健脾消肿颗粒治疗PHN大鼠的疗效观察及作用机制的初探

Effects and Mechanism of Spleen-strengthening and Detumatism Granule on the Rat of Membranous Nephropathy

目的:观察健脾消肿颗粒治疗PHN大鼠的疗效以及其作用机制。方法:选取健康雄性SPF级Wistar大鼠,适应性喂养后随机抽取正常对照组后,进行造模,正常对照组腹腔一次性注射生理盐水,1 ml/100 g,造模组腹腔一次性注射等量羊抗Fx1A抗血清。造模成功后分组,随机分为模型组、健脾消肿颗粒组。灌胃8周,分别于0周、2周、4周、8周后留取24 h尿,测定各组大鼠24 h尿蛋白定量。8周后处死大鼠取材,留取血清及组织样本。行光镜HE、PASM染色,免疫荧光等检测,观察各组大鼠肾脏一般病理形态学的改变情况。RT-PCR,免疫组化检测肾组织Podocin、Nephrin的蛋白表达量以及mRNA的表达量。结果:(1)给药2周、4周、6周、8周后,健脾消肿颗粒组24 h尿蛋白定量较模型组明显下降,差异有统计意义(P<0.05);(2)在白蛋白水平,模型组较正常对照组明显下降,差异有统计学意义(P<0.05)。(3)免疫组化:模型组、健脾消肿颗粒组WT1的表达较正常对照组均有所下降,差异有统计学意义(P<0.05);与模型组比较,健脾消肿颗粒组WT1的表达明显升高,差异有统计学意义(P<0.05)。模型组podocin的表达异常较正常对照组显著升高,差异有统计学意义(P<0.05),健脾消肿组与正常对照组差异无统计学意义(P>0.05)。(4)RT-PCR:与正常对照组比较,健脾消肿颗粒组nephrin、podocin、podoplanin各指标的表达与正常对照组差异无统计学意义(P>0.05),模型组podocin表达异常,较正常对照组显著升高,差异有统计学意义(P<0.05),其余两指标升高较正常对照组差异无统计学意义,但有显著升高趋势。结论:(1)健脾消肿颗粒疗效确切,可以降低PHN大鼠模型24 h尿蛋白,升高血浆白蛋白;(2)健脾消肿颗粒可以改善PHN大鼠病理损伤,保护足细胞并促进其修复;(3)足细胞相关蛋白nephrin、podocin等蛋白出现过表达可能仍提示足细胞损伤,蛋白表达上调或下调可能与足细胞损伤严重程度以及病情严重程度相关。PHN模型中是否存在基因突变,nephrin、podocin等足细胞蛋白的表达与氧化应激水平关系仍待进一步的验证。

Objective:To observe the therapeutic effect and mechanism of spleen-strengthening and detumatism Granule on PHN rats.Methods:Healthy male SPF Wistar rats were selected,and After adaptive feeding,the normal control group was randomly selected,The remaining rats were modeled.Normal control group was injected with 1 ml/100 g of normal saline,and the model group was injected with the same amount of anti-FX1A anti-serum.After successful modeling,the group was randomly divided into model group and spleen-strengthening and detumatism granule group.After 8 weeks of intragastric administration,24 h urine was collected after 0,2,4 and 8 weeks,respectively,and 24 h urine protein was determined in each group.After 8 weeks,the rats were sacrificed and serum and tissue samples were collected.HE,PASM staining and immunofluorescence were detected to observe the general pathologic changes of kidney in each group.The expression levels of Podocin and Nephrin protein and mRNA in renal tissue were detected by RT-PCR and immunohistochemistry.Results:(1)After 2 weeks,4 weeks and 6 weeks of administration,the 24 h urinary protein quantity in the spleen-strengthening and detumatism granule group was significantly decreased compared with the model group,and the difference was statistically significant(P<0.05);After 8 weeks of administration,24h urinary protein quantity in spleen-strengthening and detumatism granule group showed a significant trend of decrease compared with model group.(2)The level of albumin in the model group was significantly lower than that in the normal control group,and the difference was statistically significant(P<0.05).(3)Immunohistochemistry:The expression of WT1 in model group and spleen-strengthening and detumatism granule group decreased compared with normal group,and the difference was statistically significant(P<0.05),compared with model group,WT1 expression in spleen-strengthening and detumatism granule group was significantly increased,and the difference was statistically significant(P<0.05).The abnormal expression of podocin in model group was significantly higher than that in normal group,and the difference was statistically significant(P<0.05),there was no significant difference between spleen-strengthening and detumescence group and normal group(P<0.05).(4)RT-PCR:Compared with the normal control group,the expressions of nephrin,podocin and podoplanin in the spleen-strengthening and detumatism group were not significantly different from those in the normal group(P<0.05),podocin expression in model group was abnormal,which was significantly higher than that in normal group,and the difference was statistically significant(P<0.05),the other two indexes had no significant difference compared with the normal group,but there was a significant increasing trend.Conclusion:(1)Spleen-strengthening and detumatism Granule has definite curative effect,which can decrease 24h urinary protein and increase plasma albumin in PHN rat model.(2)Spleen-strengthening and detumatism Granules can improve the pathological injury of PHN rats,protect podocytes and promote their repair;(3)The overexpression of podocin-related proteins such as nephrin and podocin may still indicate podococyte injury,and the up-regulation or down-regulation of protein expression may be related to the severity of podococyte injury and the severity of disease.Whether there are gene mutations in the PHN model,the relationship between the expression of podocin,nephrin and oxidative stress level remains to be further verified.