Preliminary Study On Anti-HBV Effect Of Galactosides Modified MAP30 In Vitro And In Vivo

Hepatitis B, which is caused by hepatitis B virus, has been one of the most serious infectious diseases worldwide and brought about endless disasters to human health. The key of current clinical treatment is to eliminate hepatitis B virus. Although many anti-HBV strategies have led to certain inhibitory effect of HBV replication, the following two reasons limit the use of anti-HBV medicines. Firstly, it is impossible for current medicines to completely eliminate HBV from infected individuals. Also, the medicine will distribute evenly in human body so that there is no effective concentration in the liver. Therefore, it is essential to develop more effective and potent anti-HBV reagents.HBV is a hepatic tropic and double-strand DNA virus. The HBV lifecycle consists of transcription of covalently close circular DNA (ccc DNA) to provirus RNA, reverse transcription to negative strand DNA,synthesis of double strand DNA, and integration of HBV DNA to target hepatic cells. During the complex course, DNA topoisomerase, polymerase and integrase are needed.MAP30 (momordica anti-HIV protein of 30kDa) , a plant protein isolated from the seeds of the Momordica charantia, is one of the ribosome-inactivating proteins (RIPs). Many studies demonstrate that MAP30 can markedly inhibit HIV and HSV replication, the mechanisms of which are as following: ?MAP30 inactivates ribosome and destroys the synthesis of enzymes and proteins demanded by HBV replication. The interference of virus replication therefore results in elimination of infected cells and HBV. (2) MAP30 directly changes supercoiled DNA into relaxed or linear form, which inactivates DNA topoisomerase and interferes with virus replication and transcription. (3) MAP30 also inhibits integrase. The inhibitory effects are not only constrained to HIV.It is well known that HBV and HIV have the similar life cycle and share specific transcription and replication stage. Wang LX et al found MAP30 could inhibit secretions of HBsAg HBeAg and replication of HBV DNA from 2.2.15 cells, which indicated inhibitive effect of MAP30 on HBV replication in vitro. Based on the results above and the theory that asialoglycoprotein receptor (ASGP-R) on mammal hepatic parenchymal cell membrane can specifically identify and bind terminal galactose-modified ligand, Gal (galactose)-ABH(p-azidobenzoyl hydrazide)-MAP30 was constructed and its inhibitory effects on HBV in vitro and in vivo was studied.Gal-ABH-MAP30 was constructed by photochemical reaction and proved to be chemical coupling between MAP30 and Gal-ABH by ultravioletradiation scan. The coupling rate is 86.2%.Then Gal-ABH-MAP30 and MAP30 were radiolabeled by 125I and injected into the two groups of mouse body separately to investigate their distribution and localization. The results show that Gal-ABH-MAP30 concentrated in mouse liver much more and quicker than MAP30. There was less 125I-Gal-ABH-MAP30 in serum. Also, when Gal-ABH was delivered in advance, 125I-Gal-ABH-MAP30 taken up by liver reduced and delayed with increased renal excretion.In addition, it indicates in experiment in vitro that after Gal-ABH-MAP30 and MAP30 were added into 2.2.15 cells in culture, HBeAg and HBV DNA could be inhibited. Compared with MAP30, the inhibitory effect of Gal-ABH-MAP30 was more significant and faster. The inhibitory effect on HBeAg and HBV-DNA with 0.1μ g/ml Gal-ABH-MAP30 was roughly equal to that of 1.0 μ g/ml MAP30, which was also proved by immunohistochemistry, laser scanning confocal microscopy and in situ hybridization.At last, ayw subtype HBV transgenic mice were injected MAP30 and the galactose-modified MAP30. The results showed that both of Gal-ABH-MAP30 and MAP30 inhibited HBV replication and expression in serum and live, and the former seemed to be more effective and was deserved to be researched further.