Inhibition of hepatitis B virus via selective apoptosis modulation by Chinese patent medicine Liuweiwuling Tablet

BACKGROUND Liuweiwuling Tablet(LWWL)is a Chinese patent medicine approved for the treatment of chronic inflammation caused by hepatitis B virus(HBV)infection.Previous studies have indicated an anti-HBV effect of LWWL,specifically in terms of antigen inhibition,but the underlying mechanism remains unclear.AIM To investigate the potential mechanism of action of LWWL against HBV.METHODS In vitro experiments utilized three HBV-replicating and three non-HBV-replicating cell lines.The in vivo experiment involved a hydrodynamic injectionmediated mouse model with HBV replication.Transcriptomics and metabolomics were used to investigate the underlying mechanisms of action of LWWL.RESULTS In HepG2.1403F cells,LWWL(0.8 mg/mL)exhibited inhibitory effects on HBV DNA,hepatitis B surface antigen and pregenomic RNA(pgRNA)at rates of 51.36%,24.74%and 50.74%,respectively.The inhibition rates of LWWL(0.8mg/mL)on pgRNA/covalently closed circular DNA in HepG2.1403F,HepG2.2.15 and HepG2.A64 cells were 47.78%,39.51%and 46.74%,respectively.Integration of transcriptomics and metabolomics showed that the anti-HBV effect of LWWL was primarily linked to pathways related to apoptosis(PI3K-AKT,CASP8-CASP3 and P53 pathways).Apoptosis flow analysis revealed that the apoptosis rate in the LWWL-treated group was significantly higher than in the control group(CG)among HBV-replicating cell lines,including HepG2.2.15(2.92%±1.01%vs 6.68%±2.04%,P<0.05),HepG2.A64(4.89%±1.28%vs 8.52%±0.50%,P<0.05)and HepG2.1403F(3.76%±1.40%vs 7.57%±1.35%,P<0.05)(CG vs LWWL-treated group).However,there were no significant differences in apoptosis rates between the non-HBV-replicating HepG2 cells(5.04%±0.74%vs 5.51%±1.57%,P>0.05),L02 cells(5.49%±0.80%vs 5.48%±1.01%,P>0.05)and LX2 cells(6.29%±1.54%vs 6.29%±0.88%,P>0.05).TUNEL staining revealed a significantly higher apoptosis rate in the LWWL-treated group than in the CG in the HBVreplicating mouse model,while no noticeable difference in apoptosis rates between the two groups was observed in the non-HBV-replicating mouse model.CONCLUSION Preliminary results suggest that LWWL exerts a potent inhibitory effect on wild-type and drug-resistant HBV,potentially involving selective regulation of apoptosis.These findings offer novel insights into the anti-HBV activities of LWWL and present a novel mechanism for the development of anti-HBV medications.

Simultaneous Determination of Eight Active Components in Liuwei Wuling Tablet Using HPLC

Objective To develop an effective method for simultaneous determination of eight major components in Liuwei Wuling Tablet（LWT）. Methods Reversed phase HPLC method was used with variously improved conditions. Results Salidroside, forsythoside A, specnuezhenide, phillyrin, schisandrol A, schizandrol A, schizandrin A, and schizandrin B in LWT were successfully separated on a Kromasil 100-5-C_（18） reverse phase column（250 mm × 4.6 mm, 5 μm） using acetonitrile -0.1% phosphoric acid（gradient elution） as mobile phase. The detection wavelength was 275 nm with flow rate of 1.0 mL/min, and the column temperature was maintained at 35 ℃. The recovery rate of the method was within the range of 95.13%-104.56% and the precision（RSD） was less than 3% for all eight analytes. All the compounds showed good linearities（r^2〉0.9980） in a relatively wide concentration range. Conclusion Simultaneous quantification of the multiple components by HPLC would be a better strategy for the quality evaluation of LWT.

Spectrum-Toxicity Correlation Study Revealed the Influence of the Nine-Time Steaming and Sun Drying Method on Hepatotoxic Components of Polygoni Multiflori Radix

Objective:Polygoni Multiflori Radix(PM)is a traditional herbal medicine with repeated reports of liver injury events in recent years.We wondered whether the classical processing method,namely,nine-time steaming and sun drying(NSSD),had toxicity-attenuating effects on PM and the relationships between toxicity and times of processing,as well as with the alteration trends of its compounds.Materials and Methods:The chemical fingerprints of different PM extracts were developed using ultra-high-performance liquid chromatography.The spectrum-toxicity correlation between the chemical fingerprints and hepatocellular toxicity was analyzed with multiple correlation analysis.Results:The results suggested that the hepatotoxicity of NSSD processing products markedly decreased with the repeated steaming and sun drying,which was obviously superior to the product processed by the modern method.Comprehensive analysis revealed that the contents of cis-stilbene glycoside and emodin-8-O-β-D-glucoside related to liver injury susceptibility were reduced with the times of NSSD processing,which was consistent with the decreased trend of hepatocellular toxicity.After the five times of NSSD,the contents of them as well as the hepatotoxicity of PM were steady.Moreover,we found that the contents of catechin and physcion declined rapidly after the one time of NSSD and then remained stable until the nine times of NSSD.Based on the fact,they could be utilized to indicate whether PM products were processed by steaming and sun drying.Conclusions:This paper confirmed that the NSSD had a good influence on the toxicity attenuating to PM and found four compounds which could apply for the quality control of PM.