桦褐孔菌多糖经Nrf2信号通路对糖尿病大鼠视网膜组织氧化应激和炎症反应的抑制作用

Inhibition of oxidative stress and inflammation in retinal tissue of diabetic rats by inonotus obliquus polysaccharides via Nrf2 signaling pathway

目的探讨桦褐孔菌多糖经Nrf2信号通路对糖尿病大鼠视网膜组织氧化应激和炎症反应的抑制作用。方法选取12周龄雄性SD大鼠40只,随机分为4组正常对照组10只、糖尿病组10只、IOP组(给予大鼠桦褐孔菌多糖300 mg·kg^-1)10只和抑制剂组(给予大鼠桦褐孔菌多糖300 mg·kg^-1+锌原卟啉20 mg·kg^-1)10只。除正常对照组外,其余大鼠均采用一次性腹腔注射10 g·L^-1链脲佐菌素(60 mg·kg^-1)制作糖尿病大鼠模型,桦褐孔菌多糖采用灌胃法给药;抑制剂组在桦褐孔菌多糖灌胃前24 h将锌原卟啉经腹腔注射给大鼠。正常对照组和糖尿病组灌胃相同体积的生理盐水,并于12周后禁食不禁水12 h后进行实验。将分离到的新鲜视网膜,石蜡包埋,切片,用于HE染色和免疫组织化学染色。检测视网膜样品组织中丙二醛(MDA)、谷胱甘肽(GSH)、超氧化物歧化酶(SOD)含量;采用HE染色观察视网膜形态并检测视网膜神经节细胞密度;免疫组织化学染色观察细胞HO-1阳性染色情况;采用Western blot法检测视网膜HO-1、Nrf2和COX-2蛋白的表达。结果造模后12周,糖尿病组、IOP组、抑制剂组大鼠体质量均低于正常对照组,血糖浓度均高于正常对照组,差异均有统计学意义(均为P<0.01)。造模后,糖尿病组大鼠视网膜组织中SOD、GSH的含量均明显低于正常对照组(均为P<0.01);MDA含量明显高于正常对照组,差异有统计学意义(P<0.01)。IOP组视网膜组织中SOD、GSH含量高于糖尿病组及抑制剂组,MDA含量显著低于糖尿病组及抑制剂组,差异均有统计学意义(均为P<0.05)。正常对照组、糖尿病组、抑制剂组和IOP组视网膜神经节细胞密度分别为(2300±100)个·mm^-2、(1400±100)个·mm^-2、(1505±95)个·mm^-2、(2250±95)个·mm^-2。Western blot结果显示糖尿病组HO-1、Nrf2及COX-2蛋白的表达均高于正常对照组(均为P<0.01)。IOP组大鼠视网膜组织中HO-1、Nrf2蛋白表达高于糖尿病组,COX-2蛋白的表达低于糖尿病组(均为P<0.01);抑制剂组大鼠视网膜组织中HO-1、Nrf2蛋白表达低于IOP组,COX-2蛋白的表达高于IOP组,差异均有统计学意义(均为P<0.01)。结论桦褐孔菌多糖能够通过激活Nrf2通路降低糖尿病大鼠视网膜氧化应激及炎症反应。

Objective To explore the inhibition of oxidative stress and inflammation in retinal tissue of diabetic rats by inonotus obliquus polysaccharides(IOP)via Nrf2 signaling pathway.Methods All of 40 male SD rats aged 12 weeks were selected and randomly divided into 4 groups(10 rats in each group)control group,diabetic group,IOP group(300 mg·kg^-1 IOP was given to the rats)and inhibitor group(300 mg·kg^-1 IOP+20 mg·kg^-1 zinc protoporphyrin was given to the rats).Except for the control group,all the other groups were given 10 g·L^-1 streptozotocin(60 mg·kg^-1)once intraperitoneal injection to make the diabetic rat model.IOP was given by gavage.Zinc protoporphyrin was intraperitoneally injected to the rats in the inhibitor group 24 h before the IOP gavage.The control group and the diabetic group were given the same amount of saline,and the experiment was carried out after 12 weeks of fasting and uninhibited water for 12 h.The fresh retina was embedded in paraffin and sliced for HE staining and immunohistochemical staining.MDA,GSH and SOD contents in retinal samples were detected.The morphology of the retina was observed by HE staining and the density of retinal ganglion cells was detected;HO-1 positive staining was observed by immunohistochemical staining;Western blot was used to detect the expression of retinal HO-1,Nrf2 and COX-2 proteins.Results Twelve weeks after modeling,the body mass of rats in diabetes group,IOP group and inhibitor group were all lower than those in the control group,the blood glucose levels of rats were all higher than those in the control group,and the differences were statistically significant(all P<0.01).After modeling,the contents of SOD and GSH in retinal tissue of diabetic rats was significantly lower than those of the control group(all P<0.01),MDA content was significantly higher than that of the control group,and the difference was statistically significant(all P<0.01).The contents of SOD and GSH in retinal tissue of the IOP group was higher than those of the diabetic group and inhibitor group,and the content of MDA was significantly lower than that of diabetic group and inhibitor group,and the difference was statistically significant(all P<0.05).The retinal ganglion cell densities of the control group,diabetes group,inhibitor group and IOP group were(2300±100)cells·mm^-2,(1400±100)cells·mm^-2,(1505±95)cells·mm^-2,(2250±95)cells·mm^-2.Western blot results showed that HO-1,Nrf2 and COX-2 proteins in the diabetic group were all higher than those in the control group(all P<0.01).The protein expression levels of HO-1 and Nrf2 in the retinal tissues of rats in the IOP group was higher than those in the diabetic group,while the protein expression of COX-2 was lower than that in the diabetic group(all P<0.01).The protein expression levels of HO-1 and Nrf2 in retinal tissues of the inhibitor group was lower than those of the IOP group,and the protein expression of COX-2 was higher than that of the IOP group,and the differences were statistically significant(all P<0.01).Conclusion IOP can reduce retinal oxidative stress and inflammatory response in diabetic rats,and the mechanism is related to the activation of Nrf2 pathway.