摘要
慢性乙型肝炎(Chronic Hepatitis B,CHB)是一种全球性广泛传播的传染病,它以持续性HBV复制所致的不同严重程度的肝脏炎性病变为特征,其中部分患者最终可能发展为肝硬化、肝细胞肝癌或肝功能衰竭。乙型肝炎病毒(Hepatitis B virus,HBV)感染对全球的公共健康是极大的挑战。因此研发治疗慢性乙型肝炎的有效药物具有重要意义。
本课题利用无机合成方法合成了小分子含钒的化合物POM-2,并利用单晶X-射线衍射方法对其结构进行表征;采用细胞生物学、分子生物学及毒理学实验方法,分别检测了POM-2的体内和体外抗HBV的活性及毒性,并探讨POM-2的药物作用机制。在体外研究中以Hep G2.2.15细胞为模型,检测了POM-2对HBeAg和HBsAg分泌的抑制作用、对mRNA和细胞内以及细胞外DNA合成的活性。体内药物活性研究中以1日龄北京鸭为实验模型,检测了POM-2对DHBV DNA合成的抑制作用和POM-2对减轻肝脏炎症的作用。按照新药毒理学评价方法,检测POM-2体内和体外毒性。
研究结果显示新型多酸化合物POM-2细胞毒性较低,能够有效降低细胞培养上清液中HBeAg和HBsAg的含量,对细胞内和细胞外的DNA合成均有抑制作用,对HBV mRNA也有一定的抑制作用。体内实验结果显示,POM-2明显降低了鸭血清中的乙肝病毒载量,抑制了病毒的合成。病理实验结果表明POM-2减轻了肝脏炎症反应。体内毒理学实验结果显示POM-2属低毒化合物。本研究是在本课题组对多酸化合物抗病毒研究基础上,发现含5价钒离子多酸化合物具有抗HBV的活性,为新型非核苷类抗乙型肝炎药物开发和进一步的研究提供了实验依据。
Chronic hepatitis B is a kind of global widespread infective diseases,it has characterized by inflammatory lesions of the liver of the different severity degree due to persistence HBV copy,some of these patients may eventually become cirrhosis, hepatocellular carcinoma or liver function failure. Hepatitis B virus infection is a great challenge on the global public health, therefore, it is great significance to research and development effective drugs for the treatment of chronic hepatitis B.
The subject used cell biology, molecular biology, virology and toxicology test methods, invest the pharmacological effects of polyoxometalate POM-2 respectively from the synthesis and characterization of POM-2、anti-HBV activity in vitro and in vivo experiment and evaluation of safety. HepG 2.2.15 cell lines were used as a mode in vitro experiment to detect the inhibition effect of the POM-2 on the secretion of HBeAg and HBsAg, and on synthesis activity of mRNA and intracellular and extracellular DNA. In vivo studies to duck for the experimental model, test the effect of POM-2 on DHBV DNA synthesis. Conducted for the POM-2 toxicity in vitro and in vivo on the basis of studies in vivo and in vitro activity.
1 Characterization and structure of POM-2
POM-2 is a novel polyoxometalate pertaining to heteropolyacid synthesised by our subject group. The adicity of implicit vanadium is five, the molecular weight is 338, easy to dissolve in the water, high thermal stability, pH stability in a wide range. There is one Ce (II) ion belong to rare-earth element, two VO3- ions, three configuration water molecules, two lattice water molecules in its independent structure. Each rare-earth metal ion was coordinationed by four oxygen atoms from four VO3- ligands. Each of nine coordination Ce ion and consecutive four VO3- connected to form two dimensional layered wave-shaped structure. There are relatively strong hydrogen bonds between wave layers,these hydrogen bonds linking the consecutive two dimensiona layers to constitute the three dimensional supermolecule architecture.
2 Anti-HBV activity in vitro
The cytotoxicity of POM-2 was detected by MTT assay, the median toxic concentration of cells was calculated by linear regression equation. The results showed the CC50 of cells was 2899.21μg/mL, pertaining to low toxicity drugs. This experiment used hepatitis B virus e (s) antigen test kit to test effect on compounds on HBsAg and HBeAg in the supernatant, the results showed that inhibitory effect of POM-2 on secretion of HBsAg and HBeAg increased with the concentration of POM-2 increased. Inhibitory effect of POM-2 on secretion of HBeAg is up to highest concentration in the 5th day after treatment,which was 80μg/mL,the inhibitory rate was 91.8%; For secretory volume of HBsAg, the inhibitory effect was enhance with time extend, the concentration was 80μg/mL,the inhibitory rate was 90%; while the LAM group of the positive control group have no obvious impact on the content of HBeAg and HBsAg, compared with negative control group, significant difference was observed in the experimental group (P<0.05). The inhibitory effect of POM-2 on HBV DNA and intracellular HBV mRNA in the supernatant were detected by fluorescent quantitation Roche HBV PCR assay ,the results showed that inhibitory effect of POM-2 on extracellular HBV DNA and intracellular HBV mRNA increased with the concentration of POM-2 increased, there was good dose-effect relation between drug concentration and inhibition rate. The highest inhibitory rates of POM-2 on extracellular HBV DNA is 87.30% in its concentration of 40μg/mL, which is better than lamivudine of positive control group in its concentration of 50μg/mL ( 57.81%). However, it could not increased significantly inhibiting effect with continued high concentration of drug. The IC50 was 13.42μg/mL and IC90 was 59.47μg/mL. The inhibitory rate of POM-2 on intracellular HBV mRNA was up to 59.01%, contiguity to the positive control group. IC50 of intracellular HBV mRNA was 58.2μg/mL, which showed that POM-2 have effect on the synthesis of HBV, and there was a dose-effect relationship between the concentration and inhibitory effect ,but also that it has a certain influence for reverse transcription of HBV .
3 Anti-HBV activity in vivo
Infected with DHBV-DNA on one-day-old Beijing ducks, tested DHBV-DNA serum on the 7th day after treatment, the results showed that 30 ducklings were all positive, DHBV-DNA content of DHBV-DNA infected ducks tended to stable after physiological saline by oral, there was no difference between experiment with different points in time in the course of experiments and before experiments. It demonstrated that DHBV-DNA serum level of the DHBV-DNA infected ducklings was stable in the entire process within 32 days.
Infected with DHBV-DNA on one-day-old Beijing ducks, test DHBV-DNA serum on the 7th day after treatment, and then experimentize, the ducks were tested DHBV-DNA serum in the 10th days and the 20th days after treatment and 2 weeks after drug withdrawal. The results showed that compared with former administration in OD value, DHBV-DNA serum level was observed decreased significantly in the LAM (50mg/kg) group(P<0.01) in the 10th and 20th days after treatment and 5 days after drug withdrawal. Compare with the physiological saline control group,inhibitory rate of DHBV-DNA serum in the 10th days and the 20th days after treatment and the 5th days after drug withdrawal was observed significant difference(P<0.01). The DHBV-DNA serum contents increased obviously after drug withdrawal,which showed that there is a rebound after drug withdrawal when treatment for Hepatitis B with LAM ; the three doses groups in the experimental group, no significant difference was observed in low and middle dose group in the 10th days and the 20th days after treatment and in the 5th days after drug withdrawal.Compared with previous administration, DHBV-DNA serum level in high dose group was obsvered significant difference in the 10th days and the 20th days after treatment (P<0.05,P<0.01)。Compare in the three doses group,the physiological saline group and the LAM group each other: no significant difference was observed between high doses group and LAM group and between low dose group and physiological saline group(P>0.05), but there was significant difference in the rest every two groups(P<0.05). It noted that DHBV-DNA level of the experimental group decreased significantly with increased doses, inhibitory rate of the high dose group in the 20th days is up to the max value of 51%, although there was certain inhibitory effect on the DHBV-DNA in the middle dose group, but compare with the positive control group, the inhibition rates was lower. Which noted that POM-2 have the action of anti-HBV when it reach a certain dose in the body. Results from dot blot hybridization showed that DNA content of high dose group and LAM group were significantly decreased, but there was no significant difference between the two groups.
These results showed that DHBV-DNA content in three treatment groups, three time points after treatment by POM-2 changeed smaller, which indicated that DHBV-DNA content was maintain stable after drug withdrawal, treatment group with lamivudine had larger changes and appear rebound after drug withdrawal, but there was no HBV rebound in POM-2 after drug withdrawal. Results of pathology and electron microscopy showed that POM-2 had relief effect on liver inflammation of duck, the effect was similar to lamivudine. Which indicated that POM-2 indirectly improving the situation of liver pathology by inhibiting replication of the virus.
4 Toxicity evaluation of POM-2
The mice were divided into six doses group in acute toxicity test, there were eight mices in each group, half was male and half was female. In the six doses group, the highest dose group of POM-2 was 1256 mg/kg, mice death apeared in the 2th day of experiment. Continue to observe 2 weeks, calculate LD50 of POM-2 was 868.96mg/kg according to Crown's method that had improved. According to poison classification standards of toxicology, POM-2 was low toxic compounds.
The results of feeding rats with 90 days showed that there was no significant difference in each organ coefficient of experimental rats. The results of biochemical indicators showed that there had no changes in liver function and kidney function of rats in each experimental group. The results of blood indicators showed that POM-2 has no impact on the whole on department of leukocytic,department of red blood and hemoglobin, department of platelets.
Results of Ams experiments showed that in the effect of five doses group of POM-2 to four Salmonella typhimurium mutant strains TA97,TA98,TA100,TA102, Ams colonies number of POM-2 was less than two times compared with spontaneous colonies, and had not formed dose-response relationship. The results were negative,it showed that POM-2 has no mutagenic action.
Results of mouse bone marrow PCE nuclear showed that there were significant differences between the experimental group and the positive control group (P<0.05),there was no difference in the experimental group ( P>0.05 ) .Compared with negative control group,obvious significant difference was observed in positive control group(P<0.05),Compared with negative control group,no significant difference was observed in the high, medium and low dose group. It noted that POM-2 have no effect on mouse bone marrow PCE nuclear and POM-2 has no mutagenic action on chromosome.
Test results of mouse bone marrow lymphocyte chromosome aberrations showed that the control group (cyclophosphamide) have damage effect on chromosome,POM-2 have no damage effect on chromosome and have no teratogenic action on the mice,it would not induced chromosomal aberrations.
In a word, POM-2 as a novel polyoxometalate synthesis by our subject group,with the adicity of implicit vanadium is five,which observed inhibitory action on HBV both in vivo and in vitro experiment, its activity similar to lamivudine, and its toxicity was low.Which provided important theoretical basis, technical line and experimental methods for further pre-clinical research and clinical use of POM-2 and non-nucleoside drug for anti-hepatitis B.
引文
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