Study On The Anti-glomerular Sclerosis' Action And Mechanism Of Herbal Compound Prescription With Removing Blood Stasis And Reducing Phlegm, Dispersing Stagnated Mass

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 synthesisWe 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 mRNATGF-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.