Experimental Study Of Niuhuangshen Capsules Effect On P53、Bcl-2Gene Regulation And Cell Apoptosis Of HepG2

Objective：Based on closely related to the pathogenesis of cell proliferation andapoptosis of liver imbalance to P53, Bax, Bcl-2and apoptosis inhepatocellular carcinoma clearly related genes as a starting point, conductresearch, observe NHS containing serum vitro differentiation of humanhepatoma cell line HepG2apoptosis, explore its mechanism of action,provide theoretical and experimental basis for the study and applicationof NHS capsules for the clinical treatment of primary hepatocellularcarcinoma has opened a new idea.Methods：The use of drugs and cellular pharmacology serological methods,comparative observation(negative serum control drug, the positive controldrug CTX) NHS containing serum on apoptosis of HepG2and regulationof apoptosis-related tomb because of Bax, Bc1-2and tumor suppressorgene P53impact. Main outcome measures and testing methods are asfollows:1.Preparation of serum containing:40SD rats were randomlydivided into control group(8),CTX-containing serum group(8) and NHSreference serum containing group(24). A daily dosage of adult clinical effective dose of the drug-containing serum group,dose equivalentconversion by human and rat body surface area ratio of the translation ofthe day dose in rats, and set the final dose of the drug-containing serumgroup.Control group: the equivalent saline gavage twice a day, every5mL.Containing serum group:CTX solution(25.2mg/kg) administered twicedaily, each5mL;NHS capsules decoction(1.07g/kg) orally twice a day,every5mL; repeated administration for seven consecutive days,45minutes after the last administration, blood taken by cardiac puncturemethod of blood, the blood will be collected in the device left at roomtemperature4h,3000rpm, centrifugal15min, the serum was collected, theserum collected in a water bath at56℃for30min to inactivate, and thenin the clean the station by0.22pm microporous membrane filtersterilization, the serum to a clean tube, set at-20℃, formulated into aculture medium containing serum concentration required when needed.2.CCK-8method to check the inhibitory drugs on serumconcentrations of five HepC2cell proliferation, to find the best andeffective serum concentration of the drug the NHS.3.NHS HepG2cells after administration of the drug-containingserum48h, AO/EB and DAPI staining and morphological changesobserved under a fluorescence microscope.4.Impact of flow cytometry AnnexinV-FITC/PI double staining NHScontaining serum on HepG2cell cycle and apoptosis percentage.5.Western blotting detection NHS containing serum on HepG2cellsP53Bcl-2effect on gene expression in.6.Immunohistochemistry was used to detect the impact of the threeNHS containing serum drug concentrations effective in HepG2cellsBcl-2, Bax and proliferating cell nuclear antigen (PCNA) expression.7.Pre-treatment serum-containing drugs, the relative expressionBcl-2and Bax mRNA in HepG2cells after Real time RT-PCR testing. Results：1.CTX group and NHS various concentrations of taurine ginsengsignificantly inhibited cell proliferation,and with increasing concentrationand action time, the number and speed of proliferation of HepG2cellsdecreased,NHS articipate each concentration group with the controlgroup, the difference was statistically significance (P<0.05,P<0.01).2.By fluorescence microscopy to observe the typical morphological changes:serum containing part of the nucleus presents crease like, corrugated,nuclear chromatin with orange,some chromatin appearscondensed state;CTX serum containing group,20%NHS containing serum group visible.a large number of different levels of necrosis and apoptosis, the cell size smaller, nuclear membrane disappears,theincrease in cytoplasmic vacuoles, organelles fewer nuclear chromatin condensation,irregular,visible nuclear fragmentation, resulting in apoptosis body, and training longer, more significant change.3. HepG2cells were analyzed by flow cytometry showed significantsubdiploid peak, and the number of apoptotic cells increases the doseincreases. HepG2cells containing serum CTX,5%NHS containing serum,10%NHS containing serum and20%NHS containing serum48h afteradministration, the proportion of apoptotic cells were30.13%,11.05%,19.76%and28.32%.Cell cycle analysis shows that NHS containingserum group so that prevents cell growth in the G0/G1phase.4.Western blotting showed that: Compared with the control group,the NHS reference serum containing acting drugs in HepG2cells reality,the expression of Bcl-2protein was significantly decreased (P<0.05), asignificant increase in the expression of P53protein (P<0.05), and thereis a significant difference (P<0.05) between the two groups compared.5.Immunohistochemical results showed that: with the increase in serum concentrations of NHS administration including, PCNALIgradually decreased, indicating that NHS containing serum inhibited theproliferation of HepG2tumor cells, and with the NHS with increasedserum concentrations of the drug can significantly reduce HepG2cells thepositive expression rate of the Bcl-2protein,Bax protein was expressed ina strong,20%NHS, CTX group and10%NHS positive expression rateswere56.89%,58.33%,44.73%, compared with the control group, with asignificant difference (P<0.01, P<0.05).6. Real-time PCR to detect the expression of CT values, the analysisof each factor related apoptosis relative expression levels of mRNAdisplay, along with the expression level of NHS drug-containing serumconcentration increased Bax mRNA increased, whereas the expression ofBcl-2mRNA levels but reduced.Conclusion：1. The NHS containing serum can inhibit the proliferation of HepG2cells in a certain extent.2. The NHS containing serum can induce apoptosis in HepG2cells.3. The NHS drug-containing serum induced HepG2apoptosis of oneof the mechanisms may be upregulated the expression of pro-apoptoticgenes Bax and P53, downregulate the expression of Bcl-2related.