Effect Of Total Saponin Of Panax Ginseng On Marek's Disease Oncogenesis And Its Mechanism

Marek's Disease (MD), a highly infectious lymphoproliferative disease inchickens, is caused by an alphaherpesvirus-Marek's disease virus (MDV), resulting inT cell lymphomas in visceral organs and peripheral nerves. From 1970s, losses to thepoultry industry due to MD have been greatly limited through the use of live vaccines.But MD is a major threat to the poultry production in the world. Vaccines againstMDV are available, but immunological defeat sometimes happens, resulting in MDbreaking in some regions. And moreover, the virus is gradually becoming morevirulent. So further to explore the mechanism of MD will help us prevent and controlthis disease.MD, served as an important animal model of viral oncogenesis and the first tumorbeing prevented by vaccine, has offered important contributions to veterinary medicine,basic science and comparative oncology.At present, therapeutic methods of malignant tumor consist of operation,radiotherapy, chemotherapy, and treatment with traditional Chinese medicine.Commonly used antitumor drugs included many medicines, such as influencingnucleic acid biosynthetic drugs, and drugs interfering with the synthesis and action ofproteins, etc. However, the toxic reactions of these drugs are strong, and these drugsoften suffer from drug resistance in the clinical application. So it is necessary toexplore new antitumor drugs.Ginsenoside (GS) is the major effective ingredient of ginseng (the rhizome ofPanax ginseng C. A. Mey.). Long-term clinical practice indicate that ginseng iseffective to prevent many neoplastic diseases. Modern pharmacological researchessuggest that GS has potent antitumor activity from different routes, such as inducingapoptosis and cell differentiation, and reversing drug resistance, etc.Due to these reasons, in the present study we used the model of MD induced bythe MSB-1 cell (MD lymphoblastoid cell line) to investigate the associated pathogenicmechanism of MD, and explored the inhibitive effect of total saponin of panax ginseng(TSPG) on the growth of MSB-1 cell and its mechanism.Through drawing the growth curve of MSB-1 cell, observing the morphology ofMSB-1 cell with microscope and transmission electron microscope, making the modelof MD induced by MSB-1 cell, detecting MDV with IFA, and amplifying meq gene byPCR, we investigated the growth characteristic, morphological features, andpathogenicity of MSB-1 cell. The results showed that the population double time of1×104/ml and 1×105/ml, the initiate cell density at 37℃ and 41℃ , respectively, isabout 24 hours;MSB-1 cell has the typical lymphocytic features;after the susceptiblechickens were vaccinated with MSB-1 cell, we observed gross pathological changes invisceral organs, peripheral nerves, and eyes. The result of agar diffusion is positive,and the meq gene-oncogenic gene of MDV is amplified with PCR successfully. Theseresults suggested that MSB-1 cell induced typical MD in susceptible chickens, and thepathological changes are induced by MDV actually.Using the MD model induced by MSB-1 cell, we amplified meq gene in differentpathological tissues, and sequenced this gene. The results showed that the meq gene inMSB-1 cell and tumors of spleen is the three base pair-deleted L-meq (1 197 bp), andthe meq gene in the feather pulp is the three base pair-deleted meq (1 017 bp).Compared with the other reported meq genes, from No. 709 base pair of the standardstrain GA, a 180-bp sequence is inserted in the meq genes of CHANG L-meq, RP-1cell, Tagaki MSB-1 cell, strain BC-1, MSB-1 cell, and spleen. In all the comparedsequences, G mutated into T at No. 211 base pair of the standard strain GA exceptCHANG L-meq, and RP-1 cell;A mutated into G at No. 229 base pair of the standardstrain GA except CHANG L-meq;T mutated into C at No. 344 base pair of thestandard strain GA except CVI988/RISPENS, CHANG L-meq, and RP-1 cell;Amutated into C at No. 759 base pair. Besides, some base pairs mutated randomly. Theamino acid sequences changed accordingly. These changes of meq gene maybeparticipate in the lymphocytic transformation, proliferation and metastasis of tumorcells, and the forming of infectious viral particle.We investigated the inhibitive effect of TSPG on MSB-1 cell by MTT assay, andexplored the mechanism through agarose gel electrophoresis, flow cytometry,morphological observation by transmission electron microscope, and fluorescencedetection. The results showed that TSPG inhibited the growth of MSB-1 cell in time-and dose-dependent manners. After MSB-1 cell treated with TSPG for 72 hours, theIC50 is 340.41 μg/ml, with (340.41±33.7) μg/ml as the 95% confident interval. Flowcytometry analysis indicated that with the time of MSB-1 cell treated with TSPGextending, the proportion of cells at G0/G1 phase increased, and that of cells at G2/M+Sphase decreased, suggesting TSPG inhibited the synthesis of DNA. Agarose gelelectrophoresis showed that MSB-1 cell treated with TSPG for 48 hours had DNAladder. MSB-1 cell, treated with TSPG for 72 hours at 400 μg/ml and 600 μg/ml,became inhomogeneous, the number of microvilli decreased, chromatin shrunk andagglutinated along the nuclear membrane, and vacuoles and mitochondria increased.After MSB-1 cell, treated with TSPG for 72 hours at 400 μg/ml and 600 μg/ml, werestained with Annexin V-EGFP and PI, we observed typical apoptotic cells with greenfluorescence in 600 μg/ml group. These results suggested that TSPG inhibited thegrowth of MSB-1 cell in time-and dose-dependent manners, and the mechanism wasrelative to inhibiting the synthesis of DNA and inducing apoptosis of some MSB-1cells.We analyzed effect of TSPG on mRNA level of L-meq in MSB-1 cell usingsemi-quantitive RT-PCR. The results showed that after MSB-1 cell treated with TSPGfor 72 hours, TSPG raised the abundance of mRNA of L-meq, statistically significant(P<0.01) compared with control group. The difference of the abundance of mRNA ofL-meq between 400 μg/ml group and 600 μg/ml group is not significant (P>0.05).These results indicated that TSPG raised the abundance of mRNA of L-meqsignificantly. Maybe TSPG indirectly inhibited the action of meq gene, and inducedapoptosis through raising the abundance of mRNA of L-meq, thus inhibited the growthof MSB-1cell.Based on all the information above, we concluded that MSB-1 cell induced MDtumor in susceptible chickens;meq gene changed in different tissues, and this changemaybe participate in the lymphocytic transformation, proliferation and metastasis oftumor cells, and the forming of infectious viral particle;TSPG inhibited the growth ofMSB-1 cell, and its mechanism related with inhibiting the synthesis of DNA, inducingapoptosis of some MSB-1 cells and raising the mRNA level of L-meq in MSB-1 cells.This present study offered data for developing the antitumor resources of ginsenosides.