Experimental Study On The Effect Of JIA WEI SI NI SAN Decoction Anti-DHBV 3TC Persister And The Effect Of Anti-inflammatory And Anti-lipid Peroxidation

ObjectiveTo study the effect of anti-HBV, anti-DHBV 3TC persister and the curative effect of anti-inflammatory and anti-lipid peroxidation of the decoction JIA WEI SI NI SAN (JWSNS) decoction. Make sure the anti-HBV and restrain immunological liver injury mechanism and target of JWSNS decoction. And to provide scientific gist to the widely use of JWSNIS in the clinic. Experimental contents1. In-vivoexperimental study of the anti-DHBV effect of different combination of SI NI SAN (SNS) decoction.2. In-vivo experimental study of the anti-DHBV effect of JWSNS decoction.3. The detection of the DHBV 3TC persister.4. In-vivo experimental study of the anti-DHBV 3TC persister effect of JWSNS decoction.5. In-vitro experimental study of the anti-HBV effect of JWSNS decoction.6. Mice acute toxicity test of JWSNS.7. Protective effects of JWSNS on liver injury induced by Concanavalin A (ConA) in mice.8. Protective effects of JWSNS on liver injury induced by BCG+LPS in mice. Methods1. The 1 day old congenitally infected duck was divided to 5 groups randomly, every group was 6 ducks. Namely DHBV control group, 3TC group, Yuan Fang group, Jia Wei group and Qu Zhi group. The intragastric administration dose of SNS decoction alcohol extractive were 20g ? kg^-1 ? d^-1, 3TC was 200mg ? kg^-1 d^-1. The control group was not treated. The serum was collected in the time points of T0, T5, T10 and P3 in accordance with the time point of drugintragastric administration. The quantity of DHBV in these specimens was evaluated by dot-blot assay.2. The 1 day old congenitally infected duck was divided to 5 groups randomly, every group was 6 ducks. Namely DHBV control group, 3TC group, JWSNS large dose group, JWSNS middle dose group and JWSNS small dose group. The intragastric administration dose of JWSNS alcohol extractive were 20g ? kg"' ? d"', lOg ? kg"1 ? d"1 and 5g ? kg"1 ? d"1 respectively;3TC was 200mg ? kg"' d"';the control group was not treated. The serum was collected in the time points of TO., T5, T10 and P3 in accordance with the time point of drug intragastric administration. The quantity of DHBV in these specimens was evaluated by dot-blot assay.3. 3 sample of DHBV positive serum was taken from 2 congenitally infected ducks and the 3TC tolerated duck. Extracted the DHBV DNA, used a pair of primer which could amplify the complete genome of DHBV and amplified the DHBV DNA by PCR. 10ul output of PCR was electrophoresis by 0.8% agarose gel. Observed the result under the UV detecting instrument.4. 1 day old duck was infected DHBV DNA 3TC persister serum from the foot vine. The postnatal infected duck was bolting by dot-blot, and was used as animal model. After 13 days infection, the ducks was divided into 5 groups randomly, every group was 5-6 ducks. Namely DHBV control group, 3TC group, ADV group, JWSNS large dose group, JWSNS middle dose group and JWSNS small dose group. The intragastric administration dose of JWSNS alcohol extractive were 20g ? kg"1 ? d"', lOg ? kg"' ? d"1 and 5g ? kg"1 ? d"' respectively. 3TC was 200mg ? kg"1 d"', ADV was lOOmg ? kg"' d"'. Totally administrated 10 days. The control group was not treated. The serum was collected in the time points of TO, T5, T10 and P3 in accordance with the time point of drug intragastric administration. The quantity of DHBV in these specimens was evaluated by dot-blot assay.5. Used the 2.2. 15 cell line as cell model. JWSNS was diluted into 7 dosage groups. At the same time set cell control group. After 8 days drug administration the HBsAg and HBeAg in the supernatant was tested with ELISA assay. The toxicity of drug to the cell was evaluated with the MTT assay. Computed the TC50, IC50 and TI of JWSNS.6. NIH mice was divided into 5 groups randomly, every group was 10 mice. The dosage was 160g ? kg"1 ? d"1, 80g ? kg"1 ? d"1, 40g ? kg"' ? d"1, 20g ? kg"1 ? d"1,lOg ? kg"' ? d^' (corresponding 80 times, 40times, 20times, lOtimes, and 5 times dose per day per kilogram body weight drug consumption of 60kg adult). After 16 hours abrosia, intragastric administrated drugs one time and observed 14 days. Recorded the toxicity appearance and dead condition. Execute the mice by decapitation.7. The liver injury mice induced by ConA was used as animal model. These mice was divided into 6 groups randomly, namely normal control group, model control group, Bifendate control group, JWSNS large dose group, middle dose group and small dose group. The normal control group and model control group was intragastric administrated with normal saline 0.2ml every mice every time. The intragastric administration dose of Bifendate was 150mg ? kg"1 ? d"';JWSNS alcohol extractive were 40g ? kg"1 ? d"', 20g ? kg"1 ? d"1 and lOg ? kg"1 ? d'respectively. After 3 times intragastric administration, to excise eyeball and collected blood, separated serum to test ALT, AST;IFN-y, TNF-a;SOD, MDAandNO. Execute the mice by decapitation. Fixation the liver by 10% neutral formalin, dyed by HE means, patho-observed by light microscope. And then apply SPSS10.0 to statistical analysis the experimental data.8. The liver injury mice induced by BCG+LPS was used as animal model. These mice was divided into 6 groups randomly, namely normal control group, model control group, Bifendate control group, JWSNS large dose group, middle dose group and small dose group. The normal control group and model control group was intragastric administrated with normal saline 0.2ml every mice every time. The intragastric administration dose of Bifendate was 150mg ? kg"' ? d"';JWSNS alcohol extractive were 40g ? kg"' ? d"1, 20g ? kg"' ? d"1 and lOg ? kg"' ? d"1 respectively. After 10 days intragastric administration, to excise eyeball and collected blood, separated serum to test ALT, AST;IL-6, TNF-a;SOD, MDA and NO. Execute the mice by decapitation. Fixation the liver by 10% neutral formalin, dyed by HE means, patho-observed by light microscope. And then apply SPSS10. 0 to statistical analysis the experimental data.Result1. The virus titer of Jia Wei group was decrease markedly at T10, but the virus titer was increase again at P3, which indicated that Jia Wei decoction could anti-DHBV in-vivo. But Yuan Fang and Qu Zhi Fang didn' t have the effect of anti-DHBV. The virus titer of 3TC group was decrease markedly at T5 andT10, but but the virus titer was increase again at P3 too. The result of this experiment indicated that SNS added invigorate the spleen and dehygrosis herbs had the effect of anti-DHBV.2. The virus titer of JWSNS large dose group decreased markedly at T5, it was still at the low lever at T10, but the virus titer was increase again at P3;the virus titer of middle dose group and small dose group didn' t decrease at the hold administration course. The virus titer of 3TC group was decrease markedly at T5 and T10, but the virus titer was increase again at P3 too.3. After extracted the DHBV DNA and amplified it by PCR, 3 sample of the output of PCR was electrophoresis by 0.8% agarose gel, every one of it could be amplified about 3.0 kb DNA fragment.4. The virus titer of ADV group decreased at T5 and T10, it was still at the low lever at P3, but there was not statistic difference between TO, and it showed that the virus titer had increased, which indicated ADV couid resist DHBV, but the virus titer will increase again after drug withdrawl just like the other nucleoside retroviridase inhibitor. Large dose JWSNS could resist DHBV, it couldn' t resist DHBV 3TC persister, but we can? discern from the experiment result that the virus titer in the duck was decrease accompany with the time of drug administration extended. This? indicated us that JWSNS maybe has the effect of resist DHBV 3TC persister. Because the effect of Chinese herb to work slowly, so we can extend the time of administration or adjust the dosage or combination of JWSNS to go further study.5. The TC50 of JWSNS to 2.2.15 cell line was >200mg/ml, the IC50 of JWSNS to HBsAg, HBeAg was < 3. 12mg/ml and 43. 68mg/ml respectively, the TI of JWSNS " was >64. 12 and >4. 58 respectively.6. After administrated, the common condition of mince was well, color pattern was normal, the body weight was increase, appetite and defecation was no abnormality seen, didn' t detect evident toxic reaction, and there is no mice die.7. ATL, AST of JWSNS large and middle dosage were decrease, pathological change of hepatic tissue was lessen. Serum IFN-y and TNF-a contents of JWSNS treatment groups was lower than the model control group obviously, which indicated can resist liver injury by inhibited the activation of T celland the release of IFN- Y and TNF- a . Large and middle dose of JWSNS also can increase the SOD and decrease the MDA and NO of the ConA induced liver injury mice.8. ATL, AST of JWSNS large and middle dosage were decrease, pathological change of hepatic tissue was lessen. The contents of IL-6 and TNF- a of JWSNS large dose group was lower than the model control group obviously , which indicated can resist liver injury by inhibited the activation of T cell and the release of IFN-Y and TNF-a . Large and middle dose of JWSNS also can increase the SOD and decrease the MDA and NO of the BCG+LPS induced liver injury mice.Innovation1. Used the DHBV DNA 3TC persister strong positive serum as toxin to infect the 1 day old duck to make postnatal infected animal model. Use this model to observe the in-vivo anti-DHBV 3TC persister effect of drugs. And the animal model was established successful.2. The in-vivo experiment approved that ADV can inhibit DHBV 3TC persister, it is concord with the report of ADV has the anti-HBV 3TC effect at drug fast patient.3. Apply the treatment and decoction of Traditional Chinese Medicine at intervention 3TC persister, and used it in the in-vivo experiment. The combination principle of JWSNS is disperse the depressed liver-energy and invigorate the spleen, clear away heat and dis pelling dampness, this principle is different from the principle of invigorate the kidney, promoting blood flow, neutralize poison. And the decoction used frutex abri which is the medical herbs of Guangdong, add new substance for the modern pharmacology research of Lingnan.4. Tentative confirmation the protect mechanism of JWSNS to the liver injury was close correlated with anti-lipid peroxidation and inhibited the production of NO and decreased contents of IFN-y, TNF-a and IL-6.Conclusion1. 20g ? kg"1 ? d'1 dosage of JWSNS can inhibit HBV DNA in-vivo and in-vitro. ADV could resist DHBV, it is concord with the report of ADV has the anti-HBV 3TC effect at drug fast patient. The curative effcet of JWSNS to DHBV 3TC persister is not obvious. The in-vitro experiment showed that JWSNS could inhibit HBsAg and HBeAg which secreted by 2.2.15 cell line.2. The LD?, of JWSNS is >160g ? kg"1 ? d'1. Large dose and middle dose of JWSNS could get the favorable effect of protect liver, degraded aminopherase, anti-inflammatory, anti-lipid peroxidation and to improve the pathology change of liver tissue in two kinds of immunological liver injury mice. The protect mechanism of JWSNS to the liver injury was close correlated with anti-lipid peroxidation and inhibited the production of NO and decreased contents of IFN-Y, TNF-a and IL-6.