| Objective:This study was designed to investigate the pro-apoptotic molecular mechanism of Qianliening Capsule (QC) treatment on benign prostatic hyperplasia(BPH) and to observe the expression of key regulatory factor (Bax, Bcl-2,et al) in the mitochondrial apoptosis pathway, thus provide a theoretical basis for the clinical application of QC.Method:In the first part of the thesis,30adult male SD rats were randomly divided into blank group, model group, the Qianliening Capsule group. Model group and the Qianliening Capsule group were castrated and subcutaneously injected with testosterone propionate to generate BPH model. In the meanwhile, the Qianliening Capsule group were orally treated with Qianliening Capsule at a dose of4.5g/kg/d, while the blank group and model group were respectively orally treated with saline. After28days’ treatment, rats of each group were sacrificed and the prostatic tissues were collected to evaluate the prostatic weight (PW) and prostatic index (PI). The histological changes of prostate were obsevered by HE staining. And TUNEL staining was used to examine cell apoptosis generally. Then the expression of Bcl-2, Bax, Caspase-3in prostatictissues were determined by RT-PCR and Immunohistochemistry(IHC). In the second part of the thesis, we used bFGF, a growth factor which play a major role in the the pathogenesis of benign prostatic hyperplasia, to stimulate the normal human prostate stromal cells line WPMY-1and investigated the cellular effects of the aqueous extract of QC in the bFGF-Stimulated WPMY-1cell line. A phase-contrast microscope to Observe the morphologic changes; MTT Assay wes used to evaluate the cell viability; Detection of Apoptosis by Flow Cytometry Analysis with Annexin V/PI Staining; Measurement of Mitochonrial Membrane Potential (△Ψm) by Flow Cytometry Analysis with JC-1Staining; Analysis of Caspase Activation; RT-PCR Analysis and Western Blot Analysis were used to detect the expression of Bax、Bc1-2.Result:The first part of the thesis:(1) It was showed that QC group significantly reduced PI compared with that of Model group in BPH rats (P<0.05),(2) HE staining showed that there were significant proliferative changes in the prostate tissue of model group, and the proliferative changes of the QC group were significantly reduced compared to the model group.(3) The result of TUNEL analysis showed that QC can induce apoptosis in the prostate tissues.(4) The mRNA and protein expression of Bcl-2in the QC group were significantly decreased, compared than that in the model group (P<0.05). The mRNA and protein expression of Bax in the QC group were significantly increased, compared than that in the model group (P<0.05). The IHC socres of cleaved Caspase-3showed there were more expression in QC group then that of model group.The second part of the. thesis:The results showed that QC inhibited the growth of bFGF-Stimulated WPMY-1cells as demonstrated QC induced cell morphological changes and reduced cell viability in dose-and time-dependent manners. Furthermore, we observed that QC treatment resulted in the loss of plasma membrane asymmetry, collapse of mitochondrial membrane potential, activation of Caspase-9and Caspase-3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2.Conclusion:(1) QC can significantly improve the rat model of BPH prostatic hyperplasia, suggested the QC have a significant effect on BPH rats.(2) The expression of TUNEL, Bax, Bcl-2and cleaved Caspase-3changed showed that Qianliening Capsule can Induction prostate apoptosis in a Rat Model of Benign Prostatic Hyperplasia.(3) QC treatment resulted in the loss of plasma membrane asymmetry, collapse of mitochondrial membrane potential, activation of Caspase-9and Caspase-3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2, suggesting that inhibitory activity of QC bFGF-Stimulated WPMY-1cell growth was due to mitochondrion-mediated apoptosis, which may, in part, explain the mechanism of QC.treatment on BPH. |
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