芍药苷基于JAK2/STAT3信号通路抑制糖尿病大鼠视网膜细胞炎症的研究

Paeoniflorin inhibits retinal cell inflammation in diabetes rats based on JAK2/STAT3 signaling pathway

目的基于Janus激酶/信号传导及转录激活因子(JAK/STAT)通路探讨芍药苷抑制糖尿病大鼠视网膜细胞炎症的作用机制。方法30只SD大鼠腹腔注射链脲佐菌素建立糖尿病大鼠模型,随机分为模型组(MG)、芍药苷组(PF)、芍药苷+JAK2/STAT3通路激活剂colivelin组(COL),另设对照组(CG),每组10只。PF组以4 ng/g剂量的芍药苷腹腔注射给药,每周2次;COL组在PF组的基础上,玻璃体腔一次性注射1×10^(-4)μmol/L的colivelin 5μL;其余各组腹腔注射等体积磷酸盐缓冲液,均连续给药12周。给药后处死大鼠,取视网膜组织,苏木精-伊红(HE)染色观察视网膜形态,免疫组织化学法检测视网膜神经胶质蛋白(GFAP)表达,Western Blot法检测视网膜组织白细胞介素(IL)-6、IL-1β、肿瘤坏死因子(TNF-α)、p-JAK2、JAK2、p-STAT3和STAT3蛋白表达水平。结果(1)视网膜形态:与CG组相比,MG组视网膜神经节细胞(RGC)数量减少,排列稀疏;与MG组相比,PF组RGC数量增多;而COL组较PF组RGC数量减少。(2)GFAP:与CG组相比,MG组GFAP表达增多(t=3.748,P=0.005),贯穿视网膜全层;与MG组相比,PF组GFAP表达减少(t=3.139,P=0.013);而COL组较PF组GFAP表达增加(t=2.389,P=0.043),差异均有统计学意义。(3)炎症因子:与CG组相比,MG组视网膜IL-6、IL-1β、TNF-α表达升高(t_(IL-6)=3.640,P=0.006;t_(IL-1β)=3.635,P=0.006;t_(TNF-α)=3.163,P=0.013);与MG组相比,PF组IL-6、IL-1β、TNF-α表达降低(t_(IL-6)=2.512,P=0.036;t_(IL-1β)=2.990,P=0.017;t_(TNF-α)=2.504,P=0.036);与PF组相比,COL组IL-1β、TNF-α表达升高(t_(IL-1β)=2.872,P=0.020;t_(TNF-α)=2.636,P=0.029),差异均有统计学意义。(4)JAK2/STAT3通路:与CG组相比,MG组视网膜p-JAK2、p-STAT3表达升高(t_(p-JAK2)=3.877,P=0.004;t_(p-STAT3)=3.465,P=0.008);与MG组相比,PF组p-JAK2、p-STAT3表达降低(t_(p-JAK2)=2.516,P=0.036;t_(p-STAT3)=2.772,P=0.024);与PF组相比,COL组p-JAK2、p-STAT3表达升高(t_(p-JAK2)=2.640,P=0.029;t_(p-STAT3)=2.371,P=0.045),差异均有统计学意义。结论芍药苷能抑制糖尿病大鼠视网膜细胞炎症,可能与抑制JAK2/STAT3通路有关。

OBJECTIVE To explore the mechanism of paeoniflorin inhibiting retinal neuroinflammation in diabetic rats based on Janus kinase/signaling and activator of transcription(JAK/STAT)signaling.METHODS Thirty SD rats were intraperitoneally injected with streptozotocin to establish a diabetic rat model,and randomly divided into a model group(MG),a paeoniflorin(PF)group,a paeoniflorin+JAK2/STAT3 signaling activator colivelin(COL)group,and a control group(CG),with 10 rats in each group.The PF group was administered intraperitoneally with paeoniflorin at a dose of 4 ng/g twice a week;COL group was vitreously injected with 5μL colivelin(1×10^(-4)μmol/L)for one time on basis of PF group treatment;The other groups were intraperitoneally injected with equal volume of phosphate buffer solution,and all of them were continuously administrated for 12 weeks.After drug administration,the rats were sacrificed,and the retinal tissues were harvested and stained with hematoxylin eosin(HE)to observe the retinal morphology,immunohistochemistry to detect the expression of retinal glial protein(GFAP),and Western Blot to detect the interleukin(IL)-6,IL-1β,tumor necrosis factor(TNF-α),p-JAK2,JAK2,p-STAT3 and STAT3 protein expression levels.RESULTS(1)Retinal morphology:Compared with the CG,the MG showed a decreased number and sparse arrangement of retinal ganglion cells(RGC);Compared with the MG,the number of RGCs in the PF group was increased;However,the number of RGCs decreased in the COL group compared with the PF group.(2)GFAP:Compared with the CG,increased GFAP expression was observed in the MG(t=3.748,P=0.005)throughout the full retina;Compared with the MG,GFAP expression was reduced in the PF group(t=3.139,P=0.013);However,GFAP expression was increased in the COL group in contrast to the PF group(t=2.389,P=0.043),the differences were all statistically significant.(3)Inflammatory factors:Compared with CG,the expressions of IL-6,IL-1βand TNF-αwere increased in MG(t_(IL-6)=3.640,P=0.006;t_(IL-1β)=3.635,P=0.006;t_(TNF-α)=3.163,P=0.013);Compared with MG,the expressions of IL-6,IL-1βand TNF-αin PF group were decreased(t_(IL-6)=2.512,P=0.036;t_(IL-1β)=2.990,P=0.017;t_(TNF-α)=2.504,P=0.036);Compared with PF group,the expressions of IL-1βand TNF-αwere increased in COL group(t_(IL-1β)=2.872,P=0.020;t_(TNF-α)=2.636,P=0.029),the differences were all statistically significant.(4)JAK2/STAT3 signaling:Compared with CG,the expression of p-JAK2 and p-STAT3 in retina of MG were increased(t_(p-JAK2)=3.877,P=0.004;t_(p-STAT3)=3.465,P=0.008);Compared with MG,the expressions of p-JAK2 and p-STAT3 in PF group were decreased(t_(p-JAK2)=2.516,P=0.036;t_(p-STAT3)=2.772,P=0.024);Compared with PF group,the expression of p-JAK2 and p-STAT3 in retina of COL group were increased(t_(p-JAK2)=2.640,P=0.029;t_(p-STAT3)=2.371,P=0.045),the differences were all statistically significant.CONCLUSIONS Paeoniflorin can inhibit neuroinflammation in retinal cells of diabetic rats,which may be related to the inhibition of JAK2/STAT3 signaling pathway.