| The Shiga-like toxin type II variant (SLT-IIv) causes pig edema disease (ED) and it may cause vascular endothelial cell lesions in the intestine, subcutis and the brain, leading to edema in affected organs, neurological symptoms and eventually death. The disease spreads quickly and induces high mortality rates, resulting in substantial harm and loss to the pig industry. Many Chinese herbal medicines are widely used to prevent and cure infectious diseases, demonstrating high efficacy, lower toxicity, fewer side effects, less drug resistance and lower residual levels than most drugs currently on the market. Therefore, prevention and treatment of bacterial diseases by traditional Chinese medicine has become a research focus. QiHuang Decoction (QHD) has a good effect on edema caused by SLT-IIv, confirmed by the previous experiments in our laboratory. On the basis of it, we carried out this experiment to further investigate the mechanism of QHD.In these study, an Affymetrix mouse genome array and Two-Dimensional differential gel electrophoresis (2-D DIGE) techniques were used to investigate the mechanisms of QHD on the injury of mouse intestinal microvascular endothelial cells (MIMVECs) induced by the Shiga-like toxin type II variant (SLT-IIv). MIMVECs were challenged with 10μg/ml SLT-IIv for 12 h and then treated with QHD at a concentration of 200μg/ml for 12 h. Total RNA and proteins were extracted from cultured MIMECs for analysis by the Affymetrix GeneChip? Mouse Genome 430 2.0 microarray and 2-D DIGE. The results showed that 63 genes and 73 proteins were differentially expressed, of which 49 genes were upregulated, 14 genes downregulated, 25 protein downregulated and 48 proteins upregulated in the SLT-IIv group compared to the control group. In the QHD group, 44 genes and 19 proteins were upregulated, 84 genes and 7 proteins were downregulated, compared to the control group. In the SLT-IIv-QHD group, 72 genes were upregulated, 36 genes were downregulated, and the number of either downregulated or upregulated proteins was 63, compared to the control group. When the QHD-treated group was compared to the SLT-IIv group, expression of 74 gene was found to be increased, and all other genes were decreased, with 139 proteins upregulated and 85 downregulated. Analysis of the data demonstrated that QHD specifically and effectively reduced microvascular endothelial cell permeability induced by SLT-IIv in the treatment of pig edema disease. Then, on the basis of these study, we will make a further research about its phamacological actions.In order to further study the mechanism of QHD, we used high performance liquid chromatography (HPLC) to purify the water-soluble constituents of it. According to a certain condition of gradient elution with acetonitrile and 0.5% acetic acid solution as mobile phase, 15 water-soluble extracts were purified, and peak-7 and peak-13 were initially identified to be gallic acid and quercetin, respectively, by analytical HPLC and LC-MS. Then further study of the effect of the various components on the MIMVECs was conducted.First, to determine the effects of QHD and its extracts on the MIMVECs injury induced by SLT-IIv, the supernatant of MIMVECs, infected with SLT-IIv and then treated with QHD and its extracts for 12 h, were harvested. The results showed that QHD, QHD alcohol precipitation supernatant group(QHD-AP), QHD alcohol precipitation precipitation group(QHD-P) and peak-2, 4, 6, 11, 12, 14, and 15, could significantly (P <0.01) reduced the vitality of LDH at the indicated concentrations, while peak-3, 7, 8, 10 and 13 no significant differences compared to the SLT-IIv group. Except for the QHD-P group, the other groups at the indicated concentrations could significantly increase the SOD activity of damaged cells (P <0.01). QHD group, QHD-AP group, QHD-P group and peak-2, 3, 4, 7, 8, 11, 12, 14, and 15, can significantly decrease the content of MDA at the indicated concentrations (P <0.01), while MDA contents of peak-9 and 10 were not significantly different from the SLT-IIv group. Except for that of peak-5, GSH-Px activities of other groups at the indicated concentrations, were significantly higher than that of the SLT-IIv group (P <0.01). These results showed that almost all the ingredients of QHD can improve the SOD and GSH-Px activities, or decrease the content of MDA in MIMVECs These suggested that QHD and its ingredients can remove a lot of free radicals which damage the tissues and cells. Peak-3, 7, 8, and 11 could significantly reduce the content of NO from SLT-IIv-damaged cells (P <0.01). All indicated drugs can significantly decrease the content of ET-1 from SLT-IIv-damaged cells (P<0.01 or P<0.05 ). These data indicated that QHD can play a important role in the disease like diarrhea or edema caused by NO and ET-1. The results showed that most of the ingredients can markedly enhance the activities of SOD and GSH-Px, and reduce the LDH activity and the content of NO, ET-1, and MDA from SLT-IIv-damaged MIMVECs.Second, to explore the effects of QHD and its extracts on permeability of MIMVECs damaged by SLT-IIv, the OD values of the BSA through the Transwell were measured. The results showed that the extracts in varying degrees could reduce the MIMVECs permeability induced by SLT-IIv.Finally, the microvascular amplitude of Sanyinjiao points treated with QHD and its extracts were measured with laser Doppler flowmeter. The results showed that, except for QHD-P and peak-5, 8, 12, and 14, other indicated drugs can increase the microvascular amplitude to some extent, indicating that QHD can play an important role to improve the blood microcirculation.In conclusion, QHD and its extracts can effectively resist the effect of SLT-IIv, the mechanism of which is to improve the MIMVECs injury, reduce microvascular permeability, and improve the blood microcirculation, further curing the swine edema disease.
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