The Relationship Of Interleukin-21 With Hepatitis Virus Infection And The Screening Of Small Molecular Inhibitors Related To Liver Injury

Epidemiological survey found that infection of HBV, HCV, Aflatoxin and excessive drink are the most dangerous factors of hepatocellular carcinoma (HCC). Among them, HCC caused by chronic HBV and HCV occupied more than 80%. It usually takes more than thirty years from chronic infection to HCC. As one of the most popular malignant tumors, the prognosis of HCC is extremely bad and the survival rate is only 3%-5%. How to prevent the progress of the disease and decrease the morbidity and mortality of hepatocirrhosis and HCC has become hot issues of both doctors and scientists.PartⅠ. Relationship of interleukin 21 with HBV and HCV infectionOBJECTIVES:Interleukin 21(IL-21) has become a hot topic recently. Some scholars have examined and discussed the IL-21 and IL-21R levels in peripheral blood of AIDS patients and autoimmune hepatitis patients. However, there is no report about IL-21 level in peripheral blood of HBV and HCV patients so far. In fact, IL-21 participates in a variety of autoimmune diseases. At the same time, there is indiscerptible relation between HBV, HCV progress and immune function of the body. This study was designed to explore the relationship between IL-21 level and HBV, HCV infection by testing IL-21 level, liver functional indices, viral load and anti-nuclear antibody (ANA) of the patients.METHODS:IL-21 and ANA levels were detected by enzyme linked immunosorbent assay (ELISA). Liver functional indices including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), globulin (GLO), total bilirubin (TBIL) and direct bilirubin (DBIL) were measured by auto-biochemical analyzer. ELISA method was also used to examine HBV serum markers (HBsAg,HBsAb,HBeAg,HBeAb,HBcAb). HBV DNA was detected by FQ-PCR.RESULTS:1. The ANA and IL-21 were found to be protective for HCV patients.The positive rate of ANA in HCV positive patients with low ALT (ALT<40 U/L) was 22.4%, while that of high ALT (ALT>40 U/L) group was 15.0%, indicating that most ANA positive HCV patients had better liver function. Autoimmune antibody showed certain protective effect of hepatocyte injury and helped to control the development of HCV with the result of lower ALT. Synchronous changes were found between means of ALT and AST among high ALT, low ALT and control groups. The higher ALT level of some patients maybe correlated with the lower ANA level. The average ALT/AST ratio of high ALT (ALT>40U/L) group was greater than 1, probably because of the seriously damaged liver cells on one hand and the high fibrosis extent on the other. In this study, the average IL-21 levels of both low ALT (ALT<40 U/L) HCV patients and high ALT (ALT>40 U/L) group were lower than that of control group, and comparatively that of low ALT group was a bit higher. It was concluded that HCV infection might result in decreased IL-21 level, which was negatively correlated with the degree of hepatocellular damage, indicating HCV infection can induce cellular immune response. It was confirmed that IL-21 helped to control virus development and decrease liver cell damage. This study suggested that the expression of both ANA and IL-21 can decrease hepatocellular damage of HCV patients and the IL-21 level of ANA positive HCV patients was lower than the negative ones. Correlation analysis among ANA,IL-21,ALT,AST and ALB, showed no correlation between these parameters. So the protection effect of ANA and IL-21 should be a result of joint function by other factors.2. The IL-21 levels of both HBV carriers with HBsAg and HBeAg and HBV-related hepatocirrhosis patients were significantly lower than healthy control, but those of HBV carriers with HBsAg alone and chronic HBV patients were significantly higher.HBV patients and healthy control were separated into five groups. The average ALT of control, AsCs, AsCse, CHB and Cir group were 16.75±1.11,24.44±2.09, 23.68±2.01,201.47±36.51 and 59.28±12.97 U/L, respectively. The difference was significant (F=12.368, v=122.158, P=0.000). The mean AST of each group was 22.24±0.72,28.23±1.27,27.53±1.37,150.82±27.40 and 63.83±8.98 U/L, respectively. The difference was also significant (F=15.680, v=122.362, P=0.000). The mean ALB of each group was 47.03±0.46,45.79±0.54,46.12±0.59,43.02±0.66 and 39.57±0.94 g/L, respectively. The difference was significant (F=16.367, v=116.789, P=0.000). The mean GLO of each group was 25.57±0.62,26.61±0.61,25.94±0.65,27.96±0.48 and 30.70±0.65 g/L, respectively. Similarly, the difference was significant (F=10.395, v=109.138, P=0.000). The mean TBIL of each group was 15.20±1.44,14.13±0.80, 14.25±1.73,36.17±11.00 and 41.96±7.27μmol/L, respectively. The difference was significant (F=4.557, v=114.548, P=0.002). The mean DBIL of each group was 4.10±0.26,4.25±0.25,4.04±0.38,17.84±7.04 and 20.69±5.16μmol/L, respectively. The difference was significant (F=3.518, v=121.135, P=0.009). There was no significant difference among the mean TP level. And the average logDNA of AsCs, AsCse, CHB and Cir group was 3.76±0.19,6.80±0.28,5.41±0.18 and 3.64±0.14, respectively. The difference was significant (F=47.324, v=97.334, P=0.000). The IL-21 level of control, AsCs, AsCse, CHB and Cir group was 67.22±5.87, 117.17±23.92,47.90±7.46,112.73±18.54 and 50.18±5.87 pg/ml, respectively. The difference was significant (F=5.096, v=120.252, P=0.001). When regrouping all samples according to the status of antigen or antibody, we found that IL-21 level of HBsAb negative group (79.55±5.73 pg/ml) was notably higher than that of HBsAb positive one (62.30±6.35 pg/ml) (t=-2.016, P=0.046). IL-21 level of HBeAg negative group (90.49±10.76 pg/ml) was also notably higher than that of HBeAg positive group (66.92±5.07 pg/ml)(t=-1.982, P=0.049), and IL-21 level of HBeAb positive group (90.06±9.94 pg/ml) was notably higher than that of HBeAb negative group (66.50±3.93 pg/ml)(t=2.205,P=0.029). IL-21 value of female group was a bit higher than that of male group if all specimens regrouped by gender (t=-0.375, P=0.708). Bivariate correlation analysis of all samples showed significant correlations between ALT and AST (r=0.959, P=0.000, closely related), ALB and TP (r=0.689, P=0.000), DBIL and TBIL (r=0.988, P=0.000, closely related), and negative correlation between ALB and GLO (r=-0.413, P=0.000). There were also some weak correlations between other variables such as logDNA and ALT (r=0.328, P=0.000), logDNA and AST (r=0.302, P=0.000), GLO and TP (r=0.363, P=0.000), ALB and DBIL (r=0.333, P=0.000), ALB and TBIL (r=0.323, P=0.000) and so on. But there was no significant correlation between IL-21 and others.CONCLUSIONS:1. The positive rate of ANA in HCV positive patients with low ALT (ALT<40 U/L) was high than that of high ALT (ALT>40 U/L) group, indicating that the production of ANA was protective to liver injry.2. The average IL-21 levels of both low ALT (ALT<40 U/L) HCV patients and high ALT (ALT>40 U/L) group were lower than that of control group, indicating that HCV infection could result in decreased IL-21 level. And on the contrary, the production of IL-21 helped to control HCV development and reduce hepatocellular injury.3. The IL-21 levels of both HBV carriers with HBsAg and HBeAg and HBV-related hepatocirrhosis patients were significantly lower than healthy control, but those of HBV carriers with HBsAg alone and chronic HBV patients were significantly higher. It was concluded that HBeAg positive HBV patients had bad prognosis. The active effect of IL-21 was also validated.4. Bivariate correlation analysis of all HBV samples showed that ALT was closely related to AST (r=0.959, P=0.000), ALB was related to TP (r=0.689, P=0.000), DBIL was closely related to TBIL (r=0.988, P=0.000), and that ALB was negatively related to GLO (r=-0.413,P=0.000). PartⅡ. The screening of small molecular inhibitors related to liver injury.OBJECTIVE:To screen anti-HBV drugs in cells by HBV DNA level, and to study the inhibitory activities of drugs on HBV replication intermediates. To study the binding pattern of the compound with DNA, the cleavage effect of the compound on DNA, and the anti-tumor activity within and without cells, Futhermore, the mechanism of action was also investigated in various ways.METHODS:WST-8 method was adopted to measure cytotoxicity using CCK-8 kit. HBV DNA inside and outside cells was detected by Fluorescence Quantitative PCR Diagnostic Kit for Hepatitis B Virus DNA. Western blot was adoptive in testing HBcAg. While HBeAg and HBsAg were measured using TRFIA technique. The function of nickel complex on CT DNA was investigated by uv absorption spectrum,fluorescence spectrum,Scatchard plot and viscosity analysis. The action of nickel complex on pBR322 DNA is explored by agarose gel electrophoresis. MTT test was used to evaluate the anti-tumor activity of nickel complex in vitro. The influences of nickel complex on cell apoptosis and cell cycle were analyzed using DAPI fluorescent dying and flow cytometry.RESULTS:1. Compound XLWG-54-52 was found to be effective in reducing HBV DNA both inside cells and outside cells with the IC50 of 11.87±3.94μM and 10.84±0.60μM, respectively.HepAD38 is a cell line which can express HBsAg and HBeAg stably and replicate DNA at a high level. The cultivation time was fixed on seven days. We also screen anti-HBV drugs from a small compound library and found five compounds which inhibited HBV DNA effectively. They were XLWG-54-52, XLWG-61-3A, XLWG-61-7, XLWG-48-6 and XLWG-48-11. Among them, XLWG-54-52 was the most effective and studied further. This compound suppressed HBV DNA both inside and outside cells with the IC50 of 11.87±3.94μM and 10.84±0.60μM, respectively. The CC50 of XLWG-54-52 on HepAD38 was 0.339 mM. It did not inhibit HBeAg, HBsAg and HBcAg. XLWG-54-52 is an analogue of bicyclol. Therfore, this compiound may protect liver damage by inhibiting the HBV DNA replication.2. Nickel complex exhibited excellent performance in binding with CT DNA by insertion mode. And it could break pBR322 DNA from supercoiled form to open circular form dose-dependently through free radical mechanism.In UV absorption spectrum test, the absorption peak of nickel complex was accompanied with hypochromic effect to 18%. The binding pattern of nickel complex to DNA was presumed to be insertion. The binding constant Kb was calculated to be 2.18×105(L·mol-1). In the fluorescence spectrum experiment, the fluorescence of EB-DNA complex quenched distinctly as the concentration of nickel complex increased, thus validating that the pyrrole ring inserted into DNA double chain for binding. Fluorescent Scatchard plot further suggested the k value reduce when the concentration of nickel complex increased in the mixture. But the n value almost maintained at 0.190, indicating nickel complex inhibited the association of EB and DNA competitively. From another viewpoint, the complex inserted into DNA. When nickel complex interacted with CT DNA, the viscosity augmented with the increase of complex. Therefore, it was ascertainable that nickel complex acted with DNA through insertion. Nickel complex was found to promote the cleavage of pBR322 DNA dose-dependently from supercoiled form to open circular form by agarose gel electrophoresis. The optimal reaction temperature was 50℃. While free radical capture agent was added to the reaction system, the cleavage effect was remarkably weakened. These results indicated that nickel complex can cleave DNA chain through free radical mechanism.3. Nickel complex could inhibit HepG2 cell proliferation effectively and dose-dependently through apoptosis pathway.Nickel complex was found to inhibit HepG2 cell proliferation effectively and dose-dependently. The IC50 values of 24h,48h and 72h were calculated to be 328.68±66.31μM,389.81±52.56μM and 202.60±38.01μM, respectively. Accordingly, of the IC50S of positive control drug, cisplatin, were 70.87±9.60μM, 28.77±5.18μM and 39.97±3.66μM, respectively. Another positive control drug, 5-FU, has an IC50 of 10.35±5.78 mM,5.57±0.60 mM and 0.54±0.21 mM, respectively. Therefore, the in vitro inhibition activity of nickel complex on hepatoma cells HepG2 was better than 5-FU but weaker than cisplatin. DAPI staining showed that nickel complex treated cells displayed chromatin condensation, chromosome aggregation and nuclear fragmentation, indicating a typical morphological changes of apoptosis, which was similar to cisplatin.. The apoptosis of HepG2 cells was confirmed by FACS examination. The rate of apoptotic cells enhanced as the concentration of nickel complex increased. The cells in S phase were increased and those in G1 and G2 phases were decreased after nickel complex treatment. The higher the concentration of nickel complex, the lareger the percentage of cells in S phase. It suggested that nickel complex could prevent HepG2 cells proliferation by blocking the cell cycle shift from S phase to G2 phase, thus arresting them at S phase. This effect was similar to cisplatin.4. Sulpha compounds could inhibit HepG2 proliferation through apoptosis pathway.The anti-tumor effects of four sulpha compounds were also investigated. The IC50 of them against HepG2 cells proliferation was obtained by MTT, which was 31.48±1.49μM,13.19±2.15μM,20.11±1.48μM and 13.05±1.69μM, respectively. After DAPI staining, the concentrated chromatin and fragmented nucleus was seen in all of the four active compounds treated HepG2 cells under fluorescent-microscope, indictating that they inhibited the growth of HepG2 cells through apoptosis pathway.CONCLUSIONS:1. Compound XLWG-54-52 was found to be effective in reducing HBV DNA both inside cells and outside cells with the IC50 of 11.87±3.94μM and 10.84±0.60μM, respectively.2. The results of the UV absorption spectrum test, the fluorescence spectrum experiment and the viscosity examination all indicated the excellent character of nickel complex in binding with CT DNA by insertion mode. DNA agarose gel electrophoresis technique validated that nickel comple could break pBR322 DNA from supercoiled form to open circular form dose-dependently through free radical mechanism.3. Nickel complex could inhibit HepG2 cell proliferation effectively and dose-dependently. The IC50 at 24h,48h and 72h was 328.68±66.31μM,389.81±52.56μM, and 202.60±38.01μM, respectively. DAPI staining results showed the apoptosis mechanism. FACS examination validated that the cell cycle was blocked at S phase.4. Four sulpha compounds could inhibit HepG2 proliferation effectively through apoptosis pathway. The IC50 was 31.48±1.49μM,13.19±2.15μM,20.11±1.48μM and 13.05±1.69μM, respectively.