| Chronic glomerulonephritis (CGN), caused by a variety of factors, characterizes concealed and complex pathogenesis which is not completely clear. In recent years, miRNAs, a kind of endogenous and small non-coding RNA, is expected to be served to make certain the pathogenesis mechanism of CGN for its heavy involvement in kinds of kidney diseases. Interleukin-10(IL-10) from myriad cells is an inflammation and also an immunosuppression cytokine taking part in body’s immunological inflammatory reaction. IL-10 is secreted by mesangial cell and endothelial cells in kidney, which not only participates the mediation process for normalization of kidney function but also playing an important part in massage transduction in cells and molecules in kidney with disease. Emerging studies have showed that when kidney disease, IL-10 and miRNAs interacting each other involve in the mediation to immune response.Traditional Chinese medicine have showed great potential in the prevention and treatment to CGN. And an evident efficacy on CGN by Zhenwu decoction, a classical traditional Chinese prescription, has been observed in experiment and clinical study. However, as the new pathogenesis of CGN has been being revealed, in addition with the unclear certain mechanism of effect by Zhenwu decoction on CGN as a result of its multiple effect targets to CGN, the revelation to effect mechanism of Zhenwu decoction to CGN is necessary for providing experimental basis for its clinical application.Objective1. With establishment of the rat model with CGN, to evaluate Zhenwu decoction’ effect on CGN through detection to urine protein quantity, renal function and pathological changes of kidney, inflammatory factors in serum and inflammatory factors in kidney.2. The differentially expressed miRNAs between normal rats and rats with CGN will be gotten through application of microarray, and then with PCR to test its authenticity. And the highly expressional miRNA in MC of rat will be obtained through establishing miRNA expression profile for further relative exploration.3. With LPS to induce inflammation in MC, and then serum of rat with CGN after giving Zhenwu decoction will be applied to MC for investigating its effect on the cell proliferation, cell cycle and apoptosis.4. To transfer he mimic and inhibitor of miR-145-5p to MC, and the change in mRNA expression of miR-145-5p and IL-10 will be tested to explore relation of them. And the change in IL-10-mediated PI3K/AKT signal path, and the role of miR-145-5p at level of IL-10/AKT signal path will be studied.Methods1. The SD rats were used, and C-BSA was given by intraperitoneal injection. After 2 weeks, C-BSA was administrated through caudal vein of rats for 4 weeks to induce CGN in rats. And from the start of the fourth week, the low and high dosage of Zhenwu decoction(4.2g/kg、8.4g/kg)was given through intragastric administration for 4 weeks. Then Zhenwu decoction’s protection to renal function of CGN rats was investigated through urine protein, serum creatinine, urea nitrogen and pathological changes. And from degree of macrophagocyteion infestation in glomeruli, levels of IL10, IL-1α and IL-2 in serum, and from mRNA expression of IL-10, TNF-α, IL-2 and IL-6 in kidney, the improvement of Zhenwu decoction to CGN-caused inflammation was explored.2. 3 cases respectively from control and model groups were gained for chip hybridization after marking with miRNA. And the chip scanning image was obtained with GenePix Pro 6.0, and on the signal the differentially expressed miRNA between cases from control group and those from model group was taken. Then the up-regulated miR-145-5p and down-regulated miR-153-3p in model group were taken for authenticity test with PCR.3. The MC (mesangial cell, MC) was cultivated, and Zhenwu decoction was applied to affect MC after inducing inflammatory proliferation by LPS in MC. Then in all groups, the proliferation of MC was explored by MTT and CCK8, the cell cycle was investigated by flow cytometry after being dyed with PI, and apoptosis of MC was observed by Anexin V/FITC.4. The miR-145-5p mimic(50nM)and inhibitor(100nM)was transfected to MC,24h after, LPS was used to induce inflammation in MC, then the intervention by serum of rats had been given Zhenwu decoction was performed. Then mRNA of miR-145-5p and IL-10 was tested with PCR, and protein phosphorylation of AKT, GSK-3α, GSK-3β, p65 and p65, and Cyclin Dl protein expression were detected by Western Blot. Thus effect by miR-145-5p and Zhenwu decoction on proliferation of MC, on PI3K/AKT/GSK and NF-KappaB signal paths could be observed.Results1. Compared with rats in model group, an increased output of urine, decreased urine protein in 24h, lower serum creatinine and urea was observed in those of Zhenwu decoction group. And relative to model group, Zhenwu decoction could attenuate levels of inflammatory cell infiltration in glomerular, and inhibit glomerular hypertrophy and degeneration of renal tubular epithelial cell, as well restrain proliferation of mesangial matrix. And the raised IL-10 level and reduced levels of IL-1α, IL-2, IL-6 and TNF-α were showed in Zhenwu decoction group to model group.2. The 26 differentially expressed miRNA (fold change> 2.0) was obtained according to analysis to miRNA expression profile of renal tissues of normal and CGN rats, in which in model group,3 evident up-regulated miRNAs was showed, and 23 down-regulated miRNAs was observed, indicating miRNA plays a important role in the onset and development of CGN. On the basis of PCR test, relative to control group, miR-145-5p up-regulated 2.96 folds, and down-regulated 2.65 folds, exhibiting a common trend with chip test. Therefore, the chip test was proved to be high reliability.3. Relative to group with presence of LPS, Zhenwu decoction group could evidently inhibit MC the proliferation, stopping mitosis of MC during GO, but the effect on apoptosis of MC was not distinct.4. Compared with model group, Zhenwu decoction and miR-145-5p could restrain abnormal proliferation of MC caused by LPS and facilitate IL-10 mRNA expression, and could also inhibit phosphorylation of AKT, GSK-3α, GSK-3β and p65 proteins and descend Cyclin D1 expression.Conclusion1. Through the increase in urine volume of CGN rats, reducing in urine protein in 24h, and attenuated pathologic changes, a protection effect to renal function by Zhenwu decoction has been exhibited. And an inhibition to immuno-inflammatory responses in glomerulus demonstrated Zhenwu decoction could alleviate immune-response-caused injury.2. There were 26 differentially expressed miRNAs involving in the onset and development of CGN, and to the regulation to LPS-caused immuno-inflammatory responses in MC, miR-145-5p and Zhenwu decoction showed a similar effect, both could up-regulated IL-10 expression through restraining PI3K/AKT/GSK signal path and negative feedback control of inflammatory factor.3. miR-145-5p and Zhenwu decoction could inhibit MC abnormal proliferation. They also could restrain activation of NF-KappaB, mediatig immuno-inflammatory responses of kidney. |
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