each group).The DGP model was established by intraperitoneal injection of streptozotocin(STZ).Rats of the medication group were treated by gavage of 0.15 mg/mL mosapride citrate suspension(10 mL/kg).EA(10 Hz/50 Hz,2 mA,20 min)or Zhuang-medicine-thread moxibustion(3 cones)was applied to“Zhongwan”(CV12),bilateral“Neiguan”(PC6)and bilateral“Sanyinjiao”(SP6)of the related groups,once a day for 3 weeks.The body weight,blood glucose,gastric emptying rate and intestinal propulsion rate of rats were measured.The serum malondialdehyde(MDA)content was measured by thiobarbituric acid method,the serum supero-xide dismutase(SOD)activity was measured by xanthine oxidase method,and the serum reactive oxygen species(ROS)activity was detected by ELISA.HE staining was used to observe the pathological changes of gastric antrum.The expression of heme oxygenase-1(HO-1),nuclear factor erythroid 2-related factor 2(Nrf2),nicotin-amide adenine dinucleotide phosphate oxidases(NOX4),peroxisome proliferators activated receptor-gamma coactivator 1α(PGC-1α)proteins and mRNAs in gastric antrum was detected by Western blot and real-time quantitative PCR,respectively.Results Compared with the normal group,the body weight,gastric emptying rate,intestinal propulsion rate,serum SOD activity,the expressions of HO-1,PGC-1α,total Nrf2 proteins and mRNAs,and Nrf2 nuclear translocation in gastric antrum were decreased(P<0.01),while the blood glucose,serum MDA content and ROS activity,NOX4 protein and mRNA expressions in gastric antrum were increased(P<0.01)in the model group.Compared with the model group,the blood glucose was decreased in the EA,moxibustion and combination groups(P<0.01);the body weight,gastric emptying rate,intestinal propulsion rate,and the expressions of HO-1 and PGC-1αmRNAs in gastric antrum were all increased in the four treatment groups(P<0.01,P<0.05),while the serum MDA content and ROS activity,NOX4 protein and mRNA expressions in gastric antrum were all decreased(P<0.01);the serum SOD activity and total Nrf2 protein expression in gastric antrum were increased in medication,moxibustion and combination groups(P<0.01,P<0.05);the expressions of HO-1 and PGC-1αproteins,and Nrf2 nuclear translocation in gastric antrum were increased in medication and combination groups(P<0.05,P<0.01);the expression of Nrf2 mRNA was increased in the medication,EA and combination groups(P<0.01,P<0.05).Compared with the combination group,the body weight,gastric emptying rate and intestine propulsion rate were decreased in the medication,EA and moxibustion groups(P<0.01,P<0.05),and the blood glucose increased(P<0.01);the serum MDA content and ROS activity,NOX4 protein and mRNA expressions in gastric antrum were increased(P<0.01,P<0.05),serum SOD activity,and the expressions of total Nrf2 protein,PGC-1αprotein and mRNA,HO-1 mRNA in gastric antrum were decreased(P<0.01,P<0.05)in the EA and moxibustion groups;the expression of Nrf2 mRNA was decreased in the moxibustion group(P<0.05).HE staining showed a large number of inflammatory cell infiltration in the lamina propria and submucosa,and the gastric glands in the lamina propria were significantly expanded,the submucosa was severely edematous in the model group,which were relative milder in the four treatment groups.Conclusion EA combined with Zhuang-medicine-thread moxibustion can effectively improve the activity of antioxidant enzymes,reduce the production of lipid peroxide,and regulate the expression of antioxidant related proteins and genes,which may be one of the mechanisms in treating DGP.