| Cyclosporine A (CsA) nephropathy is characterized by renal interstitial fibrosis in the pathological changes, manifested as infiltration of inflammatory cells, extracellular matrix accumulation, tubular atrophy and peritubular capillary deficiency whose pathogenesis involves angiotensin II (Ang II), Endothelin -1 (ET-1), oxidative damage, activation of aldosterone, of which the excessive deposition of ECM is the important feature of renal interstitial fibrosis. Large number of experiments have conformed that transforming growth factor-β1 (TGF-β1) plays an important role in accumulation of ECM, which mediates glomerular sclerosis and renal interstitial fibrosis. In the process of fibrosis, there is evidence that tubular epithelial myofibroblast transdifferentiation (TEMT) plays an important part in occuring and progressing of renal tubule interstitial fibrosis, with the a-smooth muscular actin (a-SMA) as the marker for TEMT.Chronic CsA nephrotoxicity belongs to"edema","back pain","consumption","stranguria"in Traditional Chinese Medicine. Kidney and liver are injured with toxicity of drug. Retention of water dampness is induced by deficiency of kidney and spleen. Chailing decoction, combining Xiaochaihu decoction and Wuling pulvis, both originated from Treatise on Febrile and Miscellaneous Diseases written by Zhang Zhong-jing, is composed of Radix Bupleuri, Radix Scutellariae, Rhizoma Pinelliae, Radix Ginseng, Radix Glycyrrhiza, Rhizoma Zingiberis Recens, Fructus Jujubae, Polyporus, Ramulus Cinnamomi, Rhizoma Atractylodis Macrocephalae, Poria and Rhizoma Alismatis. Chailing decoction has the function of promoting diuresis and detoxicate,soothing liver and invigorating spleen, which is on the beat of pathogenesis of CsA Nephropathy. We duplicated the rat models of Chronic CsA nephrotoxicity, and studied the mechanism through observing the effect of Chailing decoction and Valsartan on TGF-β1, a-SMA and ColⅢ. Results are as follows:ExperimentⅠ: Effect of Chailing Decoction on Renal Function and Pathomorphology in Rats with CsAForty Sprague-Dawley rats were randomized equally into 4 groups: Control group, CsA group, Valsartan group and TCM group. Except those in Control group received gastrogavage with olive oil, rats in other three groups were made into CsA nephropathy model by oral infusion of CsA 30mg/kg·d for 28 days. At the same time, rats in Valsartan group and TCM group were treated respectively with Valsartan 10mg/kg·d and Chailing decoction 3g/kg·d. All animals were put into metabolism cages at each end of week and got urine and measured body weight. All animals were sacrificed on the twenty-ninth day. Kidney was harvested and the renal tissue paraffin section and specimen of serum were produced to observe the pathology changes by HE and Masson staining and detected the contents of urea nitrogen, serum creatinine and urine creatinine, calculated clearance rate.Results:1 There were no abnormal changes on spirit, activity, fur, diet, drink and urine in rats in control group. The rats in CsA group were listless, short of diet, drink and activity, while its fur turned yellow and some fell off. The rats in Valsartan group were superior to those in CsA group. The rats in TCM group were the same as those in Valsartan group. There was no obviously difference between the weights of each group(P>0.05). The urine of CsA group (19.650±9.569ml) increased compared with that of control group (15.650±2.015) after treated with CsA for 2 weeks, but there was no significant difference. After 3 weeks, the urine of CsA group (21.450±8.952) was higher than that of control group (15.400±2.514)(P<0.05), while the urine of Valsartan group (17.400±5.854) and TCM group (15.812±7.025) did not increase. There was no obviously difference compared with control group. After 4 weeks, the urine of CsA group(29.150±13.695)was significantly higher than that of control group(15.800±2.529)and treatment groups(16.700±4.939, 16.734±6.198) (P< 0.01).2 The change of pathomorphology in renal tissue: HE staining showed that the epithelial cells in renal tubule of control group arranged in neat rows without edema. The epithelial cells in renal tubule of CsA group were necrosis and abscission. There were lots of inflammatory cells infiltrating in interstitial space. The Valsartan group and the TCM group were better than CsA group. The Masson staining showed that there was no abnormal in control group but was disorder in CsA group with increased collagen in renal tubular interstitium. Valsartan group and the TCM group were better than CsA group.3 The detection of the content of urea nitrogen, serum creatinine and clearance rate: The contents of urea nitrogen (11.395±1.824 mmol/L) and serum creatinine (99.067±5.426μmol/L) in CsA group increased more obviously than those in control group (6.3450±1.053, 79.117±3.511) (P<0.01). The clearance rate of CsA group (0.2823±0.0466 ml/min) was notably lower than that of control group (0.5415±0.0257) (P<0.01). After medication, the contents of urea nitrogen (8.615±0.878, 7.160±0.679) and serum creatinine (87.817±13.189, 82.933±9.239) were reduced significantly(P<0.01), and the clearance rate in each treating groups (0.3428±0.0624, 0.3465±0.0383) was higher than that of CsA group(P<0.05) .ExperimentⅡ: Effect of Chailing Decoction on the Expression of a-SMA in Rats with CsA-induced NephropathyThe methods of CsA making, group dividing and treatment were the same as ExperimentⅠ, 28 days. The mRNA and protein expressions of a-SMA in kidneys were detected by RT-PCR, Western Blot, immunohistochemistry and flow cytometry.Results:1 The mRNA expressions of a-SMA in rats kidney determined by RT-PCR were weak in control group(0.9177±0.1239), significantly higher in CsA group (1.1950±0.0467)than those in control group (P<0.01)and sharply decreased in TCM group(0.9857±0.0760) and Valsartan group (1.0503±0.1420) (P<0.05). 2 Western Blot detection showed a small amount of protein expression of a-SMA in rats kidney (0.2766±0.1405). They were higher in CsA group (0.8788±0.3104) than those in control group (P<0.01) and notable lower in TCM group (0.5083±0.1092) and Valsartan group (0.5467±0.0737) than those in CsA group.3 The expressions of a-SMA in rats kidney detected by immunohistochemistry showed that there were a few a-SMA only in vessel smooth muscle cell, and nothing in glomeruli and tubule interstitial in control group. They expressed in cortex and tubule interstitial of corticomedullary border in CsA group. Those in TCM group and Valsartan group obviously decreased.4 The protein expressions of a-SMA in rats kidney determined by flow cytometry showed: the fluorescence index in CsA group (0.9847±0.0065) were higher than those in control group (0.8197±0.0064) (P<0.01). They sharply decreased in TCM group (0.9111±0.0068) and Valsartan group (0.9131±0.0126) compared with CsA group(P<0.01).ExperimentⅢ: Effect of Chailing Decoction on the Expression of TGF-β1 in Rats with CsA-induced NephropathyThe methods of CsA making, group dividing and treatment were the same as ExperimentⅠ, 28 days. The mRNA and protein expressions of TGF-β1 in kidneys were detected by RT-PCR and immunohistochemistry.Results:1 The mRNA expressions of TGF-β1 in rats kidney determined by RT-PCR were feeble in control group (0.7927±0.0899), significantly up-regulated in CsA group (1.1270±0.1239)compared with control group (P<0.01) and sharply down-regulated in TCM group (0.8683±0.0938) (P<0.05).2 The expressions of TGF-β1 in rats kidney detected by immunohistochemistry showed that there were weak expressions in control group but up-regulated remarkably in tubule interstitial and glomeruli in CsA group and down-regulated in TCM group and Valsartan group.ExperimentⅣ: Effect of Chailing Decoction on the Expression of ColⅢin Rats with CsA-induced Nephropathy The methods of CsA making, group dividing and treatment were the same as ExperimentⅠ, 28 days. The mRNA and protein expressions of ColⅢin kidneys were detected by RT-PCR, Western Blot and immunohistochemistry.Results:1 The mRNA expressions of ColⅢin rats kidney determined by RT-PCR in control group showed weak (0.7630±0.0557). It was significantly up-regulated in CsA group (0.9180±0.0593) than those in control group (P<0.05) and was down-regulted in TCM group (0.8107±0.0761) and Valsartan group (0.8300±0.0719).2 The protein expressions of ColⅢin rats kidney detected by Western Blot in control group were weak(0.2129±0.1089), up-regulted in CsA group (0.7334±0.1396) than those in control group (P<0.01), and notablely reduced in TCM group (0.3337±0.0802)and Valsartan group (0.4343±0.1997) than those in control group(P<0.01,P<0.05).3 The expressions of ColⅢin rats kidney detected by immunohistochemical showed a small amount in control group, pale yellow. The expressions remarkably increased in CsA group, brown orange or darkbrown. Those in TCM group and Valsartan group obviously decreased.Conclusions:1 Chailing decoction has the function of recovering and improving structure of renal tissue, reducing the expression of collagen and the degree of tuble interstitial fibrosis, decreasing urea nitrogen and serum creatinine, increasing calculated clearance rate to improve renal function.2 Chailing decoction plays a role in down-regulating the over expression of a-SMA induced by CsA, so as to retard the progress of renal interstitial fibrosis through inhibiting renal tubular epithelial phenotype transformation.3 Chailing decoction has the effect of inhibiting the over expression of TGF-β1 induced by CsA in mRNA and protein level, reducing the deposition of extracelluar matrix, delaying the procedure of renal interstitial fibrosis. 4 Chailing decoction plays a role in down-regulating the over expression of ColⅢinduced by CsA in mRNA and protein level, promoting the degradation of collagen, suppressing the deposition of collagen, inhibiting renal interstitial fibrosis, and retarding the progress of Cyclosporine A Nephropathy.
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