Study On Protection Of Radix Paeoniae Rubra And Other 10 Chinese Herbs On Injury Of Primary Bovine Mammary Epithelial Cells Induced By Lipoteichoic Acid In Vitro

Bovine mastitis is one of the common diseases in dairy cow breeding,and it prevents the development of dairy cow industry.Chinese herb has a unique and good effect in the treatment for bovine mastitis such as anti-microbial,anti-inflammatory,low toxicity,no residue bacterial drug resistance,and has a function of nutrients.In this study,an orthogonal experiment was used to screen the Chinese herb compound for dairy cow mastitis.The mammary epithelial cells were isolated and cultured by collagenase digestion method,and the mammary epithelial cells were purified by differential trypsin digestion.BMEC inflammatory model was established by Lipoteichoic acid(LTA).MTT,ELISA and q RT-PCR were used to analyze the protective and anti-inflammatory effects of Chinese herbs,such as red paeonia,on LTA induced inflammatory lesions.In order to provide a reference for the Chinese herbs treatment of dairy cow mastitis,it provides scientific basis for the research and development of Chinese herbs and related basic research.The results are as follows:(1)Five kinds of Chinese herbs were screened from 10 Chinese herbs by orthogonal test.The optimal factors of group are Radix paeoniae rubra,Scutellariae radix,Carthami flos,Radix paeoniae alba and Chrysanthemi flos,and the proportion is 3:3:3:1:2.The bacterial inhibition diameters for Escherichia coli was 17.39 ±0.42 mm,and MIC and MBC were 62.50 and 125.00 mg/m L respectively;The bacterial inhibition diameters for Staphylococcus aureus was 25.44 ± 0.38 mm,and MIC and MBC were 31.25 and 31.25 mg/m L respectively.This traditional Chinese compound is named HCJBH temporarily.(2)The b MEC were isolated and cultured by collagenase digestion method,and the b MEC were purified by differential trypsin digestion.The cultured cells were identified by measuring bovine mammary epithelial-derived keratin-keratin 18 to confirm that they were b MEC and infected with mammary gland epithelial cells with Lipoteichoic acid(LTA)to culture cells Morphological features and inflammatory factors as indicators of the occurrence and development of inflammation.The effects of LTA at different concentrations(0,10,20,40,and 80 μg / m L)on b MEC were observed at different time points(12,24 and 48 h);It was found that with the increase of LTA dose,the inhibition rate of cell vitality increased gradually,indicating that LTA of certain concentration would cause the damage of b MEC.According to24 h of LTA action cells of different concentrations,the inhibition rate of concentration of 20 μg / m L and 40 μg / m L was significantly increased(P < 0.05),and IR was close to 10%.The results of MTT,ELISA and q RT-PCR showed that the treatment of b MEC with LTA at the concentration of 40 μg / m L for 24 h obviously induced TNF-α and IL-1β protein and gene expression levels.(3)The maximum safe concentration of Radix paeoniae rubra and Radix paeoniae alba in epithelial cells was 1.0 g/L by MTT method;The maximum safe concentrations of Radix scutellariae,Carthami flos and HCJBH were 0.5 g/L;The maximum safe concentration of Chrysanthemi flos to epithelial cells was 0.25 g/L.The maximum safe concentration of Aspirin for epithelial cells was 15 μmol/L.The b MEC 12 h was treated with HCJBH and aspirin respectively.Cell viability assay showed that HCJBH with a concentration of 0.5 g/L had a protective effect on inflammatory lesion of b MEC.Aspirin with a concentration of 15 μmol/L has a strong inhibitory effect on inflammation.0.5 g/L HCJBH and 15 μmol/L Aspirin can significantly reduce the expression and gene expression of TNF-α and IL-1β.The anti-inflammatory effect of HCJBH is lower than that of aspirin.In conclusion,composed of Radix scutellariae,Radix paeoniae rubra,Chrysanthemi flos,Radix paeoniae alba and Carthami flos extract of Chinese herb formula,it has good antibacterial activity in vitro,can also inhibit Escherichia coli and Staphylococcus aureus;The LTA of 40 μg/m L treated purified b MEC 24 h,which can be used to establish the inflammatory model of b MEC.HCJBH of0.5g/L can reduce the role of LTA damage in b MEC.