Anticoliendotoxic Action Of Pulsatillae Decoction And Its Main Ingredients And Their Mechanism

Swine colibacillosis is an acute infectious disease of the alimentary canal caused by pathogenic Escherichia coli. This disease spreads quickly and induces high mortality rates, resulting in great economical loss. Although antibiotics or anti-sera can be used for treatment, the issues of drug resistance, drug residues and high costs are not optimistic. Pulsatillae Decoction (PD) is a representative prescription of Chinese herbal medicine and widely used to prevent and cure infectious diseases with high efficacy, lower toxicity, fewer side effects, less drug resistance. This research determined the antiendotoxin action of PD and its effects on gene expression of LPS-induced rat intestinal microvascular endothelial cell (MVEC), and effects of PD and its main ingredients on LPS-induced MVEC to secrete NO, ET-1, IL-1α, IL-6, IL-8, TNF-α, E-selectin, TXB2and 6-keto-PGF1α. The purpose of research was to investigate the pharmacological mechanism of PD, thus to provide a theoretical basis for clinical application of PD in controlling the diseases caused by LPS. The details are divided into eight parts as follows:ExperimentⅠObservation of anticoliendotoxin action of PD PD was extracted and condensed into the decoction at the crude drug concentration of 1 g/mL. Mice in prevention groups were treated with PD at three concentrations (0.1,0.5 and 1 g/mL) for 5 days, then challenged with 9 mg/kg lipopolysaccharide (LPS) by peritoneal injection. Mice in treatment groups were challenged with 9 mg/kg LPS by peritoneal injection, then treated with PD at three concentrations (0.1,0.5 and 1 g/mL) for 5 days. The determined items and results were as follow:compared with LPS control group, the mortality were significantly decreased in the middle-and high-dose prevention groups and high-dose treatment group; the body temperature were significantly risen in every group of treatment and prevention at 6 h and 12 h; the body weight were significantly increased in the high-dose treatment and prevention groups at 24 h; the WBC, HGB, MCH and PLT were significantly increased in the high-dose treatment group; the index of liver, spleen and intestinal were significantly decreased in the high-dose treatment group. It suggested PD had significant antiendotoxin effect.ExperimentⅡCulture and identification of rat intestinal microvascular endothelial cells Rat intestinal microvascular endothelial cell (MVEC) was isolated by mechanical compression and effects of trypsin from the jejunum of SD rat at 1 d. MVEC was cultured with complete medium containing 15% fetal bovine serum at 37℃in a cell incubator with 5% CO2. MVEC was purified by the methods of differential digestion, differential adhesion and mechanical curettage. MVEC was identified by the expression of the factorⅧ-related antigen using fluorescence immunocytochemistry and AgNO3 staining. The results confirmed that MVEC culture was successful. This method was simple, efficient and with good reproducibility. These cultured cells had ideal purity and stable nature. This provided experimental material for further research of effects of PD and LPS on secretion function of MVEC.ExperimentⅢEffects of PD on gene expression of LPS-induced MVEC MVEC was challenged with 1μg/mL LPS for 3 h, then treated with PD at a concentration of 1 mg/mL for 12 h. Total RNA of each treated group was extracted from cultured MVEC for detection by the Affymetrix Rat Genome 230 2.0 Array. The results showed that in LPS group,36 genes were upregulated and 33 genes were downregulated in comparison with Blank group; In LPS-PD group,566 genes were upregulated and 12 genes were downregulated in comparison with LPS group, and 93 genes were upregulated and 29 genes were downregulated in comparison with Blank group. These suggested that PD could simultaneously reduce the translated level of inflammatory factors, suppress the transcription of apoptosis genes, accelerate the protein synthesis of organism and enhance the energy metabolism of cells, thus play the united antiendotoxin action.ExperimentⅣReal-time RT-PCR analysis of the differently expressed genes of LPS-induced MVEC The samples from LPS group and blank control group were subjected to Real-time RT-PCR, using the chimeric fluorescence method. Primer and probe sequences were designed by Primer 5.0 software according to the mRNA sequences of five target genes and the internal control actin gene which were synthesized by Shanghai GeneCore BioTechnologies Co. Ltd. The RT-PCR ratios for the target genes Il1a,Il6, Edn1, Tnfsfll and Ptges were 9.78,1.76,1.43,2.98 and 2.12 respectively, which were very similar to the ratios determined by the gene chips (10.57,1.53,1.59,3.16 and 1.71, respectively). It suggested the conclusion of gene chip was accurate and reliable, which were further confirmed the antiendotoxin pharmacological effects of PD.ExperimentⅤEffects of PD and its ingredients on LPS-induced MVEC to secrete NO and ET-1 MVEC was challenged with 1μg/mL LPS for 3 h, then treated respectively with PD at three concentrations (1,5 and 10 mg/mL) and its seven ingredients at three concentrations (1,5 and 10μg/mL). The cells were incubated at 37℃in a cell incubator for 21 h. The supernatants were collected and analyzed the content of NO and ET-1 by ELISA kits. The results showed that as compared with LPS control group, the NO contents were significantly decreased in the middle-and high-dose groups of PD, the middle-and high-dose groups of Anemonin, the middle-and high-dose groups of Berberine and the high-dose group of Esculetin; The ET-1 contents were significantly decreased in the middle-dose group of PD, the middle-and high-dose groups of Anemonin and the middle-and high-dose groups of Esculetin. It suggested that PD may exert beneficial therapeutic actions on diarrhea, edema and haemal diseases mediated by NO and ET-1.ExperimentⅥEffects of PD and its ingredients on LPS-induced MVEC to secrete IL-1a, IL-6 and IL-8 MVEC was challenged with 1μg/mL LPS for 3 h, then treated respectively with PD at three concentrations (1,5 and 10 mg/mL) and its seven ingredients at three concentrations (1,5 and 10μg/mL). The cells were incubated at 37℃in a cell incubator for 21 h. The supernatants were collected and analyzed the content of IL-1a, IL-6 and IL-8 by ELISA kits. The results showed that as compared with LPS control group, the IL-1a contents were significantly decreased in the high-dose group of PD, the middle-and high-dose groups of Anemoside B4, the middle-and high-dose groups of Berberine and the high-dose group of Palmatine; The IL-6 contents were significantly decreased in middle-and high-dose groups of PD, the high-dose group of Anemonin, the middle-and high-dose groups of Aesculin and the high-dose group of Esculetin; The IL-8 contents were significantly decreased in high-dose group of PD, the middle-and high-dose groups of Aesculin and the middle-and high-dose groups of Esculetin. It suggested that PD may exert beneficial therapeutic actions on systemic inflammatory response syndrome, multiple organ dysfunction syndrome and immune diseases mediated by IL-1α, IL-6 and IL-8.ExperimentⅦEffects of PD and its ingredients on LPS-induced MVEC to secrete TNF-αand E-selectin MVEC was challenged with 1μg/mL LPS for 3 h, then treated respectively with PD at three concentrations (1,5 and 10 mg/mL) and its seven ingredients at three concentrations (1,5 and 10μg/mL). The cells were incubated at 37℃in a cell incubator for 21 h. The supernatants were collected and analyzed the content of TNF-αand E-selectin by ELISA kits. The results showed that as compared with LPS control group, the TNF-αcontents were significantly decreased in the middle-and high-dose groups of PD, the middle-and high-dose groups of Anemoside B4, the middle-and high-dose groups of Berberine, the middle-dose group of Jateorhizine and the high-dose group of Aesculin; The E-selectin contents were significantly decreased in the high-dose group of PD, the high-dose group of Anemoside B4, the high-dose group of Berberine and the middle-and high-dose groups of Jateorhizine. It suggested that PD may exert beneficial therapeutic actions on fever, inflammation and infected diseases mediated by TNF-a and E-selectin.ExperimentⅧEffects of PD and its ingredients on LPS-induced MVEC to secrete TXB2 and 6-keto-PGF1αMVEC was challenged with 1μg/mL LPS for 3 h, then treated respectively with PD at three concentrations (1,5 and 10 mg/mL) and its seven ingredients at three concentrations (1,5 and 10μg/mL). The cells were incubated at 37℃in a cell incubator for 21 h. The supernatants were collected and analyzed the content of TXB2 and 6-keto-PGF1αby ELISA kits. The results showed that as compared with LPS control group, the TXB2 contents were significantly decreased in the middle-and high-dose groups of PD, the high-dose group of Anemonin, the middle-dose group of Berberine, the middle-and high-dose groups of Jateorhizine and the middle-and high-dose groups of Palmatine; The 6-keto-PGF1αcontents were significantly decreased in the high-dose group of Esculetin. It suggested that PD may exert beneficial therapeutic actions on thromboxane, disseminated intravascular coagulation and shock diseases mediated by TNF-αand E-selectin.