The Study On The Immunologic Mechanism Of Antiviral Therapy Of Shu Gan Jian Pi Bu Sheng Fang On CHB Patients And HBV Transgenic Mice

1 Investigative presumptionThe mechanism of chronic hepatitis B is concerned with immunologic inadequacy, forexample, the hyporesponsiveness of T lymphocyte subset, the lower of NK activity and thelower level of Th1 cytokine, virus content of high titer in vivo and the immune tolerance ofinfected cell. Now antiviral drug to eradicate HBV has not been found yet, and the immunetolerance of HBV exactly existing on which reaction link or committed step has not beenidentified. Chinese crude drug complex prescription had favorable regulation on immunefunction of CHB organism, and had effectively facilitated the clearance of HBV. From theassociativity of CHB virus levels and immune state of the organism, now there are fewreports about the studies on Chinese crude drug complex prescription (SHU GAN JIAN PIBU SHENG FANG, SGJPBSF) on the antiviral therapy and immunological regulation forCHB patients and HBV transgenic mice.2 AimsWe observed the associativity of virus DNA levels and immune function of the organismon CHB patients using SGJPBSF combination with ADV pre-treatment and post-treatmentin this topic, studied antiviral therapy, immunological regulation and the curative effect ofcombination of TCM with Western medicine, offered the new thought and experimentalevidence for prevention and therapy of CHB. We used the vitro cell model to explore themechanism and significance of the traditional Chinese medicine inhibiting the HBVreplication too. On the basis of formerly study and research, we observed theimmunological regulation and antiviral therapy of SGJPBSF on HBV transgenic mice toexplore the curative effect of traditional Chinese medicine for HBV and functionarymechanism.3 Methods 3.1 the immunologic regulatory mechanism of SHU GAN JIAN PI BU SHENG FANGon CHB patients51 CHB patients with stagnation of liver-QI with deficiency of the spleen andasdthenic splenonephro-yang were divided into Group A, B, C according to randomnumber. A was SGJPBSF group, B was SGJPBSF plus ADV group, and C was ADV group.Each group had 17 patients. Group A was administrated SGJPBSF on the basis of theaccording to the sign and the protecting from the liver, 6 months each medication. Group Bwas administrated SGJPBSF plus ADV on the basis of the according to the sign and theprotecting from the liver. Group C was administrated ADV on the basis of the according tothe sign and the protecting from the liver. The liver function, immune function and virologyindex of all patients were detected in per-treatment and in post-treatment 6 months. Weobserved the clinic sings and symptoms of patients. The serum levels of ALT, AST, STBand A/G was detected in automatic biochemistry meter by routine biochemistry method.The serum HBV DNA titers were detected by fluorescent quantitation PCR. The serum ofHBsAg, HBeAg and anti-HBe were detected by HBV two half ELASA. The T lymphocytesubset and NK of the peripheral blood were detected by fluorescent-labeled antibody inflow cytometry. IL-2 was detected by radioimmunity.3.2 the anti-effect experiment of SGJPBSF in vitroThe 2.2.15 cell line was used as cell model. SGJPBSF was diluted into 7 dosage groups.At the same time cell control group was set. After 8 days drug administration, the HBsAgand HBeAg in the supernatant were tested with ELISA assay. The cytotoxicity of drug wasevaluated with the MTT assay, and the IC50 (50%inhibiting concentration) and TI(therapeutic index) of SGJPBSF to antigen were calculated.3.3 acute toxicity experiment of SGJPBSF to miceBefore the experiment, BALB/C mice had been randomly divided into six groups,SGJPBSF 160g/kg/d, 80g/kg/d, 40g/kg/d, 20g/kg/d, 10g/kg/d and normal control (isodosenormal sodium) to definite the safety dose range of different density SGJPBSF. After allanimals were fasted about 16 hours they were intragastricly administrated one time. Weobserved them for 5 days, recorded daily toxic symptom and death information. The deadmice must be autopsied in time and the pathological changes must be noted. The tissuemust be checked if these pathological changes were observed by naked eye. The number ofdeath, piloerection, tired to lie down, pale auricle or congest, exorbitism, walking haltingly,urination and defecation, plegia, cataphora, convulsion, carriage and anhelation at the sametime were observed. At last, all animals were sacrificed by decapitation.3.4 the immunopathogenesis intervention of SGJPBSF to HBV transgenic mice The experimental animals were divided into six groups. The normal control group were6-8 weeks age, 10 non-HBV transgenic male mice, those were intragastric administratedisodose, sterilizing, iso-osmia normal sodium. 50 HBV transgenic male mice of SPF grade,those HbsAg were positive, were randomly divided into 5 groups, model group(wereintragastric administrated isodose, sterilizing, isooosmia normal sodium), low-dose groupof SGJPBSF (were intragastric administrated 10g crude drug /kg body weight /d),middle-dose group of SGJPBSF (were intragastricly administrated 20g crude drug/kgbody weight/d), high-dose group of SGJPBSF (were intragastric administrated 40g crudedrug/kg body weight/d), and ADV group (were intragastric administrated 50g ADV/kgbody weight/d), 10 mice each group. Everyday they were administrated on time, for 21days consecutively. They were fasting for 12 hours before their blood samples were taken.Their blood samples were taken in the time points of T0, T10, T21 and P3 in accordancewith the time point of drug intragastric administration and they were sacrificed after at lastsampling. The serums were collected by centrifugalization, and were keeped under 80℃.The serum HBsAg was detected by ELASA. The serum HBV DNA titers were detected byfluorescent quantitation PCR. The spleens were taken and unicell suspension of the spleenswas prepared by secundum artem. The T lymphocyte subset of the spleen was detected byfluorescent-labeled antibody in flow cytometry. The cytokine was detected in the spleencultivation supematant, for example, IL-2 was detected by radioimmunity, and IFNγwasdetected by ELISA. The liver tissues of obviously pathological changes were taken to dopathology slice and HbcAg immunohistochemistry.4 Results4.1 the immunologic regulatory mechanism of SHU GAN JIAN PI BU SHENG FANGon CHB patientsThe symptoms of hypochondrium distending pain, abdominal distention, loose stool,feeling chill, cold limbs, depression, discomfort, tired body, debilitation, psychroalgia inthe lower abdomen, lumbar and knee in Group B were superior to other groups. Inclinically therapeutic effect, there was no significant difference in total effective rate ofthree groups. The liver functional recoveries of alanine aminotransferase, aspartateaminotransferase, total bilirubin, A/G were good in three groups, but the ALTnormalization rate in Group B (SGJPBSF plus ADV) was higher than that of in Group A(SGJPBSF) and Group C (ADV). There was significant difference in three groups about therate of negative transfer of HBV DNA, and the rate in Group B was highest. There weresignificant differences of the serum titers of HBV DNA in three groups between the pre-treatment and post-treatment, especially in Group B. Those results suggest that theeffect of inhibiting viral replication in combination medication of TCM with Westernmedicine was superior to single Chinese crude drug and single western medicine. Threetherapeutic methods all could increase the ratio of CD3~+, CD4~+ cell and decrease the ratioof CD8~+ cell, especially increasing the ratio of CD3~+ in Group A and Group B, increasingthe ratio of CD4~+ in Group A, decreasing the ratio of CD8~+ in Group B were verysignificant. When using ADV treatment, the effect of increasing the CD3~+, CD4~+ cell ratioand decreasing the CD8~+ cell ratio in single was mild. The raising of NK cytoactive andIL-2 secretion in Group B was very significantly. Group C had the norientation activationon NK, but had no significant effect on IL-2. Opposite, Group A had no significantnorientation activation on NK, but had significant upgrading effect on IL-2. The increasingof CD4~+ and NK and the decreasing of CD8~+ T cell ratio were significantly positivecorrelation to the decreasing of HBV DNA titers between the pre-treatment andpost-treatment in Group B. The increasing of CD4~+ cell ratio was significantly positivecorrelation to the decreasing of HBV DNA titers between the prior treatment andpost-treatment in Group A. There was one example drug adverse reaction in Group B andGroup C respectively.4.2 the anti-effect experiment of SGJPBSF in vitroThe TC50 of SGJPBSF to 2.2.15 cell line was 30.28mg/ml. The IC50 of SGJPBSF toHBsAg, HBeAg was 25.96mg/ml and 36.46mg/ml respectively, and the TI of SGJPBSFwas 1.17 and 0.83 respectively. It suggested SGJPBSF could inhibit the HBsAg express of2.2.15 cell line, but it could have toxic effect and could not inhibit the HBeAg express.4.3 acute toxicity experiment of SGJPBSF to miceThe mice of SGJPBSF 160g/kg/d showed some adverse effect. From the second day, themice showed seldom eating, seldom drinking water, debilitation, moving slowly, tiring tolie down, loose stool, and so on. One mouse died in 4th day and two mice died in the 5thday. Under the anatomy, the livers of three died mice were madder red and their stomachintestines were swelling and twist, possibly because too high pharmacal density could causedropsy, hemorrhage and necrosis of murine liver. The general condition was good afteradministration in the mice of SGJPBSF 80g/kg/d, 40g/kg/d, 20g/kg/d, 10g/kg/d, obviouslytoxic reaction was not found, and there was not dead animal. So all dose used in followexperiment was below 80g/kg/d.4.4 the immunopathogenesis intervention of SGJPBSF to HBV transgenic miceThe HBsAg sero-nagative rate of HBV Tg in high-dose group of SGJPBSF,middle-dose group and ADV group had no significant difference on T10, T21 after administration and P3. ADV could reduce the serum HBV DNA titers of HBV Tg, butthere was markedly rebound on 3rd after drug withdrawal. High-dose SGJPBSF andmiddle-dose both decreased the serum HBV DNA titers of HBV Tg, especially high-doseSGJPBSF, although the serum HBV DNA titers were not decreased to the level of ADVgroup, there was significant difference between the pre-treatment and post-treatment, andthere was no rebound after drug withdrawal, its effect was steady for a long time. The liverpathology change of model HBV Tg was similar to the appearance of the clinical lightlyhepatitis, and along with the increasing SGJPBSF density, the liver inflammation wasrelieved. The hepatic plate of high-dose SGJPBSF lined up in order and the degree oflymphocyte soakage was relieved. But there was still sporadic lymphocyte soakage in theconverge duct of hepatic tissue of ADV group, there was no fibroplasias, but in the centralveins surrounding there was some lightly hepatocyte adipose degeneration. Themiddle-dose, high-dose SGJPBSF and ADV all could decrease the HbcAg express in thehepatic tissue, especially high-dose SGJPBSF. SGJPBSF could up-regulate the ratio ofCD3~+, CD4~+ and CD4~+/CD8~+ cells of HBV Tg, and could down-regulate the ratio of CD8~+T cell. So it had the norientation effect of T lymphocyte immunological regulationespecially high-dose SGJPBSF. ADV had the effect of increasing cytoimmunity of HBVTg too (for example, CD3~+ cell percentage), but its function was weaker than the traditionalChinese medicine. The low-dose, middle-dose, high-dose SGJPBSF and ADV all couldincrease the serum level of IL-2 and IFN-γof HBV Tg, and there was significant differenceas compared to the model group. And the level of IL-2 and IFN-γin middle-dose, high-doseSGJPBSF had far surpassed the normal control after treatment. The ratio of CD3~+ T cell andthe level of IFN-γwere significantly positive correlation to the decreasing of HBV DNAtiters between the pre-treatment and post-treatment in middle-dose. The ratio of CD4~+,CD8~+ and CD4~+/CD8~+ T cells were significantly positive correlation to the decrease ofHBV DNA titers between the pre-treatment and post-treatment in high-dose.5 Conclusions5.1 SGJPBSF was fine immunomodulator, and it could significantly strengthen thesecretion of Th1 cytokine and could improve the lower immune function.5.2 It suggested that SGJPBSF could clear up and inhibit HBV through regulating theimmune function of organism. It was consistent with TCM theory of strengthening bodyresistance to eliminating pathogen.5.3 ADV could have the better effect of decreasing the serum HBV DNA titers thanSGJPBSF, but SGJPBSF could have the better effect of regulating immune function than ADV. United medication of SGJPBSF plus ADV is shown to have the better clinicaleffect of improving symptoms, inhibiting the HBV duplication and regulating theabnormal immune function in CHB patients.