清热解毒、凉血通络方治疗大鼠系膜增生性肾小球肾炎的实验研究
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摘要
目的:在前期实验的基础上,观察清热解毒、凉血通络方对系膜增生性肾小球肾炎(MsPGN)模型大鼠血清肌酐(SCr)、血清白蛋白(ALB)和肾组织形态学的影响,同时检测肾组织中核因子-κB(NF-κB)、B细胞淋巴瘤/白血病-2基因(Bcl-2)和肿瘤坏死因子-α(TNF-α)的表达水平,探讨清热解毒、凉血通络方治疗MsPGN的作用机制。
方法:选取雄性SD大鼠为研究对象,采用隔日口服牛血清白蛋白(BSA)、尾静脉注射葡萄球菌肠毒素B(SEB)、并皮下分点注射完全弗氏佐剂和不完全弗氏佐剂的方法制作大鼠MsPGN模型,并将大鼠随机分为空白组、模型组、清热解毒凉血通络方组和雷公藤多苷(TPG)组,以TPG作为对照,测定大鼠不同时期的SCr和血清ALB水平,光镜下观察肾组织形态学改变,以免疫组化法检测肾组织中NF-κB、Bcl-2和TNF-α的表达水平并进行相关病理分析。
结果:1.模型组实验大鼠SCr较空白组升高(P<0.05),血清ALB显著下降(P<0.01),光镜下可见轻到中度系膜增生性病变,提示MsPGN造模成功。2.清热解毒、凉血通络方和TPG自造模后6周起即可不同程度改善血清ALB下降的水平,且二者疗效相近。3.清热解毒、凉血通络方和TPG于造模后8周才明显抑制实验大鼠SCr的升高,二者疗效相近。4.清热解毒、凉血通络方和TPG都有抑制GMC增生、ECM积聚的作用。5.清热解毒、凉血通络方和TPG均显著降低肾组织中NF-κB、Bcl-2和TNF-α的表达(P<0.01),二者之间无显著性差异(P>0.05)。说明在降低肾小球NF-κB、Bcl-2和TNF-α表达方面,清热解毒、凉血通络方和TPG作用显著,二者疗效相近。6.MsPGN大鼠肾组织中NF-κB的表达水平不仅与Bcl-2的表达水平呈显著正相关(P<0.01,γ=0.734),还与TNF-α的表达水平呈显著正相关(P<0.01,γ=0.729),提示三者密切相关,相互影响。7. MsPGN大鼠肾组织中NF-κB、Bcl-2和TNF-α表达水平的升高可能与GMC增生和ECM积聚的加重呈显著正相关,SCr随之逐步升高,血清ALB的水平逐步降低。
结论:1.清热解毒、凉血通络方能够有效改善MsPGN大鼠血清ALB水平降低,抑制SCr水平升高。2.清热解毒、凉血通络方能够减轻GMC增生和ECM积聚。3.清热解毒、凉血通络方可能通过抑制肾组织中NF-κB、Bcl-2和TNF-α的表达,减轻GMC增生和ECM积聚,在MsPGN的治疗中与TPG效果相近。4.清热解毒、凉血通络方具有多靶点、多途径的整体治疗效应,在临床中具有良好的应用前景。
Objective: On the basis of previous experiments, observe the effect of“QingreJiedu and LiangxueTongluo”Recipe in treating mesangial proliferative glomerulonephritis (MsPGN) rats on serum creatinine (SCr), serum albumin (ALB), glomerular mesangial cell (GMC) and extracelluar matrix (ECM), and test the expression of nuclear factor-kappaB (NF-κB), B-cell lymphoma-2 gene (Bcl-2) and tumor necrosis factor-α(TNF-α) in glomerular mesangial area, to explore the mechanism of“QingreJiedu and LiangxueTongluo”Recipe.
Methods: Male SD rats were adopted as the study objects in the experiment. Replicated the MsPGN rats model by taking orally and time-controlled vein injecting boving serum albumin (BSA), vein injecting staphylococcus enterotoxin B (SEB), spot of division of hypodermic injecting complete Freund’s adjuvant and not complete Freund’s adjuvant. Divided 32 rats into blank group, model group, therapy group (“QingreJiedu and LiangxueTongluo”Recipe group) and comparison group (Tripterygium Glycosides group). Observed the effect of“QingreJiedu and LiangxueTongluo”Recipe and Tripterygium Glycosides (TPG) as a comparison for the rat model with MsPGN on renal function. Measured level of SCr, serum ALB in blood of different periods, observed the proliferation of GMC and ECM by light microscope, measured the expression of NF-κB, Bcl-2 and TNF-αof nephridial tissue by immunohistochemical method and took quantitative analysis.
Results: 1. There were more SCr in blood (P<0.05), but lesser serum ALB in model group than those in blank group (P<0.01), light to moderate mesangial proliferative pathological, which demonstrated the mesangial proliferative nephritis model was successfully duplicated. 2. Both therapy group and TPG group had increased serum ALB since 6th week(P<0.05), but there wasn’t obvious difference between the two (P>0.05). 3. Both therapy group and TPG group had decreased SCr in blood since 8th week (P<0.05), but there wasn’t obvious difference between them (P>0.05). 4.“QingreJiedu and LiangxueTongluo”Recipe and TPG obviously alleviated proliferation of GMC and accumulation of ECM (P<0.01). 5. Compared with model group, therapy group and comparison group could visibly decrease the NF-κB, Bcl-2 and TNF-αconcentration (P<0.01). According to the result, therapy group and comparison group played important roles in the treatment, but there was no significant difference between them (P>0.05). 6. Results of renal pathology of MsPGN rats suggested that there were significant positive correlations among NF-κB, Bcl-2 and TNF-αin renal tissue (γ>0.5, P<0.01), which prompted the increase of quantity of NF-κB in renal tissue might lead to the increased expression of both Bcl-2 and TNF-α. 7. The hyperplasia intensity of glomerulus mesangial area correlated with power of the expression of NF-κB, Bcl-2 and TNF-α. Both“QingreJiedu and LiangxueTongluo”Recipe and TPG might alleviate renal pathology damage by restraining activation of NF-κB, reducing the expression of Bcl-2 and TNF-αin renal tissue, inhibiting the proliferation of GMC and accumuliation of ECM, as well as increasing serum ALB and decreasing SCr in blood.
Conclusion: 1.“QingreJiedu and LiangxueTongluo”Recipe can prevent the reduction of SCr, and inhibite the rise of serum ALB. 2.“QingreJiedu and LiangxueTongluo”Recipe can inhibite GMC proliferation and ECM accumulation. 3. Our study indicates that“QingreJiedu and LiangxueTongluo”Recipe can alleviate the proliferation of GMC and accumuliation of ECM by inhibiting the expression of NF-κB, Bcl-2 and TNF-αof nephridial tissue, whose effect is considerable with TPG. 4.“QingreJiedu and LiangxueTongluo”Recipe can alleviate the pathological injury from different links, which reflects the therapeutic advantage of the traditional Chinese medicine compound multi-target, has a good clinical prospects.
方法:选取雄性SD大鼠为研究对象,采用隔日口服牛血清白蛋白(BSA)、尾静脉注射葡萄球菌肠毒素B(SEB)、并皮下分点注射完全弗氏佐剂和不完全弗氏佐剂的方法制作大鼠MsPGN模型,并将大鼠随机分为空白组、模型组、清热解毒凉血通络方组和雷公藤多苷(TPG)组,以TPG作为对照,测定大鼠不同时期的SCr和血清ALB水平,光镜下观察肾组织形态学改变,以免疫组化法检测肾组织中NF-κB、Bcl-2和TNF-α的表达水平并进行相关病理分析。
结果:1.模型组实验大鼠SCr较空白组升高(P<0.05),血清ALB显著下降(P<0.01),光镜下可见轻到中度系膜增生性病变,提示MsPGN造模成功。2.清热解毒、凉血通络方和TPG自造模后6周起即可不同程度改善血清ALB下降的水平,且二者疗效相近。3.清热解毒、凉血通络方和TPG于造模后8周才明显抑制实验大鼠SCr的升高,二者疗效相近。4.清热解毒、凉血通络方和TPG都有抑制GMC增生、ECM积聚的作用。5.清热解毒、凉血通络方和TPG均显著降低肾组织中NF-κB、Bcl-2和TNF-α的表达(P<0.01),二者之间无显著性差异(P>0.05)。说明在降低肾小球NF-κB、Bcl-2和TNF-α表达方面,清热解毒、凉血通络方和TPG作用显著,二者疗效相近。6.MsPGN大鼠肾组织中NF-κB的表达水平不仅与Bcl-2的表达水平呈显著正相关(P<0.01,γ=0.734),还与TNF-α的表达水平呈显著正相关(P<0.01,γ=0.729),提示三者密切相关,相互影响。7. MsPGN大鼠肾组织中NF-κB、Bcl-2和TNF-α表达水平的升高可能与GMC增生和ECM积聚的加重呈显著正相关,SCr随之逐步升高,血清ALB的水平逐步降低。
结论:1.清热解毒、凉血通络方能够有效改善MsPGN大鼠血清ALB水平降低,抑制SCr水平升高。2.清热解毒、凉血通络方能够减轻GMC增生和ECM积聚。3.清热解毒、凉血通络方可能通过抑制肾组织中NF-κB、Bcl-2和TNF-α的表达,减轻GMC增生和ECM积聚,在MsPGN的治疗中与TPG效果相近。4.清热解毒、凉血通络方具有多靶点、多途径的整体治疗效应,在临床中具有良好的应用前景。
Objective: On the basis of previous experiments, observe the effect of“QingreJiedu and LiangxueTongluo”Recipe in treating mesangial proliferative glomerulonephritis (MsPGN) rats on serum creatinine (SCr), serum albumin (ALB), glomerular mesangial cell (GMC) and extracelluar matrix (ECM), and test the expression of nuclear factor-kappaB (NF-κB), B-cell lymphoma-2 gene (Bcl-2) and tumor necrosis factor-α(TNF-α) in glomerular mesangial area, to explore the mechanism of“QingreJiedu and LiangxueTongluo”Recipe.
Methods: Male SD rats were adopted as the study objects in the experiment. Replicated the MsPGN rats model by taking orally and time-controlled vein injecting boving serum albumin (BSA), vein injecting staphylococcus enterotoxin B (SEB), spot of division of hypodermic injecting complete Freund’s adjuvant and not complete Freund’s adjuvant. Divided 32 rats into blank group, model group, therapy group (“QingreJiedu and LiangxueTongluo”Recipe group) and comparison group (Tripterygium Glycosides group). Observed the effect of“QingreJiedu and LiangxueTongluo”Recipe and Tripterygium Glycosides (TPG) as a comparison for the rat model with MsPGN on renal function. Measured level of SCr, serum ALB in blood of different periods, observed the proliferation of GMC and ECM by light microscope, measured the expression of NF-κB, Bcl-2 and TNF-αof nephridial tissue by immunohistochemical method and took quantitative analysis.
Results: 1. There were more SCr in blood (P<0.05), but lesser serum ALB in model group than those in blank group (P<0.01), light to moderate mesangial proliferative pathological, which demonstrated the mesangial proliferative nephritis model was successfully duplicated. 2. Both therapy group and TPG group had increased serum ALB since 6th week(P<0.05), but there wasn’t obvious difference between the two (P>0.05). 3. Both therapy group and TPG group had decreased SCr in blood since 8th week (P<0.05), but there wasn’t obvious difference between them (P>0.05). 4.“QingreJiedu and LiangxueTongluo”Recipe and TPG obviously alleviated proliferation of GMC and accumulation of ECM (P<0.01). 5. Compared with model group, therapy group and comparison group could visibly decrease the NF-κB, Bcl-2 and TNF-αconcentration (P<0.01). According to the result, therapy group and comparison group played important roles in the treatment, but there was no significant difference between them (P>0.05). 6. Results of renal pathology of MsPGN rats suggested that there were significant positive correlations among NF-κB, Bcl-2 and TNF-αin renal tissue (γ>0.5, P<0.01), which prompted the increase of quantity of NF-κB in renal tissue might lead to the increased expression of both Bcl-2 and TNF-α. 7. The hyperplasia intensity of glomerulus mesangial area correlated with power of the expression of NF-κB, Bcl-2 and TNF-α. Both“QingreJiedu and LiangxueTongluo”Recipe and TPG might alleviate renal pathology damage by restraining activation of NF-κB, reducing the expression of Bcl-2 and TNF-αin renal tissue, inhibiting the proliferation of GMC and accumuliation of ECM, as well as increasing serum ALB and decreasing SCr in blood.
Conclusion: 1.“QingreJiedu and LiangxueTongluo”Recipe can prevent the reduction of SCr, and inhibite the rise of serum ALB. 2.“QingreJiedu and LiangxueTongluo”Recipe can inhibite GMC proliferation and ECM accumulation. 3. Our study indicates that“QingreJiedu and LiangxueTongluo”Recipe can alleviate the proliferation of GMC and accumuliation of ECM by inhibiting the expression of NF-κB, Bcl-2 and TNF-αof nephridial tissue, whose effect is considerable with TPG. 4.“QingreJiedu and LiangxueTongluo”Recipe can alleviate the pathological injury from different links, which reflects the therapeutic advantage of the traditional Chinese medicine compound multi-target, has a good clinical prospects.
引文
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