基于pro NGF-sortilin-p75NTR凋亡复合体探讨大黄䗪虫胶囊改善DR大鼠视网膜NVU功能的机制

Discussion on the mechanism of Dahuang Zhechong Capsules for improving retinal NVU function in DR rats based on Pro NGF-sortilin-p75NTR apoptotic complex

目的:基于pro NGF-sortilin-p75NTR凋亡复合体探讨大黄䗪虫胶囊改善糖尿病视网膜病变(DR)大鼠视网膜NVU功能的机制。方法:选取60只7~8周龄SPF级雄性Wistar大鼠,随机分为空白组,模型组,大黄䗪虫胶囊低、中、高剂量组。除空白组外,其余各组均采用链佐脲菌素腹腔注射诱导DR大鼠模型。各组在DR造模成功后,同时以相应浓度大黄䗪虫胶囊灌胃8周。所有大鼠腹主动脉取血,取眼球及视网膜组织。ELISA检测血清pro NGF、p75NTR水平;RT-PCR检测大鼠视网膜组织pro NGF、sortilin、p75NTR mRNA表达;Western Blot检测大鼠视网膜中sortilin、p75NTR蛋白表达;同时应用免疫组化法检测视网膜组织sortilin水平。结果:与空白组比较,模型组大鼠血清pro NGF、p75NTR水平,视网膜组织中pro NGF、sortilin、p75NTR mRNA的表达水平及sortilin、p75NTR蛋白表达均显著升高(P<0.01,P<0.05);与模型组比较,大黄䗪虫胶囊中剂量血清pro NGF、p75NTR水平,视网膜组织pro NGF、sortilin mRNA及sortilin、p75NTR蛋白表达均显著降低(P<0.05,P<0.01)。sortilin主要表达在内核层及神经节细胞层,在模型组大鼠视网膜组织内核层及神经节细胞层强阳性表达;予大黄䗪虫胶囊中剂量干预后sortilin的阳性表达降低。结论:大黄䗪虫胶囊可通过调控pro NGF-sortilin-p75NTR凋亡复合体的表达,实现活血通络、攻逐瘀血的效果,并且可以修复DR视网膜NVU损伤。

Objective:To explore the mechanism of Dahuang Zhechong Capsules for improving retinal NVU function in rats with diabetic retinopathy(DR)based on pro NGF-sortilin-p75NTR apoptosis complex.Methods:Sixty 7-to 8-week-old SPFrated male Wistar rats were randomly divided into blank group,model group,Dahuang Zhechong Capsules low-dose,mediumdose group and high-dose group.The DR rat model was induced by intraperitoneal injection of streptozotocin in all groups except the blank group.After the successful modeling of DR,each group was simultaneously gavaged with the corresponding concentration of Dahuang zhechong Capsules for 8 weeks.Blood was collected from the abdominal aorta of all rats and retinal tissues were collected.serum pro NGF and p75NTR levels were measured by ELISA;pro NGF,sortilin and p75NTR mRNA expression in rat retinal tissues were measured by RT-PCR;sortilin and p75NTR protein expression in rat retinas were measured by Western Blot.Immunohistochemistry was also applied to detect the level of sortilin.Results:Compared with the blank group,serum pro NGF,p75NTR levels,pro NGF,sortilin,p75NTR mRNA expression levels and sortilin,p75NTR protein expression in retinal tissues of rats in the model group were significantly higher(P<0.01,P<0.05);Compared with the model group,serum pro NGF-sortilin-p75NTR levels,pro NGF-sortilin-p75NTR mRNA levels and sortilin and p75NTR protein expression in retinal tissues from medium dose group of Dahuang Zhechong Capsules were significantly lower(P<0.01,P<0.05).Sortilin was mainly expressed in the inner nuclear layer and ganglion cell layer,with strongly positive expression in the inner nuclear layer and ganglion cell layer of the retinal tissue in the model group;The positive expression of sortilin was significantly reduced after the medium dose intervention of Dahuang Zhechong Capsules.Conclusion:Dahuang Zhechong Capsules can regulate the expression of pro NGF-sortilin-p75NTR apoptotic complex to achieve the effect of activating blood circulation and attacking blood stasis,and can repair DR retinal NVU damage.