Study On The Anti-hepatitis B Virus Of Crude Extract Of Whole Scorpion And Centipede Polypeptide

Scorpion and centipede have been used as traditional Chinese medicine for thousands of years.Previous studies have shown that their venom is unique resource,which contains vast proteins and peptides which have highly targeted functions.Some venom peptides extracted from scorpion and centipede have antiviral effects,the activity of 5?10 KDa peptide fragments of scorpion and the polypeptide fragments of centipede below 5 KDa attracted our attention.This study investigated the peptides extract from scorpion's tail(peptide extract from centipede head,PEST)and centipede's head(peptide extract from centipede head,PECH)on anti-HBV activity in HepAD38 cells.The main research contents and results are as follows:1.Preparation of peptides preliminary extract from scorpion and centipede by ultrafiltration.After homogenizing the tails of scorpion and heads of centipede at 4?,used ultrafiltration tubes to perform ultrafiltration.The scorpion took the ultrafiltrate below 10 KDa and above 5 KDa as PEST,and the centipede took the ultrafiltrate below 5 KDa as PECH.After concentration in a vacuum freeze dryer,the concentration of the PEST and PECH measured were 6 mg/mL and 5.5 mg/mL,respectively.The heavy metal content test results showed that the heavy metal content of preliminary extracts did not exceed the standard.2.Cell cytotoxicity measured by MTT method.Inoculated HepAD38 cells in 96-well plates with a density of approximately 6×10~4 cells/well.After 2 days of culture,removed tetracycline and added PEST(0.125,0.25,0.5 and 1 mg/mL),PECH(0.125,0.25,0.5 and 1 mg/mL),set blank control group to add only cell culture medium,after 2 days of culture,replaced with new drug-containing culture medium and blank culture medium,continued to culture for 3 days,and then used MTT method to measure the absorbance at 570 nm of microplate reader.The seeding density of HepG2 cells was about 1×10~4 cells/well.After 12 hours of incubation,PEST and PECH were added separately.After the corresponding incubation time(24,48,72 h),the absorbance was measured by MTT method.The experimental results showed that the cell survival rate is more than 70%when PEST and PECH act on HepAD38 cells or HepG2 cells.PEST and PECH had no obvious toxicity to liver cancer cells HepAD38 and HepG2.3.Anti-hepatitis B virus effect of PEST and PECH in HepAD38 cells.After 2 days of inoculation with HepAD38 cells,tetracycline was removed,set up PEST group,PECH group,0.25?mol/L Lamivudine(LAM)group,0.01?mol/L Entecavir(ETV)group,6.86?mol/L tetracycline(Tetracycline,TEL)group and blank cell culture medium as control group.After cultured for 2 days,it was replaced and continued to culture for 3 days.Aspirated the supernatant and harvested the cells.Used HBV nucleic acid amplification(PCR)fluorescence quantitative detection kit to detect HBV DNA content of cell supernatant,used ELISA kit to detect HBV surface antigen(Hpatitis B surface antigen,HBsAg)and HBV early antigen(Hepatitis B early antigen(HBeAg)content,used fluorescence quantitative PCR to detect HBV X/S/preC/P gene expression after extracted total RNA from cells,used western blot to detect HBV core protein synthesis.The results showed that compared with control group,1 mg/mL PEST and 1 mg/mL PECH had best effect on reducing HBV DNA copy number,1mg/mL PEST significantly inhibited the secretion of HBsAg(P<0.05)and HBeAg(P<0.01),1mg/mL PECH significantly inhibited the secretion of HBsAg and HBeAg(all P<0.01),1mg/mL PEST and 1mg/mL PECH down-regulated the relative expression level of HBV P gene mRNA separately(all P<0.05);PEST has almost no effect on core protein synthesis,PECH can inhibit HBV Core protein synthesis,and the inhibitory effect is close to the positive control drugs LAM and ETV.In summary,the following conclusions:PEST and PECH had anti-HBV effects in HepAD38 Cell.The mechanism may be PEST and PECH inhibiting HBV P gene relative expression,PECH reducing HBV core protein synthesis.