Experimental Study Of Shenluotong On Controlling And Regulating Mesangial Cells, Mxtracellular Matrix And Correlated Cytokine

Propagation of Mesangial cells(MCs) and accumulation of extracellular matrix are basic pathology link of many kinds of Renal glomerular diseases. That can induce glomerular sclerosis and renal failure. Activated MCs can excessively excrete ECM and generate many kinds of mediators of inflammations, which construct extremely complicated network system and self-magnification effect. In this network system, effect of physiology and pathology of many kinds of cytokine are known as considerable domain such as transforming growth factor-β1 (TGF-β1) and connective tissue growth factor(CTGF) et cetera. Accordingly, MCs ,ECM and Correlated Cytokine become research hot spot that investigate mechanism of curative effect of traditional Chinese medicine. Shenluotong(SLT), clinic experiential recipe of professor Zhao Yuyong, can get good effect in many aspects such as delaying progression of many kinds of Renal glomerular diseases and preventing and curing glomerular sclerosis. Protophase empirical study confirmed that SLT can treat nephritis model of immunity and mesenterium hyperplasy, and can obviously lighten interstitial fibrosis of nephric tubule induced by Adriamycin. This experiment utilizing cell biology together with molecular biology technique begins with the above-mentioned three researching heatpoints to research the effect that SLT interposes MCs biology action and to strive to clarify the part of cell molecular biology mechanism of SLT'therapeutic action. Part I Culture and Identification of Mesangial cells of Rat Objective: To culture MCs of rat, and to confirm it by identification. Methods: 1. Culture of intercapillary cells of original generation Referenced Chenyipu method, ten clearing SD rats were taken, skin sterilized after anesthetization with ethylether, double kidneys taken out in the asepsis environment, cortical substance sheared and made pieces, glomerulus extracted,digested,raised,and MCs subcultivated at last. 2. Identification of Mesangial cells Cultural cells were observed with IM and TEM and detected with IHC. Results: 1. Observation of cultural cells with IM Cell bodies were in shape of Fusiform or irregular star and triangle, and had several prominences of different length stretching out from kytoplasm. 2. Observation of cultural cells with TEM Many microfilaments were seen in cells, copious rough endoplasmic reticulum and mitochondria in endochylema. 3. Detection of cultural cells with IHC Cultural cells all appeared Desmin dyeing masculine. The above-mentioned detection confirmed that the cultural cells were MCs. Part II Effect of SLT on Hyperplasia and Apoptosis of MCs Objective: Proliferative MCs, as an active metabolism cell, exorbitantly excreted groundmass and synthetized mediators of inflammation, forwardly participated in the pathological changes course of renal glomerular disease, which made MCs in the center position of glomerulus inflammation. Apoptosis played an important role when damaged glomerulus induced MCs hyperplasia. We took MCs as the target spot, studied therapeutic effect mechanism of homotherapy for heteropathy as SLT cured many kinds of Renal glomerular disease from two closely related angles of inhibiting hyperplasia and promoting apoptosis. Methods: 1. Experimental blood serum preparation Chinese materia medicaof SLT, decocted by water and filtered, then condensed to 2.5 g/ml of dried medicinal herb, were put it into refrigerator with 4 ℃for use. Blood serum group rats of SLT were lavaged twice per day, 3 ml each, for three days. One hour after the last administration, the blood of arteria cruralis was obtained; blood serum was separated, inactivated and then filtered by 0.22 μm millipore filter, then put into refrigerator with -20 ℃for use. Blank group rats were lavaged isodose distilled water, and the blood serum was extracted in the same way as Blood serum group. 2. Detection of MCs hyperplasia Cell synchrony was at resting period, added in orderly normal blood serum,Valsartan(10-7 mol·L-1),every group of SLT(1.25%,2.5%,5%SLT blood serum,supplementing blood serum to 5% by normal rat blood serum). The former two groups were added with 5% normal rat blood serum. Numerus of OD was determined with MTT after 48 h. 3. Detection of apoptosis MCs were inoculated into 75 ml culture flask of plastics. Grouping and cultivating were the same as the above-mentioned. The samples preprocessed, apoptosis of MCs were detected by flow cytometry. Expressions of Bax and Bcl-2 were detected by flow cytometry with indirect immunofluorescence. Results: 1. Effect of SLT on MCs proliferation Compared with normal control group (0.336±0.014), OD numerus of SLT groups significantly decreased(P<0.01). In all SLT groups of different dosages, OD numerus of SLT5% group (0.267±0.015) was significantly lower than that of SLT2.5% group (0.277±0.012) and SLT1.25% group (0.281±0.013) (P<0.01). OD numerus of Valsartan group (0.319±0.006), compared with normal control group, decreased with significant difference(P<0.01). OD numerus of SLT groups, compared with Valsartan group, decreased significantly (P<0.01). 2. Effect of SLT on MCs apoptosis ratio Compared with normal control group (1.99±0.25), MCs apoptosis ratio of SLT groups significantly increased (P<0.01). In groups of SLT, MCs apoptosis ratio increasedprogressively with the increasing of density, and there was significant difference between(P<0.01). Compared with normal control group, MCs apoptosis ratio of Valsartan group (7.69 ±0.54) increased significantly (P<0.01). Compared with Valsartan group, MCs apoptosis ratio of SLT1.25% group (6.14 ±0.83) decreased significantly (P<0.01), but SLT2.5% group and SLT5% group (36.37±1.48) increased significantly (P<0.01). 3. Effect of SLT on Bax expression Bax expression of groups of SLT increased, compared with normal control group(2.99±0.22), with significant difference(P < 0.01); in groups of SLT, Bax expression increased progressively with the increasing of density, and there was significant difference between (P<0.01). Bax expression of Valsartan group (7.69±0.54), compared with normal control group, increased significantly (P<0.01); compared with Valsartan group, SLT1.25% group(12.94±0.26) obviously decreased (P<0.05), but SLT5% group(18.22±0.66) increased obviously, and hadn't significant difference with SLT2.5% group. 4. Effect of SLT on Bcl-2 expression Bcl-2 expression of SLT groups all increased, compared with normal control group(5.33 ±0.14), with significant difference(P<0.01). In groups of SLT, Bcl-2 expression decreased progressively with the increasing of density of blood serum(12.10±0.19,11.17±0.46,7.93±0.45), and there was significant difference between (P<0.01). Bcl-2 expression of Valsartan group (15.32±0.44), compared with normal control group, increased significantly (P<0.01); comparing with Valsartan group, Bcl-2 expressions all decreased significantly (P<0.01). 5. Effect of SLT on Bcl-2/Bax Compared with normal control group(1.83±0.14), the ratio of Bcl-2/Bax of every group of SLT all decreased with significant difference(P<0.01),which make it clear that every group of SLT significantly decreased nati-spoptosis modulus, accordingly enhanced MCs apoptosis. In groups of SLT, Ratio of Bcl-2/Bax of SLT5% group was significantly lower than SLT2.5% group(0.75 ±0.03) and SLT1.25% group(0.94±0.01)( P<0.01). The ratio of Bcl-2/Bax of Valsartangroup(0.95 ±0.01), compared with normal control group, decreased significantly (P<0.01). Compared with Valsartan group, both ratios of SLT2.5% group and SLT5% group significantly decreased (P<0.01). Summary: 1. SLT blood serum could restrain proliferation of MCs. It had symbiosis of amount and effect. Effect of restraint of SLT blood serum was superior to Valsartan. 2. SLT can induce apoptosis of MCs, which precipitated reduction of proliferative MCs. 3. SLT could make apoptosis gene high expressing, improve and control the ratio of apoptosis gene Bax and Bcl-2, which partly elucidate mechanism of SLT inducing apoptosis. 4. The role of restraining proliferation and promoting apoptosis was one of the possible mechanisms with which this recipe cured in force proliferating glomerulopathy. Part III Effect of Shenluotong on amplification and degradation of extracellular matrix Objective: Metabolic disorder and abnormal deposit of ECM in nephridial tissue with pathological changes is an important component of the developing and changing mechanism of many glomerulopathy. ECM gradually accumulates to induce glomerular sclerosis. Activity of many ECM catabolic enzymeses and expression of inhibitive factors show misbalanced when many kinds of compositions deposit. In enzymatic system, matrix metalloproteinase(MMP) and organization inhibitive factor(TIMP) have been the hotspots in the past few years. This paper, with ECM excreted by MCs cultured in vitro with Angâ…¡, is to observe the effects of SLT on ECM , MMP-9mRNA and TIMP-1mRNA, and to discuss the protective mechanism of SLT to kidney from two closely correlative points of view of reducing ECM accumulation and strengthening ECM degradation. Methods:1. Determination of FN,Colâ…£MCs was in all resting stage. There were normal control group, Ang II group, Valsartan (Valsartan10^-7 mol·L^-1) group, low, medium and high dosage group(1.25%, 2.5%, 5%SLT blood serum,those lower than 5% were made up for by blood serum of normal rats). The former three group were added respectively with 5% serum of normal rats; except normal group, the rest were added with Angâ…¡10^–6 mol·L^-1. Supernatant was collected after 48h of incubation. FN ,Col â…£were determined with ELISA methods. 2. Detection of MMP-9mRNA, TIMP-1mRNA expression Grouping was the same as the above. Cells of all groups cultivated in 75 ml culture flask, total RNA were extracted with Trizol one-step methods. MMP-9mRNA and TIMP-1mRNA expression were determined with polymerase chain reaction(RT-PCR), and relative amount analysis was made with β-actin correcting. Results: 1. Detection of FN Compared with normal control group(71.25±2.22), the numerus of Ang II group(84.40±4.22) notably increased (P<0.01),. The numerus of all SLT groups of different dosages decreased, compared with that of Ang II group, and had significant difference(P<0.01). In SLT groups, the numerus of SLT5% group(53.94±2.24) decreased, compared with SLT2.5% group(59.27±3.33) and SLT1.25% group(60.74±2.89), and had significant difference(P<0.01). Content of FN of every dose group of SLT was lower than that of normal control group (P<0.01). Compared with normal control group and Ang II group, content of FN of Valsartan group(63.87±3.89) decreased with significant difference(P<0.01). Content of FN of SLT5% group was lower than that of Valsartan group with significant difference (P<0.01). 2. Detection of Colâ…£Compared with normal control group(22.80±3.09), the numerus of Ang II group(30.40±2.59) notably increased (P<0.01). The numerus of all SLT groups of different dosages decreased compared with that of Ang II group, and had significant difference(P<0.01). In SLT groups,the numerus of SLT1.25% group(19.75±1.50), SLT2.5% group(15.94 ±1.50) and SLT5% group(10.54 ±1.36) decreased progressively with significant difference(P<0.01). Compared with Ang II group, content of Colâ…£of Valsartan group(20.12 ±1.62) decreased with significant difference(P<0.01). Content of Colâ…£of SLT5% group and SLT2.5% group were lower than that of Valsartan group, and there was significant difference (P<0.01). 3. Detection of MMP-9mRNA Compared with normal control group(0.34±0.01), the numerus of Ang II group(0.20±0.02) decreased significantly (P<0.01). Compared with Ang II group, MMP-9mRNA expressions of SLT1.25% group(0.50±0.01), SLT2.5% group(0.55±0.05) and SLT5% group(0.71±0.06) increased with significant difference(P<0.01). In all SLT groups, SLT5% group was notably higher than SLT1.25% group and SLT2.5% group, and there was significant difference(P<0.01). Compared with Ang II group, MMP-9mRNA expression of Valsartan group(0.47±0.04) notably increased (P<0.01). MMP-9mRNA expression of SLT1.25% group and SLT2.5% group were a little higher, compared with Valsartan group, and there wasn't significant difference. MMP-9mRNA expression of SLT1.25% group increased significantly, compared with Valsartan group(P<0.01). 4. Detection of TIMP-1mRNA Compared with normal control group(0.34±0.01), TIMP-1mRNA expression of Ang II group(0.48±0.02) increased significantly (P<0.01). Compared with Ang II group, TIMP-1mRNA expressions of SLT2.5% group(0.38±0.01) and SLT5% group(0.35±0.01) decreased with significant difference(P<0.01). TIMP-1 mRNA expression of SLT1.25% group had a tendency of decreasing, but there wasn't significant difference(P<0.05). In all SLT groups, TIMP-1 mRNA expression decreased progressively with concentration increasing (P<0.01). Compared with Ang II group, TIMP-1mRNA expression of Valsartan group(0.38±0.01) decreased significantly (P<0.01). Comparing with Valsartan group, TIMP-1mRNA expression of SLT5% group tended to decrease, but there wasn't significant difference(P<0.05), TIMP-1mRNAexpression of SLT2.5% group hadn't significant difference(P<0.05), TIMP-1mRNA expression of SLT1.25% group increased significantly (P<0.01). 5. Changes of MMP-9/TIMP-1 Compared with normal control group(1.00±0.03), MMP-9/ TIMP-1 of Ang II group(0.41±0.03) decreased significantly (P<0.01). Compared with Ang II group, MMP-9/ TIMP-1 of all SLT groups of different dosages increased with significant difference (P<0.01). MMP-9/TIMP-1 of SLT5% group(2.00±0.19) increased with significant difference(P<0.01). Compared with Ang II group, MMP-9/ TIMP-1 of Valsartan group(1.24±0.19) increased significantly (P<0.01). Compared with Valsartan group, TIMP-1mRNA expression of SLT5% group and SLT2.5% group all increased, but only SLT5% group had significant difference (P<0.01). 6. Correlation analysis MMP-9mRNA expression and Contents of Colâ…£and FN of every group were made correlation analysis, and the result showed: MMP-9 and FN (r=-0.89, P<0.01), Colâ…£(r=-0.92,P<0.01)presented significant negative correlation. MMP-9/TIMP-1 ratio and Contents of Colâ…£and FN of every group were made correlation analysis, and the result showed: MMP-9/TIMP-1 ratio and FN(r=-0.80, P<0.01), Colâ…£(r=-0.92,P<0.01)presented significant negative correlation. Summary: 1. Blood serum with SLT could decrease contents of Colâ…£and FN which were major components of ECM, which suggested that SLT could reduce accumulation of ECM, and then delay the progression of Renal glomerular disease and prevent and cure glomerular sclerosis. 2. Blood serum with SLT could promote MMP-9mRNA high expression and correct misbalance of MMP-9/TIMP-1, which confirmed further the mechanism of reducing ECM with this recipe in degradation way. 3. Correlation analysis showed that SLT was able to up-regulate MMP-9mRNA expression, improve proportion disbalanceof MMP-9/TIMP-1, and that MMP-9mRNA expression, MMP-9/TIMP-1 and FN, Col â…£presented significant negative correlation, which suggested that there was close internal relationship between promoting ECM degradation and inhibiting ECM synthesis with SLT. Part â…£Effect of SLT on the expression of TGF-β₁mRNA and CTGF mRNA in MCs Objective: The expression increase of TGF-β₁ as significant medium of glomerulosclerosis accords with the increase of ECM deposit. CTGF and TGF-βpossess similar biological activity in the aspects of cell proliferation and matrix accumulation, yet CTGF as downstream factor of TGF-β₁ probably mediates negative effect of TGF-β₁. It is to explore the action of SLT interfering the expression of TGF-β₁ mRNA and CTGF mRNA in MCs, to analyse the correlation of TGF-β₁ mRNA, CTGF mRNA, ECM and MMP-9mRNA, and then to reveal the kidney-protecting mechanism of SLT inhibiting "factor of increasing evil". Methods: Ways of grouping, cell culturing, extracting of total cell RNA etc., were the same as the above. The expressions of TGF-β₁ mRNA and CTGF mRNA in cells of each group were detected with RT-PCR technique. Results: 1. Effect of SLT on the expression of TGF-β₁ mRNA RT-PCR semi quantitative analysis showed that the expression of TGF-β₁ mRNA in Angâ…¡group (0.35±0.02) heightened significantly compared with normal control group (0.23±0.02); that the expression of TGF-β₁ mRNA in SLT1.25% group (0.29±0.01), SLT2.5% group (0.25±0.01), SLT5% group (0.19±0.02) and Valsartan group (0.30±0.02) was significantly lower than that in Angâ…¡group (P<0.01); that the expression of TGF-β₁ mRNA was remarkably lower than that in Valsartan group (P<0.01); that the expression of TGF-β₁ mRNA in all SLT groups of different dosages decreased progressively with the concentration increment. 2. The correlation analysis of the expression of TGF-β₁ mRNA andthat of FN, Colâ…£and MMP-9 mRNA The results showed that the expression of TGF-β₁ mRNA and that of FN (r=0.66, P<0.01) and Colâ…£(r=0.77, P<0.01)presented significant positive correlation; that the expression of TGF-β₁ mRNA and that of MMP-9 mRNA (r=-0.69, P<0.01)presented significant negative correlation. 3. Effect of SLT on the expression of CTGF mRNA RT-PCR semi quantitative analysis showed that the expression of CTGF mRNA in Angâ…¡group (0.52±0.02) heightened significantly (P<0.01) compared with normal control group (0.31 ±0.08); that the expression of CTGF mRNA in SLT1.25% group (0.45±0.08), SLT2.5% group (0.41±0.02), SLT5% group (0.31±0.02) and Valsartan group (0.46±0.03) was significantly lower than that in Angâ…¡group (P<0.01); that the expression of CTGF mRNA in SLT2.5% group and SLT5% group was remarkably lower than that in Valsartan group (P<0.01); that the expression of CTGF mRNA in all SLT groups of different dosages decreased progressively with the concentration increment. 4. The correlation analysis of the expression of CTGF mRNA and that of FN, Colâ…£and MMP-9 mRNA The results showed that the expression of CTGF mRNA and that of FN (r=0.47, P<0.01) and Colâ…£(r=0.57, P<0.01)presented significant positive correlation; that the expression of CTGF mRNA and that of MMP-9 mRNA (r=-0.59, P<0.01) presented significant negative correlation. Summary: 1. SLT serum was able to repress high expression of TGF-β₁ mRNA produced by AngII 's stimulus in MCs and presented certain dose-effect relationship between them. The effect of SLT serum was superior to Valsartan. 2. SLT serum was able to repress high expression of CTGF mRNA produced by AngII 's stimulus in MCs and presented certain dose-effect relationship between them. The effect of SLT serum was equal or superior to Valsartan.3. The research on correlation analysis demonstrated that SLT serum repressing high expression of TGF-β1 mRNA and CTGF mRNA was closely related to its reducing the content of ECM and promoting degradation of ECM. Such results suggested that different pathways possessed synergy through which SLT exerted therapeutic effects. Conclusions: 1. SLT serum remarkably repressed the proliferation of MCs and presented certain dose-effect relationship. At the same time, SLT serum was able to induce apoptosis of MCs. Such actions were closely related to its improving proportion relationship between gene Bax and bcl-2 regulating and controlling apoptosis. SLT really possessed the action of reducing the proliferation of MCs which was demonstrated from two different angles including inhibiting proliferation and promoting apoptosis. 2. SLT serum remarkably reduced the content of FN and Colâ…£in ECM and presented certain dose-effect relationship. At the same time, SLT serum was able to up-regulate the expression of MMP-9 mRNA and to improve proportion relationship between MMP-9 and TIMP-1 in catabolic enzymes system. SLT really possessed the action of reducing the accumulation of ECM which was demonstrated from two closely associated angles including reducing the content of ECM and promoting ECM degradation. 3. SLT serum was obviously able to down-regulate the expression of TGF-β1 mRNA and CTGF mRNA. Correlation analysis confirmed that the alteration of above-mentioned two was closely related to FN and Colâ…£in ECM and their degrading factor MMP-9. The curative effect mechanism of SLT was further clarified from GF angle. 4. The hyperplasia of MCs and its epimatrix accumulation were one of the central pathogenic links which led to glomerulosclerosis and made renal glomerular disease develop to terminal stage. In this course of development , TGF-β1 and CTGF were the key factors. This subject confirmed from above-mentioned three significant aspects that the target cells were MCs through which SLT exerted therapeutic effect.