双参苓颗粒对慢性肾衰竭大鼠模型的药效及作用机制

Effect and Mechanism of Shuangshenling Granules on Chronic Renal Failure Rat Model

摘要:

目的 研究双参苓颗粒治疗慢性肾衰竭大鼠的药理作用及机制，为双参苓颗粒的临床应用提供实验室数据支持。方法 SD大鼠（150~180 g）120只，雌雄各半。取10只大鼠作为正常组，10只大鼠作为假手术组，其余采用手术方法制作大鼠5/6肾切除致慢性肾衰竭模型。术后2周，大鼠眼眶采血测定血肌酐（SCr）和血尿素氮（BUN），筛选造模成功的大鼠，并按照SCr值均匀分为模型组、肾衰宁阳性药组（0.84 g·kg^-1·d^-1）、双参苓颗粒高、中、低剂量组（4.8、2.4、1.2 g·kg^-1·d^-1），每组10只。分组后各给药组灌胃给药，10 mL·kg^-1，每日1次，连续6周。给药后第2、4、6周检测大鼠SCr、BUN、24 h尿量、尿蛋白总量（UTP）、尿肌酐（UCr）、肌酐清除率（CCr）、血清白蛋白（SAlb）、血清总蛋白含量（STP），并于试验结束后解剖动物，剥取肾脏组织进行病理学检查，免疫荧光法检测自噬相关蛋白磷酸酶及张力蛋白同源物诱导的激酶1（PINK1）、E3泛素蛋白连接酶（Parkin）和微管相关蛋白1轻链3B（LC3B）的表达水平。结果 与正常组比较，模型组大鼠SCr、BUN、24 h尿量、UTP、UCr水平均显著升高（P<0.01），SAlb、STP含量均显著降低（P<0.01），CCr呈现先升后降的趋势。大鼠肾脏组织病理学结果可见肾小球增生、萎缩，增生肾小球内有不同程度系膜细胞减少，小球内有不同程度瘀血、包氏囊内明显增宽。脏壁层细胞有移位或缺失，肾小球不完整，部分肾小球有明显损伤；肾脏髓质部分近远曲管内有大量蛋白管型，曲管内膜细胞减少、移位，有部分曲管有肿胀，间质炎性渗出，以淋巴细胞、少量中性粒细胞为主，并有大量组织细胞渗出；与模型组比较，双参苓颗粒各剂量组在给药后第2、4、6周均能明显降低大鼠SCr、BUN、24 h尿量、UTP、UCr水平（P<0.05，P<0.01）；升高SAlb、STP含量（P<0.01）；双参苓颗粒各剂量组在给药后2~6周可不同程度改善大鼠CCr并减轻大鼠肾脏病理学损伤。免疫荧光结果显示，与正常组比较，模型组大鼠肾组织中PINK1，Parkin和LC3B荧光强度均有降低；与模型组比较，双参苓颗粒各剂量组能上调PINK1、Parkin、LC3B表达水平。结论 双参苓颗粒可显著改善慢性肾衰竭大鼠肾脏功能及病理损伤，其机制可能与上调PINK1介导的自噬有关。

Abstract:

Objective To investigate the pharmacological action and mechanism of Shuangshenling granules in treating chronic renal failure in rats，providing laboratory data to support clinical application of Shuangshenling granules.Method SD rats （150-180 g），half males and half females in number，were used，with ten rats designated as the normal group，ten as the sham operation group，and the remaining rats undergoing chronic renal failure modeling induced by 5/6 nephrectomy. Two weeks after operation，serum creatinine （SCr） and blood urea nitrogen （BUN） levels were measured via orbital blood sampling to select successful model rats. Based on SCr values，the rats were evenly divided into the model group，Shenshuaining positive group （0.84 g·kg^-1·d^-1），and high，medium，and low dose groups of Shuangshenling granules （4.8，2.4，1.2 g·kg^-1·d^-1），with ten animals in each group. Each treatment group received drugs at 10 mL·kg^-1 via intragastric administration once daily for six weeks. At 2，4，6 weeks after administration，SCr，BUN，24-hour urine volume，total urinary protein （UTP），urinary creatinine （UCr），creatinine clearance rate （CCr），serum albumin （SAlb），and total serum protein （STP） were measured. Following the experiment，kidney tissues were dissected for pathological examination. The expression levels of autophagy-related proteins，including PTEN-induced kinase 1 （PINK1），E3 ubiquitin-protein ligase parkin （Parkin），and microtubule-associated protein 1 light chain 3B （LC3B），were detected by immunofluorescence.Result Compared with the normal group，the model group exhibited significantly increased levels of SCr，BUN，24-hour urine volume，UTP，and UCr （P<0.01），and decreased levels of SAlb and STP （P<0.01）. CCr showed an initial increase followed by a decrease. Histopathological results revealed glomerular hyperplasia and atrophy，with varying degrees of mesangial cell reduction，blood stasis in the glomeruli，and significant widening of Bowman's capsule. Visceral parietal layer cells were displaced or absent，leading to incomplete and damaged glomeruli. A large number of protein casts were present in the proximal and distal convoluted tubules，with reduced and displaced cells，swelling in some tubules，and interstitial inflammatory exudation predominantly comprising lymphocytes and a small number of neutrophils. Compared with the model group，all dose groups of Shuangshenling granules significantly reduced levels of SCr，BUN，24-hour urine volume，UTP，and UCr （P<0.05，P<0.01） and increased SAlb and STP levels （P<0.01） at 2，4，and 6 weeks after administration. The three dose groups also improved CCr and alleviated renal pathological injury in varying degrees at 2-6 weeks after administration. Immunofluorescence results showed that the expression levels of PINK1，Parkin，and LC3B were significantly reduced in the model group compared with the normal group，whereas all dose groups of Shuangshenling granules significantly upregulated the expression levels of PINK1，Parkin，and LC3B compared with the model group.Conclusion Shuangshenling granules significantly improved renal function and pathological injury in rats with chronic renal failure，likely through the upregulation of PINK1-mediated autophagy.