Inhibitory Effect Of Natural Drugs With Proteasome Inhibitor Activity On Primary Hepatocytes Of HBV Transgenic Mice

BACKGROUNDHepatitis B virus (HBV) replication in patients with chronic hepatitis B (CHB) could lead to liver inflammation and fibrosis. It is even associated with the development of liver cirrhosis and hepatocellular carcinoma. HBV infection is becoming a serious social public health problem. It is become a common view that the antiviral therapy displays a key role for treatment of chronic hepatitis B. Nucleotide analogs become another kind of effective drugs for HBV therapy after interferon alpha (IFN-a). Most of patients who treated with nucleotide analogs need long-term chemotherapy, because it is difficult to achieve lasting response through short-time therapy. It will be also increase the risk of drug-resistance. Along with the increase of the category of nucleotide analog, the complexity of the dug-resistance has greatly increased. So how to effective clear HBV or persistent inhibit HBV replication, reduce the occurrence of the drug-resistance is the focal and difficult point to antiviral therapy. As the further understanding to the interaction between the virus and its host cell, recently specialist proposed a new antiviral strategy:regulate host proteins that the virus depends on the various stages of its replication cycle in order to inhibit virus replication, reduce drug-resistance. Studys found that inhibitors of cyclophilin A could interrupt viral RNA replication effectively, which was used to guide the antiviral research of hepatitis C virus.Intracellular protein degradation is a very important process in organisms, including lysosomal pathway and ubiquitin-proteasome system. Lysosomal pathway is a non-selective proteolysis of intracellular proteins, occurring in the lysosomes. The ubiquitin-proteasome system is a selective process, which plays important roles in a variety of funfamental cellular processes by protein degradation selectively. A large number of research showed that virus could use the ubiquitin-proteasome pathway to evade detection by the immune system, in order to promoting the virus replication, assemble and release of the viron. It was reported that proteasome inhibitor bortezomib could effectively inhibit HBV-DNA replication, but it is not significant to the level of HBsAg in the HBV-transgenic mice. The earlier research of our laboratory found that bortezomib not only inhibit the levels of HBV-DNA, but also inhibit the expression of HBsAg on the primary HBV-transgenic mice hepatocytes. So it is really need a future study that whether the inhibition of the proteasome inhibitor have catholicity.HepG2.2.15is a best cell model that is used to evaluate the effect of drugs to HBV. HepG2.2.15derived from hepatocarcinoma cell line, which can be induced apoptosis by proteasome inhibitors through inhibit the activity of proteasome. So because of the particularity of the studied drugs, we can’t choose HepG2.2.15as a cell model in our studies. So we use primary hepatocytes which were separated from the HBV transgenic mice by new modified two-steps method. With a long term studies, we found the separated primary hepatocytes with a higher ratio of the living cells and good proliferative activity. After cultured with24h, the adherent primary hepatocytes restored the injury to cell membranes by collagenase digestion. In the first five days, cells have a good ability of albumin synthesis and stable expression of HBsAg and HBV-DNA. So the study evaluated the inhibitory effect of natural drugs with proteasome inhibitor activity use primary hepatocyes separated from HBV transgenic mice as a cell model.OBJECTIVETo evaluate the inhibitory effect of celastrol on primary hepatocytes with the modified two-steps method separated from HBV transgenic mice which were constructed by our liver center.METHODS 1) The primary hepatocytes were treated with different concentrations (20μmol·L-1、4μmol·L-1、0.8μmol·L-1,0.16μmol·L-1,0.032μmol·L-1、0.0064μmol·L-10.00128μmol·L-1,0.000256μmol·L-1) of celastrol, with the untreated cells used as the control. The cytotoxicity of celastrol on the primary hepatocytes was analyzed by MTT assay after24h.2) And then new separated hepatocytes were treated with non-toxity concentrations (0.8μmol·L-1、0.08μmol·L-1、0.008μmol·L-1、0.0008μmol·L-1) of celastrol. The changes of HBsAg and HBV-DNA in the supernatant after the drug treatment for24hours were determined with ELISA and real-PCR.RESULTS1) The TC50of celastrol on primary hepatocytes was6.490±0.802μmol·L-1. At concentrations belower than0.8μmol·L-1, celastrol showed no significant cytotoxicity to primary hepatocytes.2) HBsAg of the cells treated with celastrol was decreased markedly at the concentration of0.08μmol·L-1～0.8μmol·L-1in comparison to that of the control (P=0.000, P=0.000).And the levels of HBV-DNA was also reduced at the concentration of0.08μmol·L-1～0.8μmol·L-1in comparison to that of the control (P=0.009, P=0.044). However, when the concentration of celastrol was increased, there was a corresponding increase in inhibition in HBsAg and HBV-DNA.CONCLUSIONSThe results suggest that nature compound celastrol which with proteasome inhibitor activity can not only inhibit HBV-DNA replication, but also effectively inhibit the expression of HBsAg. It is speculated that the HBV inhibition of celastrol may be associated with it’s inhibiton effect to proteasome. Proteasome inhibitors could inhibit the proteasome activity, so that the misfolded proteins degradation was prevented. This will interfere various metabolic processes in cells. The accumulation of this interference effect leads to these cells preferentially into programmed cell death. Part two:Inhibitory effect of EGCG alone and combining with bortezomib on primary hepatocytes of HBV transgenic miceBACKGROUNDHeat shock protein, a kind of ATP-dependent molecular chaperones, plays an important role in stabilizing nascent proteins, assisting other proteins in proper folding, assembly, transporting and degradation, and keeping the integrity of the proteins function. There were many study confirmed that Hsp60、Hsp70and Hsp90play very important roles in hepatitis B virus(HBV) replication and assembly. In this study we choose natural drug—EGCG((-) epigallocatechin-3-gallate), which extracted from plants, the main component of green tea. EGCG is not only a kind of proteasome inhibitor, but also an Hsp90inhibitor. The prior study showed that EGCG has many biological activities, such as clearing free radical in the body obviously, antitumor and anti-inflammatory. It was also confirmed that EGCG could not only inhibit HBV replication in vivo and vitro but also reduce the levels of aminotransferase in serum and pathological injury of liver.Because of EGCG is an Hsp90inhibitor, it can interfere with the assembly and proteolytic of viral structural proteins so that the release and production of the viral progeny were inhibited. In addition, these substances could interfere with the later process of viral replication, such as assembly, budding, proteolytic and release, in order to regulate, interfere or obstruct protein folding. Thus EGCG could interfere with the proteolytic process of viral poly protein precursor and obstruct the activity of viral protease. Normally, these proteins without folding will be selective degradated by the ubiquitin-proteasome pathway. The proteasome inhibitor will inhibit ubiquitin-proteasome pathway so that these unfolded proteins accumulate in the cell. Thereby cell metabolism was interfered. All of these interference effects added up lead to cells with problem preferentially into programmed cell death.OBJECTIVEIn this study we use primary HBV-transgenic mice hepatocyes as a cell model to study the inhibitory effect of EGCG and antivirus mechanism. At the same time we explored the synergistical inhibitory effect of EGCG combining with bortezomib on primary hepatocytes of HBV transgenic mice.METHODS1) The primary hepatocytes was separated from HBV-Tg mice with the modified two-steps method, and purified by percoll density gradient centrifugation method. The cells were stained with trypan blue, and then counted. The cell were incubated in96-well culture plates coated by gelatin for24h in which the cultured cell density were adjusted to8×104·L-1, about100μL per well. And then the primary hepatocytes were treated with different concentrations of EGCG. The inhibitory effect of the treatment was analyzed by MTT assay.2) The new separated primary hepatocytes were incubated in96-well culture plates coated by gelatin for24h. Adding different concentration of EGCG combining with1μmol·L-1bortezomib after the cells adherented. The inhibitory effect of the treatment was analyzed by MTT assay.3) The new separated primary hepatocytes were incubated in96-well culture plates coated by gelatin. Then the hepatocytes were treated with DMSO、1μmol·L bortezomib EGCG of nontoxic and EGCG combining with1μmol·L-1bortezomib of nontoxic, with the untreated hepatocytes used as control.The hepatocytes were cultured for24h. The levels of HBsAg and HBV-DNA in the supernant were detected by ELISA and real-PCR.4) The new separated primary hepatocytes were incubated in6-well culture paletes. The cells were treated with EGCG, and extracted cell proteins at different time. Hsp90was determined by Western blot at different time and different concentrations.RESULTS1) At concentrations belower than250μmol·L-1, EGCG showed no significant cytotoxicity to primary hepatocytes. At concentrations belower than250μmol·L-1EGCG combining with1μmol·L-1bortezomib also showed no significant cytotoxicity to primary hepatocytes.2) HBsAg of the cells treated with EGCG was decreased markedly at the concentration of50μmol·L-1～250μmol·L-1in comparison to that of the control (P=0.007, P=0.000).And the levels of HBV-DNA was also reduced at the concentration of50μmol·L-1～250μmol·L-1in comparison to that of the control(P=0.019, P=0.008).3) HBsAg of the cells treated with50μmol·L-1EGCG combining with1μmol·L-1bortezomib was decreased markedly in comparison to that of the control, EGCGalone and bortezomib alone(P=0.000, P=0.002).The levels of HBV-DNA treated with250μmol·L-1EGCG combining with1μmol·L-1bortezomib was reduced markedly in comparison to that of the control, EGCGalone and bortezomib alone(P=0.034, P=0.020).4) It was confirmed by western blot that EGCG could reduce the level of Hsp90on primary hepatocytes in different concentration or at different time. However, when the concentration of EGCG and the culturetime was increased, there was a corresponding increase in inhibition.CONCLUSIONSEGCG can inhibit HBV replication and the expression on primary hepatocytes. And it is confered that the inhibition effect maybe associated with the inhibitory effect of Hsp90. Different concentrations of EGCG combining with1μmol·L-1bortezomib can inhibit HBV replication, and there is a synergistical effect when combinated with bortezomib.