| Diarrhea is a common and frequently-occurring disease in the modern large-scale breeding of livestock and poultry,which is also one of the main causes of death of young animals.The bacterial diarrhea caused by pathogenic E.coli has the characteristics of high morbidity,high mortality,and severe infectivity,which seriously restricts the development of livestock and poultry breeding industry.Polygonum hydropiper L.is a kind of annual herb of Polygonum in Polygonaceae.In veterinary clinic,the decoction of P.hydropiper has been widely used in the treatment of diarrhea in piglets,cows and horses etc with obvious effects and little side effects.The results of our previous study showed that the water extract of P.hydropiper has a significant therapeutic effect on mice with diarrhea induced by pathogenic E.coli.In order to explore of the pharmacological mechanism of P.hydropiper against diarrhea,different methods were used to establish E.coli-induced diarrhea models.The clinical symptoms of mice,the histopathological characteristics,the levels of inflammatory cytokines and mRNA expression of intestinal mucosal repair factors of different intestinal segments were used as the evaluation indexes to screen the stable and reliable modeling methods.Secondly,the effects of the water extract of P.hydropiper on the expression levels of inflammatory factors and cytochrome P450?CYPs?in the duodenum and liver of mice with diarrhea were investigated.The results of this study may lay the foundation for the further study on the mechanism P.hydropiper against diarrhea induced by E.coli and provide a theoretical basis for the clinical medication and resource development and utilization of P.hydropiper.The main research contents and results are as follows:1.Establishment of the mice diarrhea model induced by E.coli:Single and multiple intraperitoneal injections of Enteropathogenic E.coli?EPEC?were used to induce diarrhea in BALB/c mice.Four hours after the first infection,the mice showed signs of obvious diarrhea,accompanied by fatigue spirit,rough fur,and increased perioral secretion etc.The stomach flatulence was serious,the intestine tract was full of yellow watery stool and the intestinal wall became thiner,which indicated that the diarrhea models were successfully induced.The histopathological changes of the duodenum,ileum,cecum were observed and compared by HE staining;the levels of myeloperoxidase?MPO?in serum,tumor necrosis factor-? ?TNF-??and interleukin-1??IL-1??in duodenum,ileum,cecum were determined by enzyme-linked immunosorbent assay?ELISA?;the mRNA expression levels of proliferating cell nuclear antigen?PCNA?,transforming growth factor-?1?TGF-?1?and epidermal growth factor receptor?EGFR?was determined by quantitative real-time PCR?qRT-PCR?.On the first day of induction,the duodenal epithelial cells of mice in the single and multiple infection groups showed necrosis,apoptosis,inflammatory cell infiltration,tissue edema,and inflammatory cells infiltrated or exudated in the ileum and cecum;the levels of cytokines ?MPO,TNF-?,IL-1??and the mRNA expression of mucosal repair factors?PCNA,TGF-?1,EGFR?increased significantly?P<0.01,P<0.05?,which confirmed that the diarrhea model was successfully established.At day 6after induction,the duodenum of mice in the single infection group displayed slight capillary congestion or infiltration of inflammatory cells,and the structure of the ileum and cecum was almost normal;the contents of MPO,TNF-?,IL-1?and the mRNA levels of PCNA,TGF-?1,EGFR in the duodenum,ileum and cecum were not significantly different compared with the mice in the normal group.At day 9 after induction,the duodenum of mice in the multiple infection group still showed inflammatory cell infiltration and obvious edema,the ileum had some capillary congestion or a small amount of inflammatory cell infiltration,and the cecum had no obvious abnormal structure.The contents of MPO,TNF-?and IL-1?were significantly increased compared with the normal group?P<0.01?;the contents of TNF-?in the ileum and cecum were significantly different from those in the normal group?P<0.05?.The mRNA expression of PCNA,TGF-?1 and EGFR in duodenum were significantly higher than those in the normal group?P<0.01,P<0.05?,and the mRNA expression of EGFR in ileum and PCNA in cecum was significantly higher than those in the normal group?P<0.01,P<0.05?.There were no significant differences in the mRNA expression of PCNA in ileum and EGFR in cecum compared with the normal group.In summary,the diarrhea model of the single injection of E.coli has a certain degree of self-healing,but the multiple infections of E.coli could significantly prolong the disease cycle and maintain the stability of diarrhea model.Compared with the ileum and cecum,the duodenum is severely damaged after infection,which is more suitable to be used as the research material for the further exploration of pharmacological mechanism.2.The effect of P.hydropiper on the levels of inflammatory cytokines in mice with diarrhea induced by E.coli:BALB/c mice were randomly divided into 5 groups:the normal group,model group,enrofloxacin hydrochloride group(5 mg·kg-1),low-dose and high-dose groups of P.hydropiper(5 g·kg-1 and 10 g·kg-1 respectively,calculated as the crude drug amount).Except for the normal mice,the mice in the other groups were induced diarrhea by multiple intraperitoneal injections of EPEC.Two days before modeling,the mice in the administration groups were given corresponding drugs,the mice in the model group and normal group were given the same amount of saline.The histopathological characteristics of duodenum and liver were observed by HE staining;the levels of TNF-?,IL-1?,interleukin-6?IL-6?,and leukemia inhibitory factor?LIF?in duodenum and liver were determined by ELISA;the mRNA expression levels of TNF-?,IL-1?,IL-6 and LIF were determined by qRT-PCR.In the model group,there were necrosis,apoptosis of duodenal epithelial cells,inflammatory cell infiltration and tissue edema in the duodenum,and the liver cells showed necrosis,inflammatory cell infiltration,and tissue proliferation or hemorrhage.The contents and mRNA expression levels of TNF-?,IL-1?,IL-6 and LIF in the duodenum and liver were significantly increased?P<0.01,P<0.05?.The clinical symptoms of mice in the P.hydropiper groups were gradually relieved,and the damage of duodenum and liver were alleviated.The levels of TNF-?,IL-1?,IL-6 and LIF in duodenum and liver of mice in the P.hydropiper groups was reduced obviously compared with the model group?P<0.01,P<0.05?,which showed a dose-dependent relationship.It is suggested that P.hydropiper may play an anti-diarrheal role by reducing the levels of inflammatory cytokines and alleviating the damage of the duodenum and liver.3.The effect of P.hydropiper on the expression levels of cytochrome P450 enzymes in mice with diarrhea induced by E.coli:The expression levels of cytochrome P450?CYPs:CYP1A1,CYP1B1,CYP2B9,CYP2B10,CYP2C29,CYP2J6,CYP2S1,CYP3A11,CYP4B1 and CYP4B1?in the duodenum and liver were detected by qRT-PCR,ELISA and Western blot.The results of qRT-PCR showed that compared with the normal group,the mRNA expression of CYP1A1,CYP2B9,CYP2B10,CYP2C29,CYP2S1,CYP3A11 and CYP4A10 in duodenum of mice in model group was significantly reduced?P<0.01?,mRNA expression of CYP1B1 was significantly up-regulated?P<0.01?,and the mRNA expression of CYP2J6 and CYP4B1 was not statistically significant.Except that the mRNA expression of CYP4B1 in the liver was significantly decreased compared with the normal group?P<0.01?,the changes of other CYPs were similar to those of the duodenum.Compared with the model group,P.hydropiper water extract could regulate the mRNA levels of CYPs in the duodenum and liver?P<0.01,P<0.05?,and there was a dose-effect relationship.ELISA results showed that the protein contents of CYP2B9 and CYP3A11 in the duodenum decreased significantly?P<0.01,P<0.05?,and the protein content of CYP4B1remained unchanged compared with the normal group.The protein contents of CYP2B9,CYP3A11 and CYP4B1 in liver were significantly decreased?P<0.01,P<0.05?.Compared with the model group,the protein levels of CYP2B9,CYP3A11 and CYP4B1 in the duodenum and liver were significantly increased in the P.hydropiper groups?P<0.01,P<0.05?,and showed a certain dose-effect relationship.Western Blot results showed that the protein expression of CYP1A1 in the duodenum and liver of the model group was significantly decreased?P<0.01?and the protein expression of CYP1B1 was obviously up-regulated?P<0.01?compared with the normal group.The water extract of P.hydropiper could significantly up-regulate the protein expression of CYP1A1 and down-regulated the expression of CYP1B1 in the duodenum and liver?P<0.01,P<0.05?.In general,the mRNA and protein expression levels of CYPs in the duodenum and liver of mice were generally reduced in the process of E.coli infection,and P.hydropiper had a significant regulatory effect on CYPs. |
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