丹参酮ⅡA磺酸钠联合达格列净对糖尿病大鼠肾损伤的改善作用及其机制研究

Ameliorative effect and mechanism of tanshinone ⅡA sodium sulfonate combined with dapagliflozin on diabetic rats with kidney injury

目的探讨丹参酮Ⅱ磺酸钠和达格列净联合应用对糖尿病大鼠肾损伤的改善作用及其作用机制。方法将75只SD大鼠随机分为对照组、模型组、丹参酮ⅡA磺酸钠组(1 mg·kg^-1)、达格列净组(1 mg·kg^-1)、联合组,每组15只,除对照组外,其余组大鼠均采用高糖高脂饲料喂养后腹腔注射链脲佐菌素以建立糖尿病肾病(DN)模型。造模后各组大鼠进行对应处理12周,收集大鼠24 h尿液并采集血液,检测尿蛋白(UP)、血糖(BG)、血肌酐(SCr)和血尿素氮(BUN),HE 染色和TUNEL 染色检测肾组织病理学变化与细胞凋亡情况,ELISA检测血清中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)与白细胞介素-6(IL-6),检测肾组织匀浆液中超氧化物歧化酶(SOD)、丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-Px)含量,Western blot检测肾组织凋亡相关蛋白表达,免疫组化染色检测结缔组织生长因子(CTGF)与单核细胞趋化蛋白1(MCP-1)蛋白表达。结果与模型组相比,丹参酮ⅡA磺酸钠组、达格列净组、联合组大鼠的血糖水平降低,24 h UP、SCr和BUN下降,肾损伤明显改善,细胞凋亡率降低,TNF-α、IL-1β与IL-6含量下降,SOD、GSH-Px含量升高,MDA含量降低。同时,CTGF、MCP-1、Bax、Caspase-3蛋白表达降低,Bcl-2蛋白表达升高(P<0.05)。联合组除血糖含量无差异外,其他作用较丹参酮ⅡA磺酸钠组与达格列净组作用更为明显(P<0.05)。结论 1 mg·kg^-1丹参酮ⅡA磺酸钠和1 mg·kg^-1达格列净联合作用能够明显改善DN大鼠的肾组织损伤,其作用机制可能与抑制炎症反应、细胞凋亡及氧化应激反应,以及调控 CTGF与MCP-1表达相关。

Objective To investigate the improvement effect and mechanism of tanshinone Ⅱ sodium sulfonate combined with dapagliflozin on diabetic rat with kidney injury.Methods Totally 75 SD rats were randomly divided into a control group,a model group,a 1 mg·kg^-1 tanshinone ⅡA sodium sulfonate group,a 1 mg·kg^-1 dapagliflozin group and a combination group,15 rats in each group.Except for the control group,all rats were fed with high-sugar and high-fat diet,and streptozotocin was intraperitoneally injected to establish the DN model.After successful modeling,rats in each group were treated accordingly for 12 weeks.Then the 24 hour urine and blood were collected to detect the urine protein (UP),blood glucose (BG),blood creatinine (SCr) and blood urea nitrogen (BUN).HE staining and TUNEL staining were used to detect the renal histopathological changes and apoptosis.ELISA was used to detect tumor necrosis factor-α (TNF-α),interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the serum.The kit detected the content of superoxide dismutase (SOD),malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in renal tissue homogenate.Western blot was used to detect the expression of apoptosis-related proteins in the kidney tissue,and immunohistochemical staining was used to detect the expression of CTGF and MCP-1.Results Compared with that of the model group,the blood glucose level of rats decreased in the 1 mg·kg^-1 tanshinone ⅡA sodium sulfonate group,1 mg·kg^-1 dapagliflozin group and the combination group,the 24 h UP,SCr and BUN decreased,renal pathological damage was significantly improved,and the apoptosis rate was reduced.TNF-α,IL-1β,IL-6,and MDA content decreased,while SOD and GSH-Px content increased.At the same time,CTGF,MCP-1,Bax and Caspase-3 protein expression decreased,while Bcl-2 protein expression increased (P<0.05).Except for no difference in the blood glucose content in the combination group,the other effects were more obvious than those in the sodium tanshinone ⅡA sulfonate group and the dapagliflozin group (P<0.05).Conclusion The combined action of 1 mg·kg^-1 tanshinone ⅡA sodium sulfonate and 1 mg·kg^-1 dapagliflozin significantly improves the renal tissue damage in rats with diabetic nephropathy,whose mechanism may be related to the inhibition of inflammation,apoptosis and oxidative stress,as well as the regulation of CTGF and MCP-1 expression.