Effects And Mechanisms Of Rhizoma Coptidis And Berberine On Inflammatory Injury Of Intestinal Porcine Epithelial Cells Induced By LPS

Pathogenic E.coli is an important common intestinal pathogen in pigs,which can cause yellow and white scour of newborn piglets,and piglets edema disease,and seriously affects the growth and development of weaned piglets,causing an increase in feed-to-meat ratio and a decrease in economic benefits of pig breeding.LPS is one of the main toxins of E.coli disease,which can cause inflammatory damage to the liver,kidney,heart and brain of the animal body.In severe cases,it will enter the blood circulation and cause toxemia,causing animal shock and even death.For such diseases,Chinese veterinarians often use Chinese medicines,such as Rhizoma Coptis or Chinese materia medica which contain Rhizoma Coptis,to prevent and treat it.In order to research the mechanism of Rhizoma Coptis in preventing and treating E.coli intestinal diseases,the vitro inflammatory model of intestinal porcine epithelial cells(IPECs)was constructed by LPS,using aqueous extract of Coptis Chinensis(AERC)and berberine(BB)to prevent and treat it.The intervention effect and mechanism of inflammatory injury of IPECs was follows:1.After decoction of distilled water,the dried product of Coptis chinensis was extracted twice,and then concentrated by vacuum rotary evaporation into 1g crude drug/ml of Rhizoma Coptidis extract,and the content of BB in the AERC was determined by spectrophotometer to be 7% at this experience,which meet the requirements of the Pharmacopoeia.2.Using different concentrations of LPS,AERC and BB solution cultured with IPECs for 24 h,MTT assay was used to detect the cell viability of IPECs,and 5?g/mL LPS was determined to be optimal.The ideal concentration will cause the cells to shrink slightly and become round,and even see the small areas of cell shedding.0.1mg/mL,0.5mg/mL and 5mg/mL were used as the low,medium and high concentrations of the AERC,75 ?g/mL,150?g/mL,and 250?g/mL were used as the low,medium,and high concentrations of BB,respectively.3.After IPECs were co-cultured with different concentrations of AERC and BB for 4h,the mixture would be then co-cultured with 5?g/mL LPS for 1h,by means of Trizol extracting total RNA,two-step reverse transcription cDNA,Rreal-time PCR was used to detect the expression of cellular inflammatory factor mRNA.The results showed that LPS could induce the expression of IL-1?,IL-6 and TNF-? mRNA in IPECs,significantly increasing compared with the control group(P<0.01).After pretreatment,the expression of the above inflammatory cytokines decreased significantly or extremely significantly(P<0.05 or P<0.01)and was positively correlated with the drug dose.4.Different concentrations of AERC and BB were co-cultured with IPECs for 4 h,and then co-cultured with 5?g/mL LPS for 1 h.Total protein was extracted by RIPA method.Protein concentration was determined by BCA method.Western-Blot method was used to detect the expression levels of p-I?B?/I?B?,p-p65/p65,p-JNK/JNK,p-p38/p38 and p-ERK/ERK in NF-?B/MAPKs signaling pathway.These results indicated that LPS could induce the expression levels of NF-?B/MAPK signaling pathway proteins,such as p-I?B,p-p65/p65,p-JNK,p-p38 and p-ERK/ERK proteins in IPECs significantly or extremely significantly elevated(P<0.05 or P<0.01).AERC can inhibit the expression and phosphorylation of I?B?,p65 and JNK in IPECs,inhibit the expression of p38 and ERK1/2,and inhibit the induction of LPS induced activation of the inflammatory cell signaling pathway in IPECs.BB as well as can inhibit the expression and phosphorylation of I?B?,p65 and the expression of p38,ERK and JNK proteins in IPECs and inhibits LPS's induction of activation of the NF-?B/MAPKs signaling pathway in IPECs yet.In summary,AERC and BB can inhibit the downstream inflammatory cytokines,such as IL-1?,IL-6,TNF-? mRNA,by inhibiting the activation and expression of key proteins in the NF-?B/MAPK signaling pathway.The expression of LPS-induced inflammatory response in IPECs is explained,and the effect of AERC and its active component BB on LPS-induced intestinal inflammatory response in piglets and its mechanism are explained.