祛瘀化痰、散结消癥中药复方抗肾小球硬化的作用与机理研究
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摘要
肾小球硬化(GS)是各种肾小球疾病发展至终末期肾功能衰竭的一种不可逆的病理改变。目前认为,该病的形态学表现以细胞外基质(ECM)积聚和系膜细胞(MC)增殖为病理特征,系膜细胞(MC)是肾小球细胞外基质合成和降解的重要场所,MC的过度增生,ECM的合成增加、降解减少,以及相关细胞因子TGF-β1的调控失常,是引起慢性进行性肾小球病变的几个重要病理环节。
研究发现,导致肾脏ECM合成增加的主要成分之一为纤维连接蛋白(FN),FN是一种非溶性高分子糖蛋白,分布于ECM,可通过表面特异性的识别位点与ECM的其它成分如胶原、糖胺聚糖粘附,亦可与血浆蛋白如纤维蛋白结合,还可通过细胞膜表面整合蛋白与细胞结合。研究表明FN是最早出现在损伤组织中的基质蛋白成分。因此,FN的表达程度可反映ECM积聚及合成的程度。
除ECM合成外,ECM的降解对其最终过度积聚也有重要影响。而在ECM的降解过程中,基质金属蛋白酶系统MMps是调控肾脏ECM代谢的一个重要的降解酶系统(包括基质金属蛋白酶MMP和基质金属蛋白酶抑制物TIMP),其中MMP-9主要由肾小球系膜细胞等分泌,其增高的水平及持续的时间决定了肾小球损伤的范围。而MMP-9的主要抑制剂是TIMP-1,两者在ECM积聚和降解的生理平衡中起关键作用。若MMP-9表达下调,TIMP-1持续上升,使MMP-9/TIMP-1失衡,胶原降解受抑,最终导致ECM过度聚积。
此外,调控因子TGF-β1在系膜细胞增殖及肾小球硬化中亦发挥重要作用,目前已经证实,在肾小球硬化模型中,TGF-β1既能够直接刺激ECM成分,如胶原、纤维连接蛋白的形成,又能抑制ECM降解酶,促进金属蛋白酶组织抑制剂(TIMP)高表达,进而抑制ECM的降解,因此,TGF-β1增高一方面能显著促进ECM合成增加,另一方面又减少ECM降解,最终引起肾脏ECM沉积,因此,研究者认为该因子是促进ECM聚积和组织硬化最关键的细胞因子之一。
本文旨在以中医“肾络癥积”理论为依据,通过体外培养肾小球系膜细胞实验,结合临床用药经验,选取川芎、海藻、鳖甲三味药物,分别体现活血化瘀法、化痰散结法、散结消癥法,并通过相互配伍,观察其对以上所述的形成肾小球硬化的几个重要病理环节——肾小球系膜细胞过度增殖,细胞外基质合成增加、降解减少,细胞外基质相关调控细胞因子失常等的影响,综合评价上述中药抗肾小球硬化的疗效,并进一步阐述其抗肾小球硬化的具体作用机制,为日后临床运用祛瘀化痰、散结消癥中药防治“肾络癥积”之肾小球硬化提供坚实的实验研究基础和充分的科学依据。
本课题选用脂多糖(LPS)作为刺激因子,将其作用于体外培养的大鼠肾小球系膜细胞,同时设立空白对照组,实验选取川芎、鳖甲、海藻三味药物,经相互配伍,分别组成：A川芎组；B川芎+鳖甲组；C川芎+海藻组；D川芎+鳖甲+海藻组；依次体现A活血祛瘀法；B活血祛瘀、散结消癥法；C活血祛瘀、化痰散结法；D活血祛瘀、化痰、散结消癥法等四则治法,观察上述中药及治法对大鼠肾小球系膜细胞增殖、ECM及相关细胞因子TGF-β1的干预作用。研究内容主要包括以下四部分：
一、探讨祛瘀化痰、散结消癥中药复方对肾小球系膜细胞增殖的影响
实验选用体外培养的大鼠肾小球系膜细胞,经传代,取对数增长期的细胞,台盼蓝计数活细胞比例达95%以上后,以每孔5000个细胞接种于96孔细胞培养板,4h后待细胞完全贴壁,吸掉培养基,加入不含1%胎牛血清的RPMI1640培养基100μl饥饿24h,达到同步化。弃培养上清,加入LPS及各中药组。实验分空白对照组,LPS组,LPS+川芎组(1：100),LPS+川芎+鳖甲组(1：100),LPS+川芎+海藻组(1：100),LPS+川芎+鳖甲+海藻高剂量组(1：100),LPS+川芎+鳖甲+海藻中剂量组(1：500),LPS+川芎+鳖甲+海藻低剂量组(1：1000)共8组,每组设12个复孔。其中LPS浓度均为10μg／ml。将各组细胞置于37℃,5%CO2细胞培养箱中常规培养48h。以MTT比色法检测各组对肾小球系膜细胞增殖的影响。
结果显示,与空白对照组比较,脂多糖(LPS)能非常显著地促进肾小球系膜细胞异常增殖(P<0.01)；与LPS组对比,中药各组对LPS刺激下的系膜细胞增殖均有极显著的抑制作用(P<0.01)；各中药组中,以川芎+鳖甲+海藻组(高剂量)抑制肾小球系膜细胞增殖作用最强(P<0.01或P<0.05)。
因此,研究结果证实,经LPS刺激的肾小球系膜细胞增殖模型成功,活血祛瘀法,活血祛瘀、散结消瘤法,活血祛瘀、化痰散结法,活血祛瘀、化痰、散结消癥法及中药对体外培养的肾小球系膜细胞增殖均有不同程度的抑制作用,其中以活血祛瘀、化痰、散结消癥中药及治法作用最明显,且以高剂量效果最好。二、探讨祛瘀化痰、散结消癥中药复方对ECM合成调控系统的影响
本文进一步观察各中药对ECM合成中的主要成分——FN的调节,从而判断其对ECM合成调控系统的影响。
实验取对数增长期的大鼠肾小球系膜细胞,台盼蓝计数活细胞比例后,以105个细胞／ml的浓度接种于6孔细胞培养板,每孔2ml,4h后待细胞完全贴壁,吸掉培养基,加入含1%胎牛血清的RPMI1640培养基2ml饥饿24h,达到同步化。弃上清,加入LPS及各中药组,共分8组,方法及用药浓度同实验一,每组设12个复孔。其中空白对照组加完全细胞培养基2ml,LPS组每孔加入含10μg／mlLPS的完全细胞培养基2ml,LPS+各中药组每孔加入含10μg／ml LPS和相应浓度中药的完全细胞培养基2ml。置于37℃,5%CO2细胞培养箱中常规培养48h,留取细胞上清,采用酶联免疫吸附法(ELISA法)检测FN水平。
结果显示,与空白对照组比较,脂多糖(LPS)可非常明显地增高FN水平(P<0.01)；与LPS组对比,川芎组、川芎+鳖甲组、川芎+鳖甲+海藻组(高、中剂量)组能极显著或显著地降低FN水平,抑制ECM合成,延缓肾小球硬化(P<0.01或P<0.05),其中以川芎+鳖甲+海藻组(高剂量)下调ECM中FN水平,延缓肾小球硬化的作用最强(P<0.01或P<0.05)。
研究结果提示,LPS可通过增加ECM中FN的含量,加速肾小球硬化形成。活血祛瘀法,活血祛瘀、散结消癥法,活血祛瘀、化痰、散结消癥法及中药复方对体外培养的LPS刺激下肾小球系膜细胞上清中的FN水平有不同程度的下调作用,可减少ECM合成,抑制肾小球硬化；其中以活血祛瘀、化痰、散结消癥中药复方作用最明显,且作用与浓度呈剂量依赖关系。
三、探讨祛瘀化痰、散结消癥中药复方对ECM降解调控系统的影响
在上述研究结果基础上,本研究继续观察各中药对LPS刺激下肾小球系膜细胞上清中MMP-9、TIMP-1的表达及MMP-9/TIMP-1比值的调节,评价各中药对细胞外基质降解调控系统的影响。
实验分组与给药方法同实验二,采用酶联免疫吸附法(ELISA法)检测细胞上清中MMP-9、TIMP-1的表达,计算MMP-9/TIMP-1比值。
结果显示,经LPS刺激后的系膜细胞分泌MMP-9极显著减少,TIMP-1极显著增加,导致MMP-9/TIMP-1比值失调,使ECM降解明显减少,加速肾小球硬化的形成(P<0.01)。各中药组均能有效上调MMP-9的表达,下调TIMP-1的表达,提高MMP-9/TIMP-1的比值(P<0.01或P<0.05),其中川芎+鳖甲+海藻高、中剂量组,川芎组作用明显(P<0.01或P<0.05),又以川芎+鳖甲+海藻高剂量组效果最为显著；结果亦提示,川芎+鳖甲+海藻组作用强度与药物浓度成量效关系。
实验结果表明,LPS可非常显著地抑制ECM降解调控系统,导致ECM过度积聚；各中药组均能不同程度地增强LPS刺激下肾小球系膜细胞上清MMP-9的表达,并抑制LPS刺激条件下肾小球系膜细胞上清中TIMP-1的表达,从而减少对MMP-9活性的阻断,提高MMP-9/TIMP-1比值,使ECM积聚减少,发挥抗肾小球硬化的作用。但以川芎+鳖甲+海藻高剂量组作用最明显。提示活血祛瘀、化痰、散结消癥中药复方对体外培养的肾小球系膜细胞ECM降解调控系统的影响最明显,并且作用强度与用药剂量呈正相关。四、探讨祛瘀化痰、散结消癥中药复方对ECM相关调控细胞因子TGF-β1 mRNA表达的影响
ECM相关调控细胞因子TGF-β1对ECM的合成和降解起着重要调控作用。本文在完成各组中药对ECM合成与降解的研究基础上,进一步采用荧光定量PCR法,检测各中药组对LPS刺激下肾小球系膜细胞TGF-β1mRNA表达的影响,评价各组中药对ECM相关调控细胞因子TGF-β1mRNA的干预作用。
本项实验分组与给药方法同实验二,细胞用于荧光定量PCR检测。
结果显示,与空白对照组比较,LPS可极显著地刺激TGF-β1mRNA表达水平升高(P<0.01)；与LPS组对比,中药各组均能极显著或显著抑制LPS刺激后系膜细胞TGF-β1mRNA的表达(P<0.01或P<0.05)。各中药组中,川芎+鳖甲+海藻的高剂量组下调TGF-β1mRNA表达的作用趋势最明显,且川芎+鳖甲+海藻不同剂量组的作用强度呈现浓度依赖趋势。
结果证明,LPS可极显著地增加肾小球系膜细胞中TGF-β1mRNA表达水平；活血祛瘀法,活血祛瘀、散结消癥法,活血祛瘀、化痰散结法,活血祛瘀、化痰、散结消癥法及中药对体外培养的肾小球系膜细胞中TGF-β1mRNA表达均有不同程度的抑制作用。其中以川芎+鳖甲+海藻的高剂量组下调趋势最明显,且川芎+鳖甲+海藻不同剂量组的作用强度呈现浓度依赖趋势。
上述实验研究结果综合分析表明,活血祛瘀法,活血祛瘀、散结消癥法,活血祛瘀、化痰散结法,活血祛瘀、化痰、散结消癥法及相应的中药均具有一定程度干预肾小球硬化的作用,但以活血祛瘀、化痰、散结消癥中药复方——川芎+鳖甲+海藻组效果最佳,说明上述几种中药的联合配伍对体外培养LPS刺激下的大鼠肾小球系膜细胞保护作用最强,且该作用与药物浓度呈正相关,为浓度依赖型。
因此,“痰瘀同治、癥结并消”的祛瘀化痰、散结消癥中药复方具有较好的干预肾小球硬化的实验作用,而这一作用可能与以下途径有关：
①有效抑制系膜细胞增殖；②下调系膜细胞上清中FN水平,减少ECM合成；③增强系膜细胞上清中MMP-9的表达,增加ECM的降解,同时抑制TIMP-1的表达,减少对MMP-9活性的阻断,提高MMP-9/TIMP-1比值,最终减少ECM积聚；④下调系膜细胞TGF-β1mRNA的表达,可减少ECM中蛋白的含量,增加ECM降解活力,降低ECM积聚。
Glomerular sclerosis (GS) is a kind of non-reversible pathological changes due to variety of glomerular disease progressing to end-stage of renal failure. The pathological features of the disease manifested as the extracellular matrix (ECM) accumulation and mesangial cells (MC) proliferation. Mesangial cells (MC) is an important place where the glomerular extracellular matrix accomplish synthesis and degradation. MC's excessive proliferation, synthesis increasing and degradation decreasing of ECM, disordering of regulation of TGF-β1 are important pathological factors which caused chronic progressive glomerular lesions.
One of the major components of accumulation of renal ECM is fibronectin (FN). FN is a non-soluble polymer glycoprotein which is located in ECM, it can conglutinate with the other components of ECM such as collagen, glycosaminoglycan through the surface of a special recognition sites, or combine with the plasma protein such as fiber proteins, or binding to the cell through the integrase which is located in cell membrane surface. Studies have shown that FN was the first matrix protein in damage organization. Therefore, the expression of FN may reflects the accumulation level of ECM.
In addition to the synthesis of ECM, ECM's degradation also has important influence on its accumulation eventually. The matrix metalloproteinase system (MMPs) is an important degrading enzyme system to regulate metabolism of renal ECM during its degraded process (Including the matrix metalloproteinases MMP and matrix metalloproteinase inhibitor TIMP). MMP-9 is mainly secreted by glomerular mesangial cells. The level and duration of MMP-9 determine the range of glomerular damage. TIMP-1 is the main inhibitor of MMP-9, both of them play a pivotal role in physiological balance in the accumulation and degradation of ECM. The expression of MMP-9 descends while TIMP-1 continues to rise will eventually cause MMP-9/TIMP-1 imbalanced, degradation of collagen is restrained, and all of them lead to ECM accumulation.
In addition, the regulatory gene TGF-β1 plays an important role in the cells proliferation and glomerular sclerosis. In the model of glomerular sclerosis, TGF-β1 not only stimulates the ECM components such as collagen, fibronectin directly, but also inhibits degraded enzyme of ECM, promotes high expression of tissue inhibitor of metalloproteinase (TIMPS), and then, reduces the degradation of ECM. Therefore significant increasing of TGF-β1 could promote synthesis of ECM increased and decomposing of ECM decreased which lead to renal ECM accumulation. So TGF-β1 is considered as one of the most primary cytokines to promote ECM's accumulation and glomerular sclerosis.
The article mainly aims to select three Chinese medicine, Chuanxiong, Biejia, Haizao, and mutual compatibility of them based on "Mass of renal collateral" theory, clinical experience, and experiments of glomerular mesangial cells culture in vitro to observe the influence of the therapeutic principles and Chinese medicinal herbs of removing blood stasis and reducing phlegm, dispersing stagnated mass on several important pathological process of the glomerular sclerosis, including the excessive proliferation of glomerular mesangial cells, increasing of ECM's synthesis, decreasing of ECM's decomposing, disordering of ECM's regulatory gene. therefore we could conclude the anti-glomerular sclerosis'efficacy of the treatment principle and medicine, and then discuss the specific mechanism further so as to provide research base and sufficient scientific basis for using the therapeutic principles and Chinese medicinal herbs of removing blood stasis and reducing phlegm, dispersing stagnated mass blood stasis and phlegm to prevent and treat "Mass of renal collateral" glomerular sclerosis in the future.
The research has used lipopolysaccharide (LPS) as a stimulating factor to act on MCs cultured in vitro, and set up the blank control group at the same time. Chuanxiong, Biejia, Haizao and mutual compatibility of them composed four groups A Chuanxiong group, B Chuanxiong+Biejia group, C Chuanxiong+Haizao group, D Chuanxiong +Haizao+Biejia group, they reflect four therapeutic principles in turn:A remove blood stasis, B remove blood stasis, disperse stagnated mass, C remove blood stasis and reduce phlegm, D remove blood stasis and reduce phlegm, disperse stagnated mass.
Then we studied and observed the effect of above therapeutic principles and Chinese medicinal herbs on MCs'proliferation, ECM and related cytokines.
The research mainly includes the following four parts:
1. Discuss the effect of the herbal compound prescription of removing blood stasis and reducing phlegm, dispersing stagnated mass on proliferation of glomerular mesangial cell.
The study selected rat mesangial cells after cultured in vitro, chose cells in logarithmic growth phase, counted the proportion of living cells more than 95% by trypanblau, and then inoculated cells by 5000 cells per well in 96-hole cell culture inoculated board, the cells adhere to the wall completely after 4h, siphoning off medium, added RPMI1640 medium without 1% fetal calf serum 100 u 1 and made it hungered 24h for achieving synchronization, discard culture supernatant Liquid, added LPS and the Chinese Medicines. The experiment set eight groups: blank control group, LPS group, LPS+Chuanxiong group (1:100), LPS+ Chuanxiong+Biejia group (1:100), LPS+Chuanxiong+Haizao group (1:100), LPS+Chuanxiong+Biejia+Haizao high-dose group (1:100), LPS+Chuanxiong+Biejia+Haizao middle dose group (1:500), LPS+ Chuanxiong+Biejia+Haizao low dose group (1:1000), each group was re-established six holes. The concentration of LPS was 10μg/ml. Cells in all groups were placed in 37℃,5% CO2 cell incubator in normal culture 48h. And then, we observed the effect of each group on proliferation of glomerular mesangial cell by MTT colorimetric assay.
The results showed that compared with the blank control group, LPS could significantly promote glomerular mesangial cell proliferation (P <0.01); compared with the LPS group, each group of Chinese medicine significantly inhibited proliferation of mesangial cells by LPS stimulated (P<0.01), by comparison of each group, the effect of inhibition on proliferation of glomerular mesangial cells about LPS+ Chuanxiong+Biejia+Haizao group (high dose) was the strongest (P<0.01 or P<0.05).
The results confirmed that, the model of mesangial cell proliferation was success by LPS stimulation, The four therapeutic principles and herbs of removing blood stasis; removing blood stasis, disperse stagnated mass; removing blood stasis and reducing phlegm; removing blood stasis and reducing phlegm, dispersing stagnated mass could delay proliferation of glomerular mesangial cell differently, but the action of therapeutic principles and herbs of removing blood stasis and reducing phlegm, dispersing stagnated mass was the most significant, the effect of high dose group was the best.
2. Discuss the efficacy of the herbal compound prescription of removing blood stasis and reducing phlegm, dispersing stagnated mass on regulative system of ECM's synthesis
We continued to observe the regulated action of every therapeutic principle and herb group on the major component of the ECM's synthesis-FN, and then estimated the efficacy of the the above treatments and herbs on regulative system of ECM's synthesis.
The study selected rat mesangial cells in logarithmic growth phase, counted the proportion of living cells by trypanblau, and then inoculated cells by 105 cells//ml in 6-hole cell culture inoculated board,2ml/hole, the cells adhere to the wall completely after 4h, siphoning off medium, added RPMI1640 medium without 1% fetal calf serum 100μl and made it hungered 24h for achieving synchronization. Discarded culture supernatant Liquid, added LPS and the Chinese Medicines. The experiment set eight groups. The method and medicine concentration were same to that of experiment 1. Each group was re-established three holes. The blank-control group was added with complete cell culture medium 2ml, LPS group was added with 2ml complete cell culture medium containing of 10μg/ml LPS, LPS+herbs groups were added with 2ml complete cell culture medium corresponding herb concentration containing of 10μg/ml LPS. Cells in all groups were placed in 37℃,5% C02 cell incubator in normal culture 48h. Remain culture supernatant Liquid to test FN by ELISA.
The results showed that compared with the blank control group, LPS could increase FN significantly (P<0.01); Compared with the LPS group, Chuanxiong group, Chuanxiong+Biejia group, Chuanxiong+Biejia+ Haizao group (high dose, medium dose) could significant greatly or significant reduce FN, inhibit synthesis of ECM and prevent glomerular sclerosis (P<0.01 or P<0.05), among them, the action trend of reducing the level of FN and delaying the glomerular sclerosis of Chuanxiong+ Biejia+Haizao group (high-dose) is the most obvious (P<0.01 or P <0.05).
The results suggested that LPS could accelerate the formation of glomerular sclerosis by increasing the level of FN in ECM. The four therapeutic principles and herbs of removing blood stasis; removing blood stasis, disperse stagnated mass; removing blood stasis and reducing phlegm; removing blood stasis and reducing phlegm, dispersing stagnated mass could reduce FN level differently in culture supernatant liquid of mesangial cells which stimulated by LPS, decrease ECM synthesis and inhibit glomerular sclerosis; but the action of therapeutic principles and herbs of removing blood stasis and reducing phlegm, dispersing stagnated mass was the most significant, the effect depended on medicine concentration.
3. Discuss the efficacy of the herbal compound prescription of removing blood stasis and reducing phlegm, dispersing stagnated mass on regulative system of ECM's decomposing.
Based on the above studies, we continued to observe the regulated action of every therapeutic principle and herb group on the expressions of MMP-9, TIMP-1 and MMP-9/TIMP-1 in culture supernatant liquid of mesangial cells which stimulated by LPS, and then evaluated the efficacy of the above treatments and herbs on regulative system of ECM's decomposing.
In this experiment, the groups and methods were same to experiment 2. The expressions of MMP-9, TIMP-1 in culture supernatant liquid of mesangial cells were tested by ELISA and the ratio of MMP-9/TIMP-1 was calculated.
The results showed that, the secretion of MMP-9 in the mesangial cells by LPS stimulation was significantly reduced, the secretion of TIMP-1 was increased significantly, which result in the ratio of MMP-9/TIMP-1 imbalance, ECM's degradation decreased obviously and accelerated glomerular sclerosis (P<0.01). All of therapeutic principles and herb groups were able to regulate the expressions of MMP-9, TIMP-1 and the ratio of MMP-9/TIMP-1 (P<0.01 or P<0.05), among them, the action of Chuanxiong+Biejia+Haizao group (high dose, medium dose) group, Chuanxiong group were obvious, while the effect of Chuanxiong+Biejia +Haizao group (high dose) is the most obvious.
The results indicated that LPS could significantly inhibit the regulative system of ECM's decomposing, promote ECM synthesis. All of herb groups were able to increase the expression of MMP-9 and inhibit the expression of TIMP-1 and reduce the interdiction of MMP-9's activity, enhance the ratio of MMP-9/TIMP-1, so that reduce the accumulation of ECM and produce anti-glomerular sclerosis action. While the action of Chuanxiong+Biejia+Haizao group (high dose) is strongest. Thus, the efficacy therapeutic principles and herbs of removing blood stasis and reducing phlegm, dispersing stagnated mass on regulative system of ECM's decomposing of mesangial cells which stimulated by LPS was most obvious, the effect was proportional to the dosage.
4. Discuss the effect of the herbal compound prescription of removing blood stasis and reducing phlegm, dispersing stagnated mass on the expression of ECM's regulatory gene TGF-β1 mRNA
TGF-β1 plays an important role in regulation of synthesis and degradation of ECM. Based on the completed study of the synthesis and degradation of ECM, we chose fluorescent quantitative PCR method to detect the effect of each therapeutic principle and herb group on the expression of TGF-β1 mRNA in mesangial cells which stimulated by LPS, and evaluated the intervention role of each therapeutic principle and herb group on TGF-β1 mRNA in regulative system of ECM's decomposing.
In this experiment, the groups and methods were same to experiment 2. The cells were used for fluorescence quantitative PCR.
The results showed that LPS could significantly stimulate TGF-β1 mRNA increased compared with the blank control group (P<0.01); Compared with the LPS group, all of herb groups could significantly inhibit the expression of TGF-β1 mRNA in mesangial cells by LPS-stimulated (P<0.01 or P<0.05). Among them, the trend of action of lowering high expression of TGF-β1 mRNA in Chuanxiong+Biejia+Haizao group (high dose) was the most obvious. The effects of different dosage of Chuanxiong+ Biejia+Haizao group ware proportional to the dosage.
The results indicated that LPS could increase the level of TGF-β1 mRNA in glomerular mesangial cells; The four therapeutic principles and herbs of removing blood stasis;removing blood stasis, disperse stagnated mass; removing blood stasis and reducing phlegm; removing blood stasis and reducing phlegm, dispersing stagnated mass have the significant inhibition of TGF-β1 mRNA in vitro cultured glomerular mesangial cells. Among them, the trend of action of lowering high expression of TGF-β1 mRNA in Chuanxiong+Biejia+Haizao group (high dose) was the most obvious. The effects of different dosage of Chuanxiong+Biejia+Haizao group ware proportional to the dosage.
According as above results, we concluded that medicinal herbs and therapy of removing blood stasis, therapy of removing blood stasis, dispersing stagnated mass, therapy of removing blood stasis and reducing phlegm, therapy of removing blood stasis and reducing phlegm, dispersing stagnated mass could able to interfere with glomerular sclerosis to some extent, but the action of Chuanxiong+Biejia+Haizao group and the therapy of removing blood stasis and reducing phlegm, dispersing stagnated mass is the most obvious. It illustrated that mutual compatibility of above-mentioned medicinal herbs and therapies has the strongest protective effect of rat glomerular mesangial cells in vitro which stimulated by LPS. The action is correlated with drug concentration, and has the character of concentration-dependence.
Therefore, the herbal compound prescription of removing blood stasis and reducing phlegm, dispersing stagnated mass may has the interference on glomerular sclerosis with "Mass of renal collateral". The effect may be related to the following mechanisms:
①Inhibit the proliferation of mesangial cells effectively;②Reduce FN level in supernatants liquid of mesangial cell and decrease synthesis of ECM;③Enhance the expression of MMP-9 to increase the degradation of ECM, at the same time, inhibit the expression of TIMP-1 to reduce the interdiction of MMP-9 activity, enhance the ratio of MMP-9/TIMP-1, so that lower the accumulation of ECM;④Decrease the expression of TGF-β1 mRNA in mesangial cells by mesangial cells to reduce protein content of ECM, increase the activity of ECM degradation, and lower ECM's accumulation.
研究发现,导致肾脏ECM合成增加的主要成分之一为纤维连接蛋白(FN),FN是一种非溶性高分子糖蛋白,分布于ECM,可通过表面特异性的识别位点与ECM的其它成分如胶原、糖胺聚糖粘附,亦可与血浆蛋白如纤维蛋白结合,还可通过细胞膜表面整合蛋白与细胞结合。研究表明FN是最早出现在损伤组织中的基质蛋白成分。因此,FN的表达程度可反映ECM积聚及合成的程度。
除ECM合成外,ECM的降解对其最终过度积聚也有重要影响。而在ECM的降解过程中,基质金属蛋白酶系统MMps是调控肾脏ECM代谢的一个重要的降解酶系统(包括基质金属蛋白酶MMP和基质金属蛋白酶抑制物TIMP),其中MMP-9主要由肾小球系膜细胞等分泌,其增高的水平及持续的时间决定了肾小球损伤的范围。而MMP-9的主要抑制剂是TIMP-1,两者在ECM积聚和降解的生理平衡中起关键作用。若MMP-9表达下调,TIMP-1持续上升,使MMP-9/TIMP-1失衡,胶原降解受抑,最终导致ECM过度聚积。
此外,调控因子TGF-β1在系膜细胞增殖及肾小球硬化中亦发挥重要作用,目前已经证实,在肾小球硬化模型中,TGF-β1既能够直接刺激ECM成分,如胶原、纤维连接蛋白的形成,又能抑制ECM降解酶,促进金属蛋白酶组织抑制剂(TIMP)高表达,进而抑制ECM的降解,因此,TGF-β1增高一方面能显著促进ECM合成增加,另一方面又减少ECM降解,最终引起肾脏ECM沉积,因此,研究者认为该因子是促进ECM聚积和组织硬化最关键的细胞因子之一。
本文旨在以中医“肾络癥积”理论为依据,通过体外培养肾小球系膜细胞实验,结合临床用药经验,选取川芎、海藻、鳖甲三味药物,分别体现活血化瘀法、化痰散结法、散结消癥法,并通过相互配伍,观察其对以上所述的形成肾小球硬化的几个重要病理环节——肾小球系膜细胞过度增殖,细胞外基质合成增加、降解减少,细胞外基质相关调控细胞因子失常等的影响,综合评价上述中药抗肾小球硬化的疗效,并进一步阐述其抗肾小球硬化的具体作用机制,为日后临床运用祛瘀化痰、散结消癥中药防治“肾络癥积”之肾小球硬化提供坚实的实验研究基础和充分的科学依据。
本课题选用脂多糖(LPS)作为刺激因子,将其作用于体外培养的大鼠肾小球系膜细胞,同时设立空白对照组,实验选取川芎、鳖甲、海藻三味药物,经相互配伍,分别组成：A川芎组；B川芎+鳖甲组；C川芎+海藻组；D川芎+鳖甲+海藻组；依次体现A活血祛瘀法；B活血祛瘀、散结消癥法；C活血祛瘀、化痰散结法；D活血祛瘀、化痰、散结消癥法等四则治法,观察上述中药及治法对大鼠肾小球系膜细胞增殖、ECM及相关细胞因子TGF-β1的干预作用。研究内容主要包括以下四部分：
一、探讨祛瘀化痰、散结消癥中药复方对肾小球系膜细胞增殖的影响
实验选用体外培养的大鼠肾小球系膜细胞,经传代,取对数增长期的细胞,台盼蓝计数活细胞比例达95%以上后,以每孔5000个细胞接种于96孔细胞培养板,4h后待细胞完全贴壁,吸掉培养基,加入不含1%胎牛血清的RPMI1640培养基100μl饥饿24h,达到同步化。弃培养上清,加入LPS及各中药组。实验分空白对照组,LPS组,LPS+川芎组(1：100),LPS+川芎+鳖甲组(1：100),LPS+川芎+海藻组(1：100),LPS+川芎+鳖甲+海藻高剂量组(1：100),LPS+川芎+鳖甲+海藻中剂量组(1：500),LPS+川芎+鳖甲+海藻低剂量组(1：1000)共8组,每组设12个复孔。其中LPS浓度均为10μg／ml。将各组细胞置于37℃,5%CO2细胞培养箱中常规培养48h。以MTT比色法检测各组对肾小球系膜细胞增殖的影响。
结果显示,与空白对照组比较,脂多糖(LPS)能非常显著地促进肾小球系膜细胞异常增殖(P<0.01)；与LPS组对比,中药各组对LPS刺激下的系膜细胞增殖均有极显著的抑制作用(P<0.01)；各中药组中,以川芎+鳖甲+海藻组(高剂量)抑制肾小球系膜细胞增殖作用最强(P<0.01或P<0.05)。
因此,研究结果证实,经LPS刺激的肾小球系膜细胞增殖模型成功,活血祛瘀法,活血祛瘀、散结消瘤法,活血祛瘀、化痰散结法,活血祛瘀、化痰、散结消癥法及中药对体外培养的肾小球系膜细胞增殖均有不同程度的抑制作用,其中以活血祛瘀、化痰、散结消癥中药及治法作用最明显,且以高剂量效果最好。二、探讨祛瘀化痰、散结消癥中药复方对ECM合成调控系统的影响
本文进一步观察各中药对ECM合成中的主要成分——FN的调节,从而判断其对ECM合成调控系统的影响。
实验取对数增长期的大鼠肾小球系膜细胞,台盼蓝计数活细胞比例后,以105个细胞／ml的浓度接种于6孔细胞培养板,每孔2ml,4h后待细胞完全贴壁,吸掉培养基,加入含1%胎牛血清的RPMI1640培养基2ml饥饿24h,达到同步化。弃上清,加入LPS及各中药组,共分8组,方法及用药浓度同实验一,每组设12个复孔。其中空白对照组加完全细胞培养基2ml,LPS组每孔加入含10μg／mlLPS的完全细胞培养基2ml,LPS+各中药组每孔加入含10μg／ml LPS和相应浓度中药的完全细胞培养基2ml。置于37℃,5%CO2细胞培养箱中常规培养48h,留取细胞上清,采用酶联免疫吸附法(ELISA法)检测FN水平。
结果显示,与空白对照组比较,脂多糖(LPS)可非常明显地增高FN水平(P<0.01)；与LPS组对比,川芎组、川芎+鳖甲组、川芎+鳖甲+海藻组(高、中剂量)组能极显著或显著地降低FN水平,抑制ECM合成,延缓肾小球硬化(P<0.01或P<0.05),其中以川芎+鳖甲+海藻组(高剂量)下调ECM中FN水平,延缓肾小球硬化的作用最强(P<0.01或P<0.05)。
研究结果提示,LPS可通过增加ECM中FN的含量,加速肾小球硬化形成。活血祛瘀法,活血祛瘀、散结消癥法,活血祛瘀、化痰、散结消癥法及中药复方对体外培养的LPS刺激下肾小球系膜细胞上清中的FN水平有不同程度的下调作用,可减少ECM合成,抑制肾小球硬化；其中以活血祛瘀、化痰、散结消癥中药复方作用最明显,且作用与浓度呈剂量依赖关系。
三、探讨祛瘀化痰、散结消癥中药复方对ECM降解调控系统的影响
在上述研究结果基础上,本研究继续观察各中药对LPS刺激下肾小球系膜细胞上清中MMP-9、TIMP-1的表达及MMP-9/TIMP-1比值的调节,评价各中药对细胞外基质降解调控系统的影响。
实验分组与给药方法同实验二,采用酶联免疫吸附法(ELISA法)检测细胞上清中MMP-9、TIMP-1的表达,计算MMP-9/TIMP-1比值。
结果显示,经LPS刺激后的系膜细胞分泌MMP-9极显著减少,TIMP-1极显著增加,导致MMP-9/TIMP-1比值失调,使ECM降解明显减少,加速肾小球硬化的形成(P<0.01)。各中药组均能有效上调MMP-9的表达,下调TIMP-1的表达,提高MMP-9/TIMP-1的比值(P<0.01或P<0.05),其中川芎+鳖甲+海藻高、中剂量组,川芎组作用明显(P<0.01或P<0.05),又以川芎+鳖甲+海藻高剂量组效果最为显著；结果亦提示,川芎+鳖甲+海藻组作用强度与药物浓度成量效关系。
实验结果表明,LPS可非常显著地抑制ECM降解调控系统,导致ECM过度积聚；各中药组均能不同程度地增强LPS刺激下肾小球系膜细胞上清MMP-9的表达,并抑制LPS刺激条件下肾小球系膜细胞上清中TIMP-1的表达,从而减少对MMP-9活性的阻断,提高MMP-9/TIMP-1比值,使ECM积聚减少,发挥抗肾小球硬化的作用。但以川芎+鳖甲+海藻高剂量组作用最明显。提示活血祛瘀、化痰、散结消癥中药复方对体外培养的肾小球系膜细胞ECM降解调控系统的影响最明显,并且作用强度与用药剂量呈正相关。四、探讨祛瘀化痰、散结消癥中药复方对ECM相关调控细胞因子TGF-β1 mRNA表达的影响
ECM相关调控细胞因子TGF-β1对ECM的合成和降解起着重要调控作用。本文在完成各组中药对ECM合成与降解的研究基础上,进一步采用荧光定量PCR法,检测各中药组对LPS刺激下肾小球系膜细胞TGF-β1mRNA表达的影响,评价各组中药对ECM相关调控细胞因子TGF-β1mRNA的干预作用。
本项实验分组与给药方法同实验二,细胞用于荧光定量PCR检测。
结果显示,与空白对照组比较,LPS可极显著地刺激TGF-β1mRNA表达水平升高(P<0.01)；与LPS组对比,中药各组均能极显著或显著抑制LPS刺激后系膜细胞TGF-β1mRNA的表达(P<0.01或P<0.05)。各中药组中,川芎+鳖甲+海藻的高剂量组下调TGF-β1mRNA表达的作用趋势最明显,且川芎+鳖甲+海藻不同剂量组的作用强度呈现浓度依赖趋势。
结果证明,LPS可极显著地增加肾小球系膜细胞中TGF-β1mRNA表达水平；活血祛瘀法,活血祛瘀、散结消癥法,活血祛瘀、化痰散结法,活血祛瘀、化痰、散结消癥法及中药对体外培养的肾小球系膜细胞中TGF-β1mRNA表达均有不同程度的抑制作用。其中以川芎+鳖甲+海藻的高剂量组下调趋势最明显,且川芎+鳖甲+海藻不同剂量组的作用强度呈现浓度依赖趋势。
上述实验研究结果综合分析表明,活血祛瘀法,活血祛瘀、散结消癥法,活血祛瘀、化痰散结法,活血祛瘀、化痰、散结消癥法及相应的中药均具有一定程度干预肾小球硬化的作用,但以活血祛瘀、化痰、散结消癥中药复方——川芎+鳖甲+海藻组效果最佳,说明上述几种中药的联合配伍对体外培养LPS刺激下的大鼠肾小球系膜细胞保护作用最强,且该作用与药物浓度呈正相关,为浓度依赖型。
因此,“痰瘀同治、癥结并消”的祛瘀化痰、散结消癥中药复方具有较好的干预肾小球硬化的实验作用,而这一作用可能与以下途径有关：
①有效抑制系膜细胞增殖；②下调系膜细胞上清中FN水平,减少ECM合成；③增强系膜细胞上清中MMP-9的表达,增加ECM的降解,同时抑制TIMP-1的表达,减少对MMP-9活性的阻断,提高MMP-9/TIMP-1比值,最终减少ECM积聚；④下调系膜细胞TGF-β1mRNA的表达,可减少ECM中蛋白的含量,增加ECM降解活力,降低ECM积聚。
Glomerular sclerosis (GS) is a kind of non-reversible pathological changes due to variety of glomerular disease progressing to end-stage of renal failure. The pathological features of the disease manifested as the extracellular matrix (ECM) accumulation and mesangial cells (MC) proliferation. Mesangial cells (MC) is an important place where the glomerular extracellular matrix accomplish synthesis and degradation. MC's excessive proliferation, synthesis increasing and degradation decreasing of ECM, disordering of regulation of TGF-β1 are important pathological factors which caused chronic progressive glomerular lesions.
One of the major components of accumulation of renal ECM is fibronectin (FN). FN is a non-soluble polymer glycoprotein which is located in ECM, it can conglutinate with the other components of ECM such as collagen, glycosaminoglycan through the surface of a special recognition sites, or combine with the plasma protein such as fiber proteins, or binding to the cell through the integrase which is located in cell membrane surface. Studies have shown that FN was the first matrix protein in damage organization. Therefore, the expression of FN may reflects the accumulation level of ECM.
In addition to the synthesis of ECM, ECM's degradation also has important influence on its accumulation eventually. The matrix metalloproteinase system (MMPs) is an important degrading enzyme system to regulate metabolism of renal ECM during its degraded process (Including the matrix metalloproteinases MMP and matrix metalloproteinase inhibitor TIMP). MMP-9 is mainly secreted by glomerular mesangial cells. The level and duration of MMP-9 determine the range of glomerular damage. TIMP-1 is the main inhibitor of MMP-9, both of them play a pivotal role in physiological balance in the accumulation and degradation of ECM. The expression of MMP-9 descends while TIMP-1 continues to rise will eventually cause MMP-9/TIMP-1 imbalanced, degradation of collagen is restrained, and all of them lead to ECM accumulation.
In addition, the regulatory gene TGF-β1 plays an important role in the cells proliferation and glomerular sclerosis. In the model of glomerular sclerosis, TGF-β1 not only stimulates the ECM components such as collagen, fibronectin directly, but also inhibits degraded enzyme of ECM, promotes high expression of tissue inhibitor of metalloproteinase (TIMPS), and then, reduces the degradation of ECM. Therefore significant increasing of TGF-β1 could promote synthesis of ECM increased and decomposing of ECM decreased which lead to renal ECM accumulation. So TGF-β1 is considered as one of the most primary cytokines to promote ECM's accumulation and glomerular sclerosis.
The article mainly aims to select three Chinese medicine, Chuanxiong, Biejia, Haizao, and mutual compatibility of them based on "Mass of renal collateral" theory, clinical experience, and experiments of glomerular mesangial cells culture in vitro to observe the influence of the therapeutic principles and Chinese medicinal herbs of removing blood stasis and reducing phlegm, dispersing stagnated mass on several important pathological process of the glomerular sclerosis, including the excessive proliferation of glomerular mesangial cells, increasing of ECM's synthesis, decreasing of ECM's decomposing, disordering of ECM's regulatory gene. therefore we could conclude the anti-glomerular sclerosis'efficacy of the treatment principle and medicine, and then discuss the specific mechanism further so as to provide research base and sufficient scientific basis for using the therapeutic principles and Chinese medicinal herbs of removing blood stasis and reducing phlegm, dispersing stagnated mass blood stasis and phlegm to prevent and treat "Mass of renal collateral" glomerular sclerosis in the future.
The research has used lipopolysaccharide (LPS) as a stimulating factor to act on MCs cultured in vitro, and set up the blank control group at the same time. Chuanxiong, Biejia, Haizao and mutual compatibility of them composed four groups A Chuanxiong group, B Chuanxiong+Biejia group, C Chuanxiong+Haizao group, D Chuanxiong +Haizao+Biejia group, they reflect four therapeutic principles in turn:A remove blood stasis, B remove blood stasis, disperse stagnated mass, C remove blood stasis and reduce phlegm, D remove blood stasis and reduce phlegm, disperse stagnated mass.
Then we studied and observed the effect of above therapeutic principles and Chinese medicinal herbs on MCs'proliferation, ECM and related cytokines.
The research mainly includes the following four parts:
1. Discuss the effect of the herbal compound prescription of removing blood stasis and reducing phlegm, dispersing stagnated mass on proliferation of glomerular mesangial cell.
The study selected rat mesangial cells after cultured in vitro, chose cells in logarithmic growth phase, counted the proportion of living cells more than 95% by trypanblau, and then inoculated cells by 5000 cells per well in 96-hole cell culture inoculated board, the cells adhere to the wall completely after 4h, siphoning off medium, added RPMI1640 medium without 1% fetal calf serum 100 u 1 and made it hungered 24h for achieving synchronization, discard culture supernatant Liquid, added LPS and the Chinese Medicines. The experiment set eight groups: blank control group, LPS group, LPS+Chuanxiong group (1:100), LPS+ Chuanxiong+Biejia group (1:100), LPS+Chuanxiong+Haizao group (1:100), LPS+Chuanxiong+Biejia+Haizao high-dose group (1:100), LPS+Chuanxiong+Biejia+Haizao middle dose group (1:500), LPS+ Chuanxiong+Biejia+Haizao low dose group (1:1000), each group was re-established six holes. The concentration of LPS was 10μg/ml. Cells in all groups were placed in 37℃,5% CO2 cell incubator in normal culture 48h. And then, we observed the effect of each group on proliferation of glomerular mesangial cell by MTT colorimetric assay.
The results showed that compared with the blank control group, LPS could significantly promote glomerular mesangial cell proliferation (P <0.01); compared with the LPS group, each group of Chinese medicine significantly inhibited proliferation of mesangial cells by LPS stimulated (P<0.01), by comparison of each group, the effect of inhibition on proliferation of glomerular mesangial cells about LPS+ Chuanxiong+Biejia+Haizao group (high dose) was the strongest (P<0.01 or P<0.05).
The results confirmed that, the model of mesangial cell proliferation was success by LPS stimulation, The four therapeutic principles and herbs of removing blood stasis; removing blood stasis, disperse stagnated mass; removing blood stasis and reducing phlegm; removing blood stasis and reducing phlegm, dispersing stagnated mass could delay proliferation of glomerular mesangial cell differently, but the action of therapeutic principles and herbs of removing blood stasis and reducing phlegm, dispersing stagnated mass was the most significant, the effect of high dose group was the best.
2. Discuss the efficacy of the herbal compound prescription of removing blood stasis and reducing phlegm, dispersing stagnated mass on regulative system of ECM's synthesis
We continued to observe the regulated action of every therapeutic principle and herb group on the major component of the ECM's synthesis-FN, and then estimated the efficacy of the the above treatments and herbs on regulative system of ECM's synthesis.
The study selected rat mesangial cells in logarithmic growth phase, counted the proportion of living cells by trypanblau, and then inoculated cells by 105 cells//ml in 6-hole cell culture inoculated board,2ml/hole, the cells adhere to the wall completely after 4h, siphoning off medium, added RPMI1640 medium without 1% fetal calf serum 100μl and made it hungered 24h for achieving synchronization. Discarded culture supernatant Liquid, added LPS and the Chinese Medicines. The experiment set eight groups. The method and medicine concentration were same to that of experiment 1. Each group was re-established three holes. The blank-control group was added with complete cell culture medium 2ml, LPS group was added with 2ml complete cell culture medium containing of 10μg/ml LPS, LPS+herbs groups were added with 2ml complete cell culture medium corresponding herb concentration containing of 10μg/ml LPS. Cells in all groups were placed in 37℃,5% C02 cell incubator in normal culture 48h. Remain culture supernatant Liquid to test FN by ELISA.
The results showed that compared with the blank control group, LPS could increase FN significantly (P<0.01); Compared with the LPS group, Chuanxiong group, Chuanxiong+Biejia group, Chuanxiong+Biejia+ Haizao group (high dose, medium dose) could significant greatly or significant reduce FN, inhibit synthesis of ECM and prevent glomerular sclerosis (P<0.01 or P<0.05), among them, the action trend of reducing the level of FN and delaying the glomerular sclerosis of Chuanxiong+ Biejia+Haizao group (high-dose) is the most obvious (P<0.01 or P <0.05).
The results suggested that LPS could accelerate the formation of glomerular sclerosis by increasing the level of FN in ECM. The four therapeutic principles and herbs of removing blood stasis; removing blood stasis, disperse stagnated mass; removing blood stasis and reducing phlegm; removing blood stasis and reducing phlegm, dispersing stagnated mass could reduce FN level differently in culture supernatant liquid of mesangial cells which stimulated by LPS, decrease ECM synthesis and inhibit glomerular sclerosis; but the action of therapeutic principles and herbs of removing blood stasis and reducing phlegm, dispersing stagnated mass was the most significant, the effect depended on medicine concentration.
3. Discuss the efficacy of the herbal compound prescription of removing blood stasis and reducing phlegm, dispersing stagnated mass on regulative system of ECM's decomposing.
Based on the above studies, we continued to observe the regulated action of every therapeutic principle and herb group on the expressions of MMP-9, TIMP-1 and MMP-9/TIMP-1 in culture supernatant liquid of mesangial cells which stimulated by LPS, and then evaluated the efficacy of the above treatments and herbs on regulative system of ECM's decomposing.
In this experiment, the groups and methods were same to experiment 2. The expressions of MMP-9, TIMP-1 in culture supernatant liquid of mesangial cells were tested by ELISA and the ratio of MMP-9/TIMP-1 was calculated.
The results showed that, the secretion of MMP-9 in the mesangial cells by LPS stimulation was significantly reduced, the secretion of TIMP-1 was increased significantly, which result in the ratio of MMP-9/TIMP-1 imbalance, ECM's degradation decreased obviously and accelerated glomerular sclerosis (P<0.01). All of therapeutic principles and herb groups were able to regulate the expressions of MMP-9, TIMP-1 and the ratio of MMP-9/TIMP-1 (P<0.01 or P<0.05), among them, the action of Chuanxiong+Biejia+Haizao group (high dose, medium dose) group, Chuanxiong group were obvious, while the effect of Chuanxiong+Biejia +Haizao group (high dose) is the most obvious.
The results indicated that LPS could significantly inhibit the regulative system of ECM's decomposing, promote ECM synthesis. All of herb groups were able to increase the expression of MMP-9 and inhibit the expression of TIMP-1 and reduce the interdiction of MMP-9's activity, enhance the ratio of MMP-9/TIMP-1, so that reduce the accumulation of ECM and produce anti-glomerular sclerosis action. While the action of Chuanxiong+Biejia+Haizao group (high dose) is strongest. Thus, the efficacy therapeutic principles and herbs of removing blood stasis and reducing phlegm, dispersing stagnated mass on regulative system of ECM's decomposing of mesangial cells which stimulated by LPS was most obvious, the effect was proportional to the dosage.
4. Discuss the effect of the herbal compound prescription of removing blood stasis and reducing phlegm, dispersing stagnated mass on the expression of ECM's regulatory gene TGF-β1 mRNA
TGF-β1 plays an important role in regulation of synthesis and degradation of ECM. Based on the completed study of the synthesis and degradation of ECM, we chose fluorescent quantitative PCR method to detect the effect of each therapeutic principle and herb group on the expression of TGF-β1 mRNA in mesangial cells which stimulated by LPS, and evaluated the intervention role of each therapeutic principle and herb group on TGF-β1 mRNA in regulative system of ECM's decomposing.
In this experiment, the groups and methods were same to experiment 2. The cells were used for fluorescence quantitative PCR.
The results showed that LPS could significantly stimulate TGF-β1 mRNA increased compared with the blank control group (P<0.01); Compared with the LPS group, all of herb groups could significantly inhibit the expression of TGF-β1 mRNA in mesangial cells by LPS-stimulated (P<0.01 or P<0.05). Among them, the trend of action of lowering high expression of TGF-β1 mRNA in Chuanxiong+Biejia+Haizao group (high dose) was the most obvious. The effects of different dosage of Chuanxiong+ Biejia+Haizao group ware proportional to the dosage.
The results indicated that LPS could increase the level of TGF-β1 mRNA in glomerular mesangial cells; The four therapeutic principles and herbs of removing blood stasis;removing blood stasis, disperse stagnated mass; removing blood stasis and reducing phlegm; removing blood stasis and reducing phlegm, dispersing stagnated mass have the significant inhibition of TGF-β1 mRNA in vitro cultured glomerular mesangial cells. Among them, the trend of action of lowering high expression of TGF-β1 mRNA in Chuanxiong+Biejia+Haizao group (high dose) was the most obvious. The effects of different dosage of Chuanxiong+Biejia+Haizao group ware proportional to the dosage.
According as above results, we concluded that medicinal herbs and therapy of removing blood stasis, therapy of removing blood stasis, dispersing stagnated mass, therapy of removing blood stasis and reducing phlegm, therapy of removing blood stasis and reducing phlegm, dispersing stagnated mass could able to interfere with glomerular sclerosis to some extent, but the action of Chuanxiong+Biejia+Haizao group and the therapy of removing blood stasis and reducing phlegm, dispersing stagnated mass is the most obvious. It illustrated that mutual compatibility of above-mentioned medicinal herbs and therapies has the strongest protective effect of rat glomerular mesangial cells in vitro which stimulated by LPS. The action is correlated with drug concentration, and has the character of concentration-dependence.
Therefore, the herbal compound prescription of removing blood stasis and reducing phlegm, dispersing stagnated mass may has the interference on glomerular sclerosis with "Mass of renal collateral". The effect may be related to the following mechanisms:
①Inhibit the proliferation of mesangial cells effectively;②Reduce FN level in supernatants liquid of mesangial cell and decrease synthesis of ECM;③Enhance the expression of MMP-9 to increase the degradation of ECM, at the same time, inhibit the expression of TIMP-1 to reduce the interdiction of MMP-9 activity, enhance the ratio of MMP-9/TIMP-1, so that lower the accumulation of ECM;④Decrease the expression of TGF-β1 mRNA in mesangial cells by mesangial cells to reduce protein content of ECM, increase the activity of ECM degradation, and lower ECM's accumulation.
引文
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