复方鱼腥草对糖尿病小鼠肾损伤和JAK/STAT-SOCS-1负反馈调节研究
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摘要
目的研究复方鱼腥草3种提取部位及其完整成分对db/db小鼠肾损害的保护作用,通过研究各提取物对JAK2/STAT3通路及其下游蛋白的影响探讨复方鱼腥草防治糖尿病肾病的可能机制。方法 1、提取复方鱼腥草水提物、醇提物、挥发油成分,并将3种成分按照所得质量比配制得到复方鱼腥草完整成分。2、选取8只db/m小鼠为正常组;将56只db/db小鼠分为模型组、盐酸二甲双胍组、AG490组、复方鱼腥草水提物组、复方鱼腥草醇提物组、复方鱼腥草挥发油组以及复方鱼腥草完整成分组,每组8只,给药8周,酶联免疫试剂盒法检测血浆转化生长因子-β1(TGF-β1)、纤连蛋白(FN)、胰高血糖素样肽-1(GLP-1)、抑胃肽(GIP)水平,HE染色观察小鼠肾脏病理变化。3、以RT-PCR法检测各组小鼠肾组织中的JAK2 mRNA、STAT3mRNA、SOCS-1 mRNA表达的变化情况;以Western Blot法检测肾组织中的JAK2、p-JAK2、STAT3、pSTAT3、SOCS-1、c-fos及c-jun蛋白表达变化,运用免疫荧光法检测肾组织c-fos、c-jun蛋白的表达。结果给药8周后,与正常组比较,模型组血浆中TGF-β1、FN、GIP水平升高(P <0.05),GLP-1水平下降(P <0.05);与模型组比较,治疗组小鼠血浆中TGF-β1、FN、GIP、GLP-1水平及肾组织病理改变均有所改善。模型组小鼠肾组织p-JAK2、p-STAT3蛋白表达及JAK2、STAT3 mRNA表达均比正常组增高(P <0.05);与模型组相比,各给药组小鼠肾组织p-JAK2,p-STAT3蛋白表达均有降低,醇提物组、挥发油组及完整成分组小鼠肾组织SOCS-1蛋白显著升高(P <0.05);各给药组的JAK2、STAT3基因表达变化不显著(P> 0.05)。AG490组及完整成分组的c-fos、c-jun在肾小管及肾间质的表达均有不同程度下降(P <0.05)。结论复方鱼腥草对糖尿病肾损伤具有改善作用,其保护肾脏的作用可能与调节GLP-1、GIP水平,降低FN、TGF-β1的分泌,减少ECM聚积,维持肾脏结构、功能的完整性有关。其改善糖尿病肾损伤的分子机制可能通过SOCS负反馈调节JAK/STAT信号转导通路相关基因及蛋白表达,从而抑制降低下游原癌基因c-fos、c-jun的表达,减少炎症因子的活化有关。
Objective To study the renoprotective effects of different extracts from compound Houttuyniae herba and its mechanism through JAK/STAT-SOCS-1 signaling pathway. Methods 1. The complete extracts of compound Houttuyniae herba were prepared by mixing the volatile oils,aqueous extracts and alcohol extracts according to the mass ratio of the acquired extracts from herbs. 2. Eight db/m mice were assigned to control group,and 56 db/db mice were randomized into 7 group with 8 animals in each group:model group,Metformin hydrochloride group,AG490 group, Water extract group, Ethanol extract group, Volatile oil group, and Mixture group(mixture of above three extract) of compound Houttuyniae herba. After treating the mice for 8 weeks, 2 mL of blood was collected from the abdominal aorta after anesthesia and kidney tissues were quickly removed. All the left kidney tissues were fixed in 10% formalin,and the right ones were stored at-80℃ for PCR and Western Blot detection.Serum level of FN,TGF-β1,GLP-1,GIP were measured by ELISA. The total RNA and protein were collected and RT-PCR method was used to detect the effect on mRNA expression of JAK2,STAT3,SOCS1 and Western Blot method was used to detect the protein expression of JAK2,p-JAK2,STAT3,p-STAT3,SOCS1,c-fos and cjun. Immunofluorescence was used to detect the protein expression of c-fos,c-jun in kidney tissue. Results After 8 weeks, the mRNA levels of p-JAK2, p-STAT3, JAK2 and STAT3 in mice kidney tissue were significantly increased in the model group(P < 0.05). Compared with the model group,mRNA and protein levels of p-JAK2,pSTAT3 of mice in all treatment groups were lowered;the mRNA and protein levels of SOCS-1 in volatile oils and complete extracts groups mice were significantly increased(P < 0.05),with no change in those of JAK2,STAT3 in all treatment groups. Expression levels of c-fos, c-jun in kidney tubule and renal interstitium of mice in all treatment groups decreased to varies degrees. Conculsion Compound Houttuynia has beneficial effects on diabetic renal injury,which may regulate GLP-1 and GIP secretion,and the molecular mechanism may be through negative feedback regulation of the SOCS on the expression of JAK/STAT signal transduction pathway related genes and proteins, thus inhibit the expression of downstream oncogenes c-fos and c-jun, reduce the activation of inflammatory cytokines,decreases the secretion of FN and accumulation of ECM.
Objective To study the renoprotective effects of different extracts from compound Houttuyniae herba and its mechanism through JAK/STAT-SOCS-1 signaling pathway. Methods 1. The complete extracts of compound Houttuyniae herba were prepared by mixing the volatile oils,aqueous extracts and alcohol extracts according to the mass ratio of the acquired extracts from herbs. 2. Eight db/m mice were assigned to control group,and 56 db/db mice were randomized into 7 group with 8 animals in each group:model group,Metformin hydrochloride group,AG490 group, Water extract group, Ethanol extract group, Volatile oil group, and Mixture group(mixture of above three extract) of compound Houttuyniae herba. After treating the mice for 8 weeks, 2 mL of blood was collected from the abdominal aorta after anesthesia and kidney tissues were quickly removed. All the left kidney tissues were fixed in 10% formalin,and the right ones were stored at-80℃ for PCR and Western Blot detection.Serum level of FN,TGF-β1,GLP-1,GIP were measured by ELISA. The total RNA and protein were collected and RT-PCR method was used to detect the effect on mRNA expression of JAK2,STAT3,SOCS1 and Western Blot method was used to detect the protein expression of JAK2,p-JAK2,STAT3,p-STAT3,SOCS1,c-fos and cjun. Immunofluorescence was used to detect the protein expression of c-fos,c-jun in kidney tissue. Results After 8 weeks, the mRNA levels of p-JAK2, p-STAT3, JAK2 and STAT3 in mice kidney tissue were significantly increased in the model group(P < 0.05). Compared with the model group,mRNA and protein levels of p-JAK2,pSTAT3 of mice in all treatment groups were lowered;the mRNA and protein levels of SOCS-1 in volatile oils and complete extracts groups mice were significantly increased(P < 0.05),with no change in those of JAK2,STAT3 in all treatment groups. Expression levels of c-fos, c-jun in kidney tubule and renal interstitium of mice in all treatment groups decreased to varies degrees. Conculsion Compound Houttuynia has beneficial effects on diabetic renal injury,which may regulate GLP-1 and GIP secretion,and the molecular mechanism may be through negative feedback regulation of the SOCS on the expression of JAK/STAT signal transduction pathway related genes and proteins, thus inhibit the expression of downstream oncogenes c-fos and c-jun, reduce the activation of inflammatory cytokines,decreases the secretion of FN and accumulation of ECM.
引文
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[13]王海颖,陆敏,修彦凤.鱼腥草改善糖尿病模型大鼠结缔组织生长因子与胰岛素抵抗的实验研究[J].中国新药杂志,2009,18(16):1540-1544.
[14]CHENG L,MENG X B,LU S,et al.Evaluation of hypoglycemic efficacy of tangningtongluo formula,a traditional Chinese Miao medicine,in two rodent animal models[J].J Diabetes Res,2014,2014(2014):1-12.
[15]PEI F,LI B Y,ZHANG Z,et al.Beneficial effects of phlorizin on diabetic nephropathy in diabetic db/dbmice[J].Journal of Diabetes&Its Complications,2014,28(5):596-603.
[16]胡乃军.胃腺癌中原癌基因C-jun、C-fos和磷酸酶及张力蛋白同源蛋白的表达及意义[J].中国老年学杂志,2016,(13):3208-3210.
[17]王玥.大黄酸对糖尿病大鼠肾组织中MMP-9/TIMP-1比值及AP-1表达的影响[D].长沙:中南大学,2010.
[18]王小琴,向少伟,唐庆.肾安提取液对糖尿病小鼠肾脏AP-1和TGF-β-1表达的影响[J].中国中西医结合肾病杂志,2010,(12):1057-1061,1138.
[2]胡丹.糖尿病肾病的治疗进展[J].医疗装备,2017,(4):188-189.
[3]BANES A K,SéAN S,JENKINS J,et a1.Angiotensin 2blocrade prevents hyperglycemia-induced activation of JAK and STAK proteins in diabetic rat kidney glomemli[J].Am J physical renal physical,2004,286(4):F653-F659.
[4]赵松,史永红,段惠军.JAK/STATs信号途径参与高糖诱导的肾小管上皮细胞转分化[J].中国病理生理杂志,2008,24(2):325-329.
[5]MARRERO M B,BANES BERCELI A K,STERN D M,et al.Role of the JAK/STAT signaling pathway in diabetic nephropathy[J].Am J Physiol Renal Physiol,2006,290(4):F762-F800.
[6]张晓东,方敬爱.细胞因子信号转导抑制分子与肾脏疾病[J].中国中西医结合肾病杂,2011,12(11):1021-1023.
[7]段军,仝小林.消渴病的初始病机及其演变浅谈[J].北京中医,2007(3):157-159.
[8]刘吾英.糖尿病患者的饮食调养[J].医学信息(中旬刊),2010,5(1):127.
[9]唐德才,吴庆光.中药学[M].3版.北京:人民卫生出版社,2018:87.
[10]唐德才,吴庆光.中药学[M].3版.北京:人民卫生出版社,2018:52-53.
[11]王海颖,修彦凤.鱼腥草改糖尿病大鼠尿白蛋白与胰岛素抵抗的实验研究[J].中药新药与临床药理,2008,19(88):12-14.
[12]王海颖,修彦凤,孙康德.鱼腥草改善糖尿病大鼠血脂与胰岛素抵抗的实验研究[J].中国中医基础医学杂志,2009,15(1):72-73.
[13]王海颖,陆敏,修彦凤.鱼腥草改善糖尿病模型大鼠结缔组织生长因子与胰岛素抵抗的实验研究[J].中国新药杂志,2009,18(16):1540-1544.
[14]CHENG L,MENG X B,LU S,et al.Evaluation of hypoglycemic efficacy of tangningtongluo formula,a traditional Chinese Miao medicine,in two rodent animal models[J].J Diabetes Res,2014,2014(2014):1-12.
[15]PEI F,LI B Y,ZHANG Z,et al.Beneficial effects of phlorizin on diabetic nephropathy in diabetic db/dbmice[J].Journal of Diabetes&Its Complications,2014,28(5):596-603.
[16]胡乃军.胃腺癌中原癌基因C-jun、C-fos和磷酸酶及张力蛋白同源蛋白的表达及意义[J].中国老年学杂志,2016,(13):3208-3210.
[17]王玥.大黄酸对糖尿病大鼠肾组织中MMP-9/TIMP-1比值及AP-1表达的影响[D].长沙:中南大学,2010.
[18]王小琴,向少伟,唐庆.肾安提取液对糖尿病小鼠肾脏AP-1和TGF-β-1表达的影响[J].中国中西医结合肾病杂志,2010,(12):1057-1061,1138.