Anti-inflammatory Mechanism Of Mixture Of Scutellaria Baicalensis And Radix Et Rhizoma Rhei On LPS-induced RAW264.7 Cell Inflammation

“Scutellaria baicalensis-Radix Et Rhizoma Rhei” herbal pair is always used in prescription such as Sanhuang prescriptions(Xiexin Decoction and its derivatives Yiqing Tablets,Yiqing Capsules,Yiqing Granules and Sanhuang Tablets)and Niuhuang Jiedu Tablets,Fangfeng Tongsheng Pills,Xiaoer Qingre San,Niuhuang Shangqing Pills,Daochi Pills.Combining Scutellaria baicalensis with Radix Et Rhizoma Rhei,can promote the effect of clearing fire exert by Scutellaria baicalensis,in line with compatibility of drugs which belong to seven emotion.In vivo and vitro experiments proved that Radix Et Rhizoma Rhei and Scutellaria baicalensis had a regulatory effect on various inflammations.However,the anti-inflammatory mechanisms of Water extract of Scutellaria baicalensis and Radix Et Rhizoma Rhei combination has not been elucidated.Therefore,mouse macrophage cell was used to establish an vitro inflammation model induced by Lipopolysaccharide(LPS)and explored the anti-inflammatory effect of “Scutellaria baicalensis-Radix Et Rhizoma Rhei” drug and its related mechanisms.Provide a theoretical basis for the research and development of drugs for the treatment of inflammatory diseases in livestock and poultry.In this experiment,the safe concentration of “Scutellaria baicalensis-Radix Et Rhizoma Rhei” drug on RAW264.7 cells was determined by CCK8,the levels of IL-6,IL-1β and TNF-α in the cell supernatant were detected by ELISA,and the m RNA expressions of IL-6,IL-1β and TNF-α in the cells were detected by RT-q PCR.Western blot,RT-q PCR and Griess methods were used to detect the effects of “Scutellaria baicalensis-Radix Et Rhizoma Rhei” on i NOS protein and m RNA expressions and NO release in the cell supernatants.The results showed that 9.375 mg/m L “Scutellaria baicalensis-Radix Et Rhizoma Rhei” has a significant inhibitory effect on cell viability regardless of 3 h,6 h and12 h.However,“Scutellaria baicalensis-Radix Et Rhizoma Rhei” drug at concentrations below 9.375 mg/m L had no significant effects on cell viability.To avoid experimental errors,1000,500,and 250 μg/m L were selected as the subsequent experimental concentrations.Compared with the blank group,LPS stimulated RAW264.7 macrophages to release a large amount of IL-6,IL-1β,TNF-α and NO,and significantly promoted the synthesis of i NOS,indicating that LPS induced the inflammation model in RAW264.7cells,and “Scutellaria baicalensis-Radix Et Rhizoma Rhei” drug can inhibit the excessive secretion of IL-6,IL-1β,TNF-α and the relative expression of m RNA in RAW264.7 cells and the drug pair can reduce i NOS expression and NO production in RAW264.7 cells stimulated by LPS.The results suggest that "Scutellaria baicalensis-Radix Et Rhizoma Rhei" medicine has protective effect on LPS-induced inflammatory response in RAW264.7 cells.To further explore its mechanism of action,western blot was used to detect the effects of “Scutellaria baicalensis-Radix Et Rhizoma Rhei” on TLR4,p-IκB-α and p-P65 in the NF-κB pathway,the phosphorylated proteins p-P38,p-JNK,and p-ERK in the MAPK pathway and the expression of p-STAT1 and p-STAT3 in the JAK-STAT pathway of RAW264.7 cells induced by LPS.The cellular immunofluorescence was used to examine the effect of “Scutellaria baicalensis-Radix Et Rhizoma Rhei” on the nuclear translocation of NF-κB P65 protein in RAW264.7 cells induced by LPS.In addition,the “Scutellaria baicalensis-Radix Et Rhizoma Rhei” drug pair and the NF-κB inhibitor JSH-23 were used to detect the effects of NF-κB on the inflammation in the LPS-induced RAW264.7 cells.The results showed that the “Scutellaria baicalensis-Radix Et Rhizoma Rhei” had a significant inhibitory effect on the proteins expressions of TLR4,and increased the proteins expressions of IκB-α and P65,and inhibited the phosphorylated proteins expressions of p-IκB-α,p-P65,p-P38,p-JNK,p-ERK,p-STAT1 and p-STAT3,and the“Scutellaria baicalensis-Radix Et Rhizoma Rhei” has a significant inhibitory effect on the nuclear translocation of NF-κB P65.Meanwhile,JSH-23(NF-κB inhibitor)administration significantly down-regulated the protein expression of p-P65 and the content of IL-6,IL-1β and TNF-α.In summary,the “Scutellaria baicalensis-Radix Et Rhizoma Rhei” drug pair had a significant inhibitory effect on the expressions and the secretions of inflammatory factors by inhibiting the activation of NF-κB,MAPK and STAT pathways,thereby achieving anti-inflammatory effects.These results suggest that the "Scutellaria baicalensis-rhubarb" drug pair is a potential anti-inflammatory drug.