| Transmissible gastroenteritis (TGE), one of the main diseases causing early death of piglets, is a highly contagious disease of swine caused by transmissible gastroenteritis virus (TGEV). Usage of the vaccination and western medicine is the major method for the prevention of TGE. However, both vaccination and western medicine exist deficiencies. For example, vaccines could result in frequent immunity failure and western medicine could produce drug residues, both of which pose a serious threat to food security and human health. Under this circumstances the exploitation of innovative, safe, efficient and environment protectional Chinese traditional veterinary medicine becomes the urgent priority. Therefore, considering the Swine Intestinal epithelial Cells (SIEC) as the object of research, this study assessed and analyzed the cell cycle, related cell signal transduction pathways, porcine aminopeptidase N (pAPN) and anti-virus factors applying methods of flow cytometry, western blot, qRT-PCR and fluorescence microscope to explore the intervention mechanism of Chinese herbal compound on the apoptosis of SIEC induced by TGEV, which laied the foundation for the clinical application of Chinese herbal compound for preventing and controlling of infectious diseases of livestock and poultry.Results:1. The maximum safe concentration of Chinese herbal compound treated SIEC determined by microscopic observation combined with MTT assay:6.25mg/mL for compound shenzhu,1.5625mg/mL for compound lianweng and 3.125mg/mL for compound shenfu.2. Cell survival was assessed by MTT assay. The results showed that the three Chinese herbal compounds could all inhibit the absorption effects of TGEV on SIEC and PK-15 cells in vitro.3. Proportion of the cell cycle was analyzed by flow cytometry. The results showed that Chinese herbal compounds could change cell cycle arrest induced by TGEV at G2/M phase. The gene expression of cyclins was evaluated by qRT-PCR. The results showed that the expression of cyclinA and cyclinB1 was markedly up-regulated by Chinese herbal compounds (P<0.05). Observation by fluorescence microscope showed that the morphologic characteristics of SIEC that had been treated with Chinese herbal compounds before infected with TGEV were integral, cell structures were clear presenting long spindle-shaped and the cell nucleus structures were clear with uniform pigmenting of chromatin and unobvious apoptosis in most cells.4. The detection of Western blot assay showed that the expression levels of Caspase-8, Caspase-9, Caspase-3, Cyt c and Bid protein in TGEV-infected SIEC were down regulated. The Western blot and qRT-PCR results indicated that the Chinese herbal compounds could extremely and significantly increase the transcription of Fas, Bcl-2 mRNA (P<0.01) and the expression of relative protein. The results also indicated that the Chinese herbal compounds could extremely and significantly decrease the transcription of Fasl and Bax mRNA (P<0.01) and the expression of relative protein. The caspase inhibitor group could decrease the expression of Caspase-8, Caspase-9, Caspase-3 protein in SIEC compared with the TGEV group.5. The gene expression of pAPN and glucose-regulated protein 78 (GRP78) was evaluated by qRT-PCR. The results proofed that the Chinese herbal compounds could decrease the expression level of pAPN and positively regulate GRP78.6. The gene expression of TNF-α, IFN-β, TLR3 and NF-kB was detected by qRT-PCR. The results displayed that the TNF-a expression was increased by Chinese herbal compounds with higher expression than the control group. The IFN-P expression was increased by Chinese herbal compounds with higher transcription quantity than the TGEV group. Moreover, the expression of TLR3 in Chinese herbal compound groups at 24,48 and 72 hours were all extremely and significantly less than the control group (P<0.01), but higher than that of TGEV group. However, the NF-kB expression at all phases were all remarkably increased by Chinese herbal compounds (P<0.05) compared with control group.Conclusions:1. Chinese herbal compounds with suitable concentrations (compound shenzhu: 6.25mg/mL, compound lianweng:1.5625mg/mL, compound shenfu:3.125mg/mL) were safe and could protect cells by inhibiting the absorption effects of TGEV on SIEC and amending typical CPE phenomenon in the TGEV-infected SIEC.2. Chinese herbal compounds could positively regulate the expression of key apoptosis factors (Fas/Fas1、Bcl-2/Bax、Cyt-c、Bid),cyclins (cyclinA、cyclinB1) and TGEV receptor (pAPN) in SIEC, through which the caspase activity was inhibited, cell cycle was improved and TGEV adsorption and replication were interrupted. Finally, the apoptosis of SIEC infected by TGEV was inhibited by Chinese herbal compounds.3. Chinese herbal compounds could suppress viral genome replication or disturb the protein synthesis of virus by improving the transcription levels of antivirus factors (IFN-β、TNF-α、TLR3、NF-κB). Finally, the proliferation of TGEV was inhibited by Chinese herbal compounds.The results of the overall study indicated that Chinese herbal compounds could prevent SIEC from TGEV infecting. |
PDF Full Text Request