基于炎症研究白芍总苷抗糖尿病肾病作用及分子机制
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摘要
背景与目的：
最近的研究表明糖尿病发病与机体慢性低度炎症反应和固有免疫系统的激活有关，这一发现更新了以前认为糖尿病仅为代谢紊乱性疾病的概念。糖尿病肾病（Diabetic nephropathy, DN）是严重威胁人类健康的疾病，DN患者随着糖尿病发病率及患病率增加而逐年增多。DN的发病机制及干预治疗措施一直是近年来的研究热点。糖代谢紊乱是DN发病基础，由此产生的肾血流动力学改变、非酶糖基化反应、多元醇通路激活、炎症反应、氧化应激、足细胞损伤、肾小管上皮细胞转分化、蛋白激酶C激活、脂代谢紊乱及遗传背景等都与DN有关。炎症反应与DN的发展有着密切的关系，在很多DN临床及动物实验的研究中发现趋化因子、粘附分子、前炎症因子表达异常。糖尿病的实验研究发现一些药物如霉酚酸酯、甲氨蝶呤、红霉素等可通过抑制炎症反应来防止糖尿病肾损伤的发生。DN进展缓慢，早期不易觉察，目前多以出现微量白蛋白尿做为早期DN的标志。DN一旦进入到显性白蛋白尿期，即临床糖尿病肾病期，则病情发展迅速，所以目前强调只有对早期DN进行合理干预才能有效降低DN患者进入终末期肾衰的风险。白芍总苷（Total glucosides of paeony,TGP）是中药白芍的有效提取物，主要含有芍药苷、羟基芍药苷、芍药花苷、芍药内酯苷、苯甲酰芍药苷等成分,其药理作用有抗炎、抗氧化与免疫调节活性。TGP在类风湿性关节炎的治疗方面已经取得了显著的疗效，在实验性肝损伤、系统性红斑狼疮、肾小球肾炎的研究中亦取得了较好的结果,且无明显的毒副作用。本研究通过建立早期DN大鼠模型并观察经TGP干预的疗效，进一步探讨TGP能否通过干预炎症反应、氧化应激损伤、足细胞损伤、肾小管上皮转分化、细胞内信号转导等多种途径来治疗DN。
研究方法
建立链脲佐菌素糖尿病大鼠模型，将50只大鼠随机分为正常对照组、模型组、TGP给药组，TGP分为三个剂量组（50，100，200mg/kg.d）每日灌胃给药，对照组和模型组每日给予等量溶媒灌胃，干预治疗8周。留取大鼠血、尿、肾组织标本，观察以下指标包括肾重、肾重/体重比、血糖、血肌酐、尿肌酐、尿量、肾脏病理形态学、肾脏超微结构、肾组织总抗氧化能力（T-AOC）、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-PX)等。间接免疫荧光法观察nephrin在肾小球内的分布特点。免疫组化法检测转化生长因子β1（TGF-β1）、Toll样受体（Toll-like-receptor, TLR）2、TLR4、骨桥蛋白（OPN）、α-平滑肌肌动蛋白（α-SMA）、E-钙粘附蛋白（E-cad）、波形蛋白(Vim)、ED-1、增殖细胞核抗原（PCNA）、信号转导和转录活化因子3（STAT3）、磷酸化STAT3(p-STAT3)、NF-κB-p-p65等蛋白在肾组织内的表达分布情况。免疫组化双染检测PCNA/ED-1、p-STAT3/ED-1、TLR2/ED-1、TLR4/ED-1阳性细胞在肾组织内的分布情况。Western印迹法检测肿瘤坏死因子α（TNF-α）、细胞间粘附分子-1（ICAM-1）、白细胞介素-1（IL-1）、NF-κB-p65、Ⅳ型胶原、3-硝基酪氨酸（3-NT）、nephrin、磷酸化p38MAPK(p-p38)，磷酸化JAK2（p-JAK2）、磷酸化STAT3（p-STAT3）等蛋白在肾组织内的表达情况。
结果
模型组大鼠24h尿白蛋白排泄率(UAER)明显高于对照组(P<0.01)，TGP给药组UAER较模型组明显降低(P<0.01)。模型组大鼠表现为血糖升高、体重下降、相对肾重增加，TGP(50，100，200mg/kg.d）给药对大鼠血糖升高与体重下降没有明显的影响(P﹥0.05)。模型组大鼠肾小球体积明显高于对照组（P<0.01），TGP(100，200mg/kg.d）给药组肾小球体积较模型组明显减小(P<0.05, P<0.01)。TGP给药组肾脏超微结构的病变较模型组明显改善。TGP(100，200mg/kg.d）给药组肾小管-间质损伤指数(TII)明显低于模型组(P<0.05, P<0.01)。TGP(50，100，200mg/kg.d）给药组肾组织内ICAM-1、TNF-α、IL-1、NF-κB-p65、Ⅳ型胶原等蛋白表达水平较模型组均有明显下降，其中ICAM-1分别下降47.9%、60.4%、72.9%，TNF-α分别下降46.5%、86%、93.8%，IL-1分别下降77.7%、86.4%、88.6%，NF-κB-p65分别下降38.4%、53%、59%，Ⅳ型胶原分别下降47.9%、60.4%、72.9%。模型组大鼠肾组织中TLR2、TLR4表达明显高于对照组（P<0.01）。与模型组相比，TGP（50、100、200mg/kg·d）给药8周对肾小球TLR2表达无明显影响，肾小管-间质TLR2表达明显低于模型组（P<0.01）。TGP（50、100、200mg/kg·d）给药组肾小球与肾小管-间质TLR4表达均明显低于模型组（P<0.01）。
与对照组相比、模型组肾组织内T-AOC、SOD、CAT活性明显降低(P<0.01)，TGP(200mg/kg.d)给药组T-AOC、SOD、CAT活性明显高于模型组(P<0.01)。TGP(50，100，200mg/kg.d）给药组肾组织内3-NT、p-p38蛋白表达水平较模型组明显下降，NT分别下降41.2%、43.8%、57.5%，p-p38分别下降30.8%、24%、60.1%。免疫荧光发现对照组肾小球内nephrin呈线状均匀分布，模型组nephrin表达明显减小、呈颗粒状不均匀分布，TGP给药组nephrin表达部分恢复。TGP(50，100，200mg/kg.d）给药组nephrin蛋白表达较模型组分别增加25.2%、89.2%、73.9%。
模型组大鼠肾小管-间质E-cad蛋白表达明显低于对照组(P<0.01)，TGP （50，100，200mg/kg.d）给药组肾小管-间质E-cad蛋白表达明显高于模型组(P<0.01)；模型组肾小管-间质α-SMA蛋白表达明显高于对照组(P <0.01)，TGP （50，100，200mg/kg.d）给药组肾小管-间质α-SMA蛋白表达明显低于模型组(P<0.01)；模型组肾小管-间质Vim蛋白表达明显高于对照组(P <0.01)，TGP （50，100，200mg/kg.d）给药组肾小管-间质Vim蛋白表达明显低于模型组(P<0.01)；模型组肾小管-间质OPN蛋白表达明显高于对照组(P<0.01)，TGP（100，200mg/kg.d）给药组肾小管-间质OPN蛋白表达明显低于模型组(P<0.01)。
模型组大鼠肾组织内PCNA+细胞数、ED-1+细胞数、p-STAT3+细胞数、PCNA+/ED-1+细胞数、p-STAT3+/ED-1+细胞数、TLR2+/ED-1+细胞数、TLR4+/ED-1+细胞数、NF-κB-p-p65+细胞数均明显高于对照组(P<0.01)。TGP(50，100，200mg/kg.d）给药组PCNA+、ED-1+、p-STAT3、PCNA+/ED-1+、p-STAT3+/ED-1+、TLR2+/ED-1+、TLR4+/ED-1+、NF-κB-p-p65+阳性细胞数均较模型组明显减少(P <0.01)。模型组肾组织内p-JAK2、 p-STAT3蛋白表达均显著高于对照组(P <0.01)，TGP(50，100，200mg/kg.d）给药组使p-JAK2蛋白分别下降22.3%、50.5%、43.2%，使p-STAT3蛋白分别下降20.9%、61.1%、42.3%。
结论
TGP对STZ诱导糖尿病大鼠的早期肾损害有明显的保护作用。TGP可能是通过以下多种机制如抑制肾内炎症反应、抗氧化应激损伤、抑制肾小管上皮细胞转分化、抑制肾间质纤维化、抑制巨噬细胞浸润及增殖活化、抑制JAK2/STAT3信号通路的活性等多种途径发挥肾保护作用。
Background and objective
The notion that chronic low-grade inflammation and activation of the innateimmune system are closely involved in the pathogenesis of diabetes mellitus hassubstantially changed our vision of this disease as a metabolic disorder in the past years.Diabetic nephropathy (DN) is a serious threat to human health. With the increase in theincidence and the prevalence of diabetes, DN is becoming more common. Pathogenesisand therapeutic intervention in DN have been the research focus in recent years.Disorder of glucose metabolism is the fundamental step in DN, resulting renalhemodynamic changes， non-enzymatic glycosylation reaction, polyol pathwayactivation, inflammation, oxidative stress, and podocyte injury, renal tubular epithelialcell transdifferentiation，protein kinase C activation, lipid metabolism disorders andgenetic background etc, are all associated with DN. Development of DN is slow andimperceptible in early stage; microalbuminuria apperance is used to be a sign of earlyDN. A number of experimental and clinical studies have demonstrated the significantrole of various inflammatory molecules in the setting of DN, including chemokines,adhesion molecules, and proinflammatory cytokines. Several works in the setting ofexperimental DN using different drugs, such as mycophenolate mofetil, methotrexate,and erythromycin, have shown that prevention of the development or amelioration ofrenal injury in diabetes was associated with anti-inflammatory actions. When diabeticpatients progress into albuminuria stage, DN becomes irreversible. So the current effortswere stressed that giving the early DN reasonable intervention to reduce the risk of entering end-stage renal failure. Paeonia lactiflora pall is a kind of Chinese traditionalherbal medicine. Effective parts and chemical constituents of TGP have been extractedand purified, their structures have been identified. TGP contains96.2%of paeoniflorinand other components such as hydroxy-paeoniflorin, glycosides of peony flower, peonylactone glycosides, benzoyl paeoniflorin etc. TGP have been extensively proven toexhibit anti-inflammatory, antioxidative, antihepatic injury and immunoregulatoryactivities without evident toxic or side-effects. In this study we established the early DNrat model and investigated the efficacy of the TGP, further explored the possiblemechanisms involving in the TGP renoprotective effects were through interfering withthe inflammatory response, oxidative stress injury, podocyte injury, renal tubularepithelial transdifferentiation and intracellular signal transduction.
Method
Diabetes was induced by peritoneal injection of streptozotocin in rats.50rats wererandomly divided into normal group, control diabetic group, TGP administration groupthat was divided into three dose groups (50,100,200mg/kg.d) with daily gavaged,normal group and control diabetic group were given the same amount of solvent foreight weeks. Specimens of rat blood, urine, kidney tissue were collected, detectionindex including kidney weight, kidney weight/body weight ratio, blood glucose, serumcreatinine, urine creatinine,24h urinary albumin excretion rate (UAER), renal pathology,renal ultrastructure, renal tissue total antioxidant capacity (T-AOC), superoxidedismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX) using theappropriate method for testing. Indirect immunofluorescence was used to detect nephrindistribution in glomeruli. Immunohistochemical assays of transforming growth factorβ1(TGF-β1), Toll-like-receptor2(TLR2), TLR4, osteopontin protein (OPN), α-smoothmuscle actin (α-SMA), E-calcium adhesion proteins (E-cad), vimentin (Vim), ED-1,proliferating cell nuclear antigen (PCNA), signal transduction and transcription-activating factor3(STAT3), phosphorylation of STAT3(p-STAT) proteinexpressions in renal tissue. Double immunohistochemical assays of PCNA/ED-1,p-STAT3/ED-1, TLR2/ED-1, TLR4/ED-1positive cells in the renal tissue distribution.Western blotting detection of tumor necrosis factor-α (TNF-α), intercellular adhesionmolecule-1(ICAM-1), interleukin-1(IL-1), NF-κB p65, collagen type IV,3-nitrotyrosine (NT), nephrin, phosphorylation of p38MAPK (p-p38), phosphorylationof JAK2(p-JAK2) phosphorylation of STAT3(p-STAT3) protein expression in renaltissue.
Results：
In the control diabetic group, UAER was significantly higher than the normalgroup (P<0.01); UAER in the three TGP administration groups was significantly lowerthan the control diabetic group (P<0.01). The rats in control diabetic group manifestedas hyperglycemia, weight loss, increased relative kidney weight, TGP (50,100,200mg/kg.d) administration eight weeks had no signifcant effect in preventing the modelrats hyperglycemia and weight loss (P﹥0.01). The glomerular volume (VG) in thecontrol diabetic group was significantly higher than the normal group, in TGP (100,200mg/kg.d) administration groups the VGwere significantly lower than the controldiabetic group (P<0.05, P<0.01). In the TGP (100,200mg/kg.d) administration groupsthe tubulointerstitial injury index (TII) was significantly lower than the control diabeticgroup (P<0.05, P<0.01). The rats renal tissue expressions of ICAM-1, TNF-α, IL-1,NF-κB-p65, type IV collagen protein compared with the control diabetic group weresignificantly reduced by TGP (50,100,200mg/kg.d) administration, ICAM-1weredown47.9%,60.4%,72.9%, respectively; and TNF-α were down46.5%,86%,93.8%respectively; IL-1, were down77.7%,86.4%,88.6%, respectively; NF-κB-p65weredown38.4%,53%,59%, respectively; type IV collagen were down47.9%,60.4%,72.9%, respectively. Compared with the control diabetic group, TGP (50,100,200mg/ kg.d) administration eight weeks had no significant effect on glomerular TLR2expression, tubular-interstitial TLR2expression was significantly lower than that in thecontrol diabetic group (P<0.01). The glomerular and tubular-interstitial TLR4expressions were significantly lower by TGP (50,100,200mg/kg.d) administrationcompared with the control diabetic group (P<0.01).
The renal tissue T-AOC, SOD and CAT activity in the control diabetic groupdecreased significantly compared with the normal group (P<0.01). T-AOC, SOD andCAT activity in TGP (200mg/kg.d) administration were significantly higher than thecontrol diabetic group (P<0.01). The renal tissue expression of3-NT, p-p38proteinlevels were significantly reduced by TGP (50,100,200mg/kg.d) administrationcompared with the control diabetic group,3-NT was down41.2%,43.8%,57.5%,respectively; p-p38was down30.8%,24%and60.1%, respectively. The nephrin waslinearly uniformly distributed in normal group glomeruli. Nephrin expression wassignificantly reduced, showing granular uneven distribution in control diabetic group.Nephrin expression was partially restored in the glomeruli by TGP administration.Compared with the control diabetic group the nephrin protein expression were up25.2%,89.2%,73.9%, respectively in the TGP (50,100,200mg/kg.d) administration group.
The tubulointerstitial E-cad protein expression in the control diabetic group wassignificantly lower than that in the normal group (P<0.01), tubulointerstitial E-cadprotein expression was significantly higher than that in the control diabetic group byTGP (50,100,200mg/kg.d)8weeks administration (P<0.01). The tubulointerstitialα-SMA, Vim and OPN protein expression in the control diabetic group weresignificantly higher than the normal group (P<0.01), tubulointerstitial α-SMA, Vim andOPN protein expressions were significantly lower than that in the control diabetic groupby TGP (50,100,200mg/kg.d)8weeks administration (P <0.01).
The PCNA, ED-1, p-STAT3, PCNA/ED-1, p-STAT3/ED-1, TLR2/ED-1,TLR4/ED-1positive staining cell number in the control diabetic group were significantly higher than that in the normal group (P<0.01). The PCNA, ED-1, p-STAT3,PCNA/ED-1, p-STAT3/ED-1positive staining cell number in TGP (50,100,200mg/kg.d) administered group were significantly lower than that in the control diabetic group(P<0.01). p-JAK2, p-STAT3protein expressions in renal tissue of the control diabeticgroup were significantly higher than that in the normal group (P<0.01). By TGP (50,100,200mg/kg.d) administration, the p-JAK2protein expressions were down22.3%,50.5%,43.2%, respectively; p-STAT3protein expressions were down20.9%,61.1%,42.3%, respectively compared to the control diabetic group.
Conclusionsdf
TGP has a significant protective effect on early renal damage in STZ-induceddiabetic rats. The TGP plays a renoprotective effect possiblely by following a variety ofmechanisms, such as the inhibition of intrarenal inflammatory response, anti-oxidativestress damage, inhibition of renal tubular epithelial cell transdifferentiation, inhibition ofrenal interstitial fibrosis, inhibition of macrophage infiltration and proliferation andactivation, inhibition of JAK2/STAT3signaling pathways.
最近的研究表明糖尿病发病与机体慢性低度炎症反应和固有免疫系统的激活有关，这一发现更新了以前认为糖尿病仅为代谢紊乱性疾病的概念。糖尿病肾病（Diabetic nephropathy, DN）是严重威胁人类健康的疾病，DN患者随着糖尿病发病率及患病率增加而逐年增多。DN的发病机制及干预治疗措施一直是近年来的研究热点。糖代谢紊乱是DN发病基础，由此产生的肾血流动力学改变、非酶糖基化反应、多元醇通路激活、炎症反应、氧化应激、足细胞损伤、肾小管上皮细胞转分化、蛋白激酶C激活、脂代谢紊乱及遗传背景等都与DN有关。炎症反应与DN的发展有着密切的关系，在很多DN临床及动物实验的研究中发现趋化因子、粘附分子、前炎症因子表达异常。糖尿病的实验研究发现一些药物如霉酚酸酯、甲氨蝶呤、红霉素等可通过抑制炎症反应来防止糖尿病肾损伤的发生。DN进展缓慢，早期不易觉察，目前多以出现微量白蛋白尿做为早期DN的标志。DN一旦进入到显性白蛋白尿期，即临床糖尿病肾病期，则病情发展迅速，所以目前强调只有对早期DN进行合理干预才能有效降低DN患者进入终末期肾衰的风险。白芍总苷（Total glucosides of paeony,TGP）是中药白芍的有效提取物，主要含有芍药苷、羟基芍药苷、芍药花苷、芍药内酯苷、苯甲酰芍药苷等成分,其药理作用有抗炎、抗氧化与免疫调节活性。TGP在类风湿性关节炎的治疗方面已经取得了显著的疗效，在实验性肝损伤、系统性红斑狼疮、肾小球肾炎的研究中亦取得了较好的结果,且无明显的毒副作用。本研究通过建立早期DN大鼠模型并观察经TGP干预的疗效，进一步探讨TGP能否通过干预炎症反应、氧化应激损伤、足细胞损伤、肾小管上皮转分化、细胞内信号转导等多种途径来治疗DN。
研究方法
建立链脲佐菌素糖尿病大鼠模型，将50只大鼠随机分为正常对照组、模型组、TGP给药组，TGP分为三个剂量组（50，100，200mg/kg.d）每日灌胃给药，对照组和模型组每日给予等量溶媒灌胃，干预治疗8周。留取大鼠血、尿、肾组织标本，观察以下指标包括肾重、肾重/体重比、血糖、血肌酐、尿肌酐、尿量、肾脏病理形态学、肾脏超微结构、肾组织总抗氧化能力（T-AOC）、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-PX)等。间接免疫荧光法观察nephrin在肾小球内的分布特点。免疫组化法检测转化生长因子β1（TGF-β1）、Toll样受体（Toll-like-receptor, TLR）2、TLR4、骨桥蛋白（OPN）、α-平滑肌肌动蛋白（α-SMA）、E-钙粘附蛋白（E-cad）、波形蛋白(Vim)、ED-1、增殖细胞核抗原（PCNA）、信号转导和转录活化因子3（STAT3）、磷酸化STAT3(p-STAT3)、NF-κB-p-p65等蛋白在肾组织内的表达分布情况。免疫组化双染检测PCNA/ED-1、p-STAT3/ED-1、TLR2/ED-1、TLR4/ED-1阳性细胞在肾组织内的分布情况。Western印迹法检测肿瘤坏死因子α（TNF-α）、细胞间粘附分子-1（ICAM-1）、白细胞介素-1（IL-1）、NF-κB-p65、Ⅳ型胶原、3-硝基酪氨酸（3-NT）、nephrin、磷酸化p38MAPK(p-p38)，磷酸化JAK2（p-JAK2）、磷酸化STAT3（p-STAT3）等蛋白在肾组织内的表达情况。
结果
模型组大鼠24h尿白蛋白排泄率(UAER)明显高于对照组(P<0.01)，TGP给药组UAER较模型组明显降低(P<0.01)。模型组大鼠表现为血糖升高、体重下降、相对肾重增加，TGP(50，100，200mg/kg.d）给药对大鼠血糖升高与体重下降没有明显的影响(P﹥0.05)。模型组大鼠肾小球体积明显高于对照组（P<0.01），TGP(100，200mg/kg.d）给药组肾小球体积较模型组明显减小(P<0.05, P<0.01)。TGP给药组肾脏超微结构的病变较模型组明显改善。TGP(100，200mg/kg.d）给药组肾小管-间质损伤指数(TII)明显低于模型组(P<0.05, P<0.01)。TGP(50，100，200mg/kg.d）给药组肾组织内ICAM-1、TNF-α、IL-1、NF-κB-p65、Ⅳ型胶原等蛋白表达水平较模型组均有明显下降，其中ICAM-1分别下降47.9%、60.4%、72.9%，TNF-α分别下降46.5%、86%、93.8%，IL-1分别下降77.7%、86.4%、88.6%，NF-κB-p65分别下降38.4%、53%、59%，Ⅳ型胶原分别下降47.9%、60.4%、72.9%。模型组大鼠肾组织中TLR2、TLR4表达明显高于对照组（P<0.01）。与模型组相比，TGP（50、100、200mg/kg·d）给药8周对肾小球TLR2表达无明显影响，肾小管-间质TLR2表达明显低于模型组（P<0.01）。TGP（50、100、200mg/kg·d）给药组肾小球与肾小管-间质TLR4表达均明显低于模型组（P<0.01）。
与对照组相比、模型组肾组织内T-AOC、SOD、CAT活性明显降低(P<0.01)，TGP(200mg/kg.d)给药组T-AOC、SOD、CAT活性明显高于模型组(P<0.01)。TGP(50，100，200mg/kg.d）给药组肾组织内3-NT、p-p38蛋白表达水平较模型组明显下降，NT分别下降41.2%、43.8%、57.5%，p-p38分别下降30.8%、24%、60.1%。免疫荧光发现对照组肾小球内nephrin呈线状均匀分布，模型组nephrin表达明显减小、呈颗粒状不均匀分布，TGP给药组nephrin表达部分恢复。TGP(50，100，200mg/kg.d）给药组nephrin蛋白表达较模型组分别增加25.2%、89.2%、73.9%。
模型组大鼠肾小管-间质E-cad蛋白表达明显低于对照组(P<0.01)，TGP （50，100，200mg/kg.d）给药组肾小管-间质E-cad蛋白表达明显高于模型组(P<0.01)；模型组肾小管-间质α-SMA蛋白表达明显高于对照组(P <0.01)，TGP （50，100，200mg/kg.d）给药组肾小管-间质α-SMA蛋白表达明显低于模型组(P<0.01)；模型组肾小管-间质Vim蛋白表达明显高于对照组(P <0.01)，TGP （50，100，200mg/kg.d）给药组肾小管-间质Vim蛋白表达明显低于模型组(P<0.01)；模型组肾小管-间质OPN蛋白表达明显高于对照组(P<0.01)，TGP（100，200mg/kg.d）给药组肾小管-间质OPN蛋白表达明显低于模型组(P<0.01)。
模型组大鼠肾组织内PCNA+细胞数、ED-1+细胞数、p-STAT3+细胞数、PCNA+/ED-1+细胞数、p-STAT3+/ED-1+细胞数、TLR2+/ED-1+细胞数、TLR4+/ED-1+细胞数、NF-κB-p-p65+细胞数均明显高于对照组(P<0.01)。TGP(50，100，200mg/kg.d）给药组PCNA+、ED-1+、p-STAT3、PCNA+/ED-1+、p-STAT3+/ED-1+、TLR2+/ED-1+、TLR4+/ED-1+、NF-κB-p-p65+阳性细胞数均较模型组明显减少(P <0.01)。模型组肾组织内p-JAK2、 p-STAT3蛋白表达均显著高于对照组(P <0.01)，TGP(50，100，200mg/kg.d）给药组使p-JAK2蛋白分别下降22.3%、50.5%、43.2%，使p-STAT3蛋白分别下降20.9%、61.1%、42.3%。
结论
TGP对STZ诱导糖尿病大鼠的早期肾损害有明显的保护作用。TGP可能是通过以下多种机制如抑制肾内炎症反应、抗氧化应激损伤、抑制肾小管上皮细胞转分化、抑制肾间质纤维化、抑制巨噬细胞浸润及增殖活化、抑制JAK2/STAT3信号通路的活性等多种途径发挥肾保护作用。
Background and objective
The notion that chronic low-grade inflammation and activation of the innateimmune system are closely involved in the pathogenesis of diabetes mellitus hassubstantially changed our vision of this disease as a metabolic disorder in the past years.Diabetic nephropathy (DN) is a serious threat to human health. With the increase in theincidence and the prevalence of diabetes, DN is becoming more common. Pathogenesisand therapeutic intervention in DN have been the research focus in recent years.Disorder of glucose metabolism is the fundamental step in DN, resulting renalhemodynamic changes， non-enzymatic glycosylation reaction, polyol pathwayactivation, inflammation, oxidative stress, and podocyte injury, renal tubular epithelialcell transdifferentiation，protein kinase C activation, lipid metabolism disorders andgenetic background etc, are all associated with DN. Development of DN is slow andimperceptible in early stage; microalbuminuria apperance is used to be a sign of earlyDN. A number of experimental and clinical studies have demonstrated the significantrole of various inflammatory molecules in the setting of DN, including chemokines,adhesion molecules, and proinflammatory cytokines. Several works in the setting ofexperimental DN using different drugs, such as mycophenolate mofetil, methotrexate,and erythromycin, have shown that prevention of the development or amelioration ofrenal injury in diabetes was associated with anti-inflammatory actions. When diabeticpatients progress into albuminuria stage, DN becomes irreversible. So the current effortswere stressed that giving the early DN reasonable intervention to reduce the risk of entering end-stage renal failure. Paeonia lactiflora pall is a kind of Chinese traditionalherbal medicine. Effective parts and chemical constituents of TGP have been extractedand purified, their structures have been identified. TGP contains96.2%of paeoniflorinand other components such as hydroxy-paeoniflorin, glycosides of peony flower, peonylactone glycosides, benzoyl paeoniflorin etc. TGP have been extensively proven toexhibit anti-inflammatory, antioxidative, antihepatic injury and immunoregulatoryactivities without evident toxic or side-effects. In this study we established the early DNrat model and investigated the efficacy of the TGP, further explored the possiblemechanisms involving in the TGP renoprotective effects were through interfering withthe inflammatory response, oxidative stress injury, podocyte injury, renal tubularepithelial transdifferentiation and intracellular signal transduction.
Method
Diabetes was induced by peritoneal injection of streptozotocin in rats.50rats wererandomly divided into normal group, control diabetic group, TGP administration groupthat was divided into three dose groups (50,100,200mg/kg.d) with daily gavaged,normal group and control diabetic group were given the same amount of solvent foreight weeks. Specimens of rat blood, urine, kidney tissue were collected, detectionindex including kidney weight, kidney weight/body weight ratio, blood glucose, serumcreatinine, urine creatinine,24h urinary albumin excretion rate (UAER), renal pathology,renal ultrastructure, renal tissue total antioxidant capacity (T-AOC), superoxidedismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX) using theappropriate method for testing. Indirect immunofluorescence was used to detect nephrindistribution in glomeruli. Immunohistochemical assays of transforming growth factorβ1(TGF-β1), Toll-like-receptor2(TLR2), TLR4, osteopontin protein (OPN), α-smoothmuscle actin (α-SMA), E-calcium adhesion proteins (E-cad), vimentin (Vim), ED-1,proliferating cell nuclear antigen (PCNA), signal transduction and transcription-activating factor3(STAT3), phosphorylation of STAT3(p-STAT) proteinexpressions in renal tissue. Double immunohistochemical assays of PCNA/ED-1,p-STAT3/ED-1, TLR2/ED-1, TLR4/ED-1positive cells in the renal tissue distribution.Western blotting detection of tumor necrosis factor-α (TNF-α), intercellular adhesionmolecule-1(ICAM-1), interleukin-1(IL-1), NF-κB p65, collagen type IV,3-nitrotyrosine (NT), nephrin, phosphorylation of p38MAPK (p-p38), phosphorylationof JAK2(p-JAK2) phosphorylation of STAT3(p-STAT3) protein expression in renaltissue.
Results：
In the control diabetic group, UAER was significantly higher than the normalgroup (P<0.01); UAER in the three TGP administration groups was significantly lowerthan the control diabetic group (P<0.01). The rats in control diabetic group manifestedas hyperglycemia, weight loss, increased relative kidney weight, TGP (50,100,200mg/kg.d) administration eight weeks had no signifcant effect in preventing the modelrats hyperglycemia and weight loss (P﹥0.01). The glomerular volume (VG) in thecontrol diabetic group was significantly higher than the normal group, in TGP (100,200mg/kg.d) administration groups the VGwere significantly lower than the controldiabetic group (P<0.05, P<0.01). In the TGP (100,200mg/kg.d) administration groupsthe tubulointerstitial injury index (TII) was significantly lower than the control diabeticgroup (P<0.05, P<0.01). The rats renal tissue expressions of ICAM-1, TNF-α, IL-1,NF-κB-p65, type IV collagen protein compared with the control diabetic group weresignificantly reduced by TGP (50,100,200mg/kg.d) administration, ICAM-1weredown47.9%,60.4%,72.9%, respectively; and TNF-α were down46.5%,86%,93.8%respectively; IL-1, were down77.7%,86.4%,88.6%, respectively; NF-κB-p65weredown38.4%,53%,59%, respectively; type IV collagen were down47.9%,60.4%,72.9%, respectively. Compared with the control diabetic group, TGP (50,100,200mg/ kg.d) administration eight weeks had no significant effect on glomerular TLR2expression, tubular-interstitial TLR2expression was significantly lower than that in thecontrol diabetic group (P<0.01). The glomerular and tubular-interstitial TLR4expressions were significantly lower by TGP (50,100,200mg/kg.d) administrationcompared with the control diabetic group (P<0.01).
The renal tissue T-AOC, SOD and CAT activity in the control diabetic groupdecreased significantly compared with the normal group (P<0.01). T-AOC, SOD andCAT activity in TGP (200mg/kg.d) administration were significantly higher than thecontrol diabetic group (P<0.01). The renal tissue expression of3-NT, p-p38proteinlevels were significantly reduced by TGP (50,100,200mg/kg.d) administrationcompared with the control diabetic group,3-NT was down41.2%,43.8%,57.5%,respectively; p-p38was down30.8%,24%and60.1%, respectively. The nephrin waslinearly uniformly distributed in normal group glomeruli. Nephrin expression wassignificantly reduced, showing granular uneven distribution in control diabetic group.Nephrin expression was partially restored in the glomeruli by TGP administration.Compared with the control diabetic group the nephrin protein expression were up25.2%,89.2%,73.9%, respectively in the TGP (50,100,200mg/kg.d) administration group.
The tubulointerstitial E-cad protein expression in the control diabetic group wassignificantly lower than that in the normal group (P<0.01), tubulointerstitial E-cadprotein expression was significantly higher than that in the control diabetic group byTGP (50,100,200mg/kg.d)8weeks administration (P<0.01). The tubulointerstitialα-SMA, Vim and OPN protein expression in the control diabetic group weresignificantly higher than the normal group (P<0.01), tubulointerstitial α-SMA, Vim andOPN protein expressions were significantly lower than that in the control diabetic groupby TGP (50,100,200mg/kg.d)8weeks administration (P <0.01).
The PCNA, ED-1, p-STAT3, PCNA/ED-1, p-STAT3/ED-1, TLR2/ED-1,TLR4/ED-1positive staining cell number in the control diabetic group were significantly higher than that in the normal group (P<0.01). The PCNA, ED-1, p-STAT3,PCNA/ED-1, p-STAT3/ED-1positive staining cell number in TGP (50,100,200mg/kg.d) administered group were significantly lower than that in the control diabetic group(P<0.01). p-JAK2, p-STAT3protein expressions in renal tissue of the control diabeticgroup were significantly higher than that in the normal group (P<0.01). By TGP (50,100,200mg/kg.d) administration, the p-JAK2protein expressions were down22.3%,50.5%,43.2%, respectively; p-STAT3protein expressions were down20.9%,61.1%,42.3%, respectively compared to the control diabetic group.
Conclusionsdf
TGP has a significant protective effect on early renal damage in STZ-induceddiabetic rats. The TGP plays a renoprotective effect possiblely by following a variety ofmechanisms, such as the inhibition of intrarenal inflammatory response, anti-oxidativestress damage, inhibition of renal tubular epithelial cell transdifferentiation, inhibition ofrenal interstitial fibrosis, inhibition of macrophage infiltration and proliferation andactivation, inhibition of JAK2/STAT3signaling pathways.
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