Effects Of Medical Ozone On Cellular Immunity In Chronic Viral Hepatitis B Patients

BackgroundHepatitis B virus is one of the most serious and prevalent health problems, affecting more than 2 billion people worldwide.Although highly effective vaccines against hepatitis B virus have been available since 1982,there are still more than 350 million chronic carriers,75%of whom reside in the Asia Pacific region.People with hepatitis B are at increased risk of developing hepatic decompensation,cirrhosis,and hepatocellular carcinoma.The estimated worldwide mortality is 0.5 to 1.2 million deaths a year.In china,there are 120 million people with HBV infection,out of which 20 to 30 million people show chronic hepatitis B virus infection.Chronic infection remains a challenging global health problem.The outcome of HBV infection is closely related with host's immune status.The immune tolerance and defect of specific cytotoxic T cell(CTL) response are the important reasons of chronic HBV infection.There are about five immunological characteristics for chronic HBV infection.Firstly,in chronic HBV infection,it exists immune tolerance to some extent,and there is a large number of virus antigens in peripheral blood.The long-term stimulation of high concentration of viral antigens probably induced more Treg cells,and expression of immunosuppressive molecules such as PD-1 and CTLA-4 were increased,all of which may cause immune tolerance and chronic HBV infection.Secondly,The less quantity of antigen-presenting cells,especially dendritic cells(DC) and functional defects may affect the interaction of DC-T cells,so that the expression of HLA-â…¡by HBsAg-induced T cells and T cells proliferation weakened,which should not activate Th cells to produce cytokines,or cytotoxicity of cytotoxic T cell(CTL).Thirdly,Patterns of HBV-specific TCR CDR3 are narrow and its peak shape is bias,and proliferation of T cells were monoclonal or oligoclonal,so that the spectral of identification of HBV antigen and response was narrow.Fourthly,Th1/Th2 response is being imbalance.The cytokines type secreted by T cells at the site of virus replication may affect the final outcome of HBV infection.When specific Th1 cell is dominant,they promote the cellular immunity which is in favor of the removal of virus;and when Th2 cell is dominant,although it's in favor of production of antibodies to HBV,easily lead to chronic infection.For chronic HBV infection,Th1-type cytokines(IFN-γ, TNF-α,IL-2,etc.) is relatively short,and the Th2-type cytokines(IL-4,IL-10,etc.) are being relatively high levels,thereby maintaining the chronic infection status.Fifthly,anti-virus-related cytokines(TNF-αand IFN-γ,etc.) may exist gene mutation, genetic polymorphism or signal transduction abnormalities.An improved understanding of hepatitis B virology,immunology,and the natural course of chronic infection,has identified hepatitis B virus replication as the key driver of immune-mediated liver injury and disease progression.The approval of potent oral antiviral agents has revolutionized hepatitis B treatment since 1998. Conventional and pegylated interferon alfa and nucleotide analogues(lamivudine, adefovir,telbivudine and entecavir,etc.) are widely authorized treatments,and monotherapy with these drugs greatly suppresses virus replication,reduces hepatitis activity,and halts disease progression.However,hepatitis B virus is rarely eliminated, and drug resistance is a major drawback during long term therapy.The development of new drugs and strategies is needed to improve treatment outcomes. In recent years,medical ozone for the treatment of hepatitis B has become a new kind of exploration.Medical ozone is a gas mixed ozone with medical oxygen,which can be used to treat a variety of diseases such as rheumatism,ulcerative colitis and protrusion of irtervertebral disc.The reaction of ozone with human whole blood could generate reactive oxygen species(ROS,mainly H₂O₂) and lipid peroxidation products (LOPs),the role of these two products is to separately act on the various components of the blood(monocytes,platelets,red blood cell,WBC) and endothelial cells and other organs,resulting in a variety of effects.In recent years,hydrogen peroxide (H₂O₂) is considered an important intracellular messenger in animal and plant cells, so ozone plays an indirect role through its reaction products.At the end of 2004 Chinese mainland start of ozone treatment for viral hepatitis B and has shown initial signs of efficacy,medical ozone treatment of chronic hepatitis B is expected to become a new strategy.However,the mechanism of treatment is not clear.Previous studies have inferred immunoregulatory role of medical ozone,but many things still need to be further confirmed,and therefore the study of immune mechanism of medical ozone has important clinical significance.Objective1.To explore the mechanism of cell-mediated immunity of medical ozone for the CHB treatment.2.To provide evidence of basic research for the treatment of CHB with medical ozone and promote the wide range of clinical applications of medical ozone.3.To look for new therapeutic strategies for the treatment of CHB and improve therapeutic effects.Methods1.IFN-γand IL-4 levels of PBMC culture supernatant after acting by medical ozone with Enzyme-linked immunosorbent assay(ELISA) in CHB patients:There are 11 cases of CHB patients in this study,eight cases of healthy donors as control.PBMCs are separated with density gradient centrifugation,and then add medical ozone of five concentrations(4, 10,20,40,80μg / mL) in the supernatants of PBMCs with the method of effervescency,then cultivated in 37℃with 5%CO₂ cell culture incubator for 72 hours.We set up medical oxygen(O2,acting with effervescency),positive (to PHA stimulation),negative and double-complex-hole self-control group. The IFN-γand IL-4 concentrations in PBMC culture supernatants are detected wirh enzyme-linked immunosorbent assay(ELISA).The experimental operation carried out in accordance with the kit instructions.2.Detection of proportions of IFN-γand IL-4 positive cells in CD4⁺ and CD8⁺ T cells with flow cytometry(FCM) in CHB patients:There are 8 cases of CHB patients in this study,six cases of healthy donors as control,PBMCs are separated with density gradient centrifugation,and then add medical ozone of five concentrations(4,10,20,40,80μg / mL) in the supernatants of PBMCs,after the medical ozone reacting completely,add protein transporter blockers(Brefeldin A),at 37℃with 5%CO2 incubator cultivate the cells 6 hours,set up medical oxygen(O2),positive(to PHA stimulation),negative and double-complex-hole self-control group.The cell surface molecules CD4 and CD8 and intracellular cytokines IFN-γand IL-4 are stained by different fluorescent dyes labeled antibody.The percentage of IFN-γand IL-4 positive cells of CD4 + and CD8 + T lymphocytes is detected with flow cytometry (FCM).The results are analyzed using CellQuest software.3.In this study,2×8 factorial design is used,which compared the overall differences of two groups(CHB patients and healthy volunteers) or eight kinds of different treatment(5 experimental groups and 3 control groups). Differences in the overall comparison between the two groups or eight different treatment groups are compared by factorial design analysis of variance data;the overall differences of treatment group within the two groups is in comparison by one-way ANOVA;between two groups comparison of each experimental group with control group use two independent samples t test;comparisons in each experimental group with control group use paired t test.All data are analyzed using SPSS 13.0 software for statistical analysis,and the testing standard less than 0.05(P＜0.05) is considered significant difference.Results1.The concentrations and percentages of IFN-γand IL-4 of CD8 and CD4 positive T cells were significantly higher in positive control group than that of other treatment groups(P＜0.05).2.The IFN-γconcentration of CHB patients was significantly higher than that of healthy volunteers(F = 12.097,P = 0.001),while IL-4 concentration was slightly lower than that of healthy volunteers in the overall level,though there was no significant difference(F = 1.822,P = 0.179).The overall level of IFN-γand IL-4 concentrations between each treatment groups was significantly different(P＜0.05) for both CHB patients and health volunteers. Low concentration(4μg/mL,10μg/mL) Ozone can slightly stimulate IFN-γand IL-4 secretion,while high concentration(40μg/mL,80μg/mL) has inhibitory effect.3.For CHB patients,the proportion of IFN-γ-positive cells within CD8 positive T cells was significantly higher than that of healthy volunteers(F=5.101,P= 0.026),and the proportion of IFN-γ-positive CD4 T cells was higher than healthy control group(except the positive control group),though there was no significant difference(F=0.034,P = 0.854).The percentage of IFN-γ-positive cells within CD8 positive T cells was significant both in CHB patients and healthy donors(P = 0.001 and P = 0.043),and the percentage of IFN-γ-positive cells within CD4 positive T cells was not significant(P＞0.05). The percentage of IL-4 -positive cells within CD8 and CD4 positive T cells were not significant(P＞0.05) in the overall between CHB patients and healthy donors,and there is no significant trend.The percentage of IL-4 -positive cells within CD8 and CD4 positive T cells were not significant(P＞0.05) for CHB patients,while that was significant for healthy donors(P = 0.019 and P = 0.009).When medical ozone concentration increased,the proportion of IFN-γand IL-4 positive cells gradually decreased,which shows that low concentrations of medical ozone had a slight stimulating effect on cytokine secretion of PBMC,while high concentrations inhibited it.Conclusions1.Low concentration of medical ozone in patients with CHB can enhance the function of cytokines secretion in PBMC,strengthens the body's cellular immune responses and will help to clear the virus;while high concentration of medical ozone shows inhibitory effect for the secretion of cytokines in PBMC.This discovery provides a theoretical basis for ozone application in clinical treatment.The effect of medical ozone on HBV-specific cellular immunity remains to be further researched.2.Effects of medical ozone on cytokines secretion of PBMC are closely related to ozone dose and the ozone response medium,and that is worth concerning.3.Even though this study found a very interesting phenomenon,it still needs many basic research,as well as multi-center randomized controlled clinical trial to further confirm the role of ozone.Medical ozone treatment of viral hepatitis is a new attempt and ozone treatment is an endless exploration.4.Though some comparisons could not form a significant difference,and some conclusions are not yet certain enough.That was possibly owing to our small sample.So we still need to carry out larger sample studies to get more meaningful results.