Protective Effect Of Rhizoma Coptis On LPS-induced Injury Of Rat Liver Cells

Severe trauma,infection can lead to uncontrolled inflammatory reaction,and ultimately lead to endotoxemia and liver function failure.The results show that Rhizoma Coptis(Rhizoma Coptis RC)has antibacterial,anti-inflammatory,anti-tumor,analgesic,antioxidant,hypoglycemic,liver detoxification and many other biological activities,but the protection of liver cell injury caused by the LPS and the mechanism has not been reported.So this experiment with Rhizoma Coptis(RC)water extract for drug trials,dexamethasone(DEX)as a positive control drug,cultured rat liver cells(BRL cells)as target cells by lipopolysaccharide(LPS)construction of liver cell injury model,following several aspects of the study: CCK-8 method was used to screen the concentration of no toxicity and the damage concentration of LPS,Cell survival rate was detected when RC and LPS were co acting on BRL cells at 24 h by CCK-8 method,the apoptosis rate of BRL cells was tested by flow cytometry,two signaling pathway of NF kappa B,IRF3 in TLR4 and variation of the expression of inflammatory factor were detected by RT-PCR,NF kappa B p65 protein nuclear transfer was observed by fluorescence microscope.The results are as follows:(1)Screening of non-toxic concentration of RC and the concentration of LPS damage: The non-toxic concentration of RC was 0.175 mg/mL,the damage concentration of LPS was 0.1 mg/mL;At the same time,it was found that the concentration of RC in mg/mL 0.0625~0.125 could promote the increment of BRL cells.Once again,it is considered that the 0.1 RC mg/mL promotes the proliferation of the best.(2)Detection of cell viability in BRL cells treated with LPS and RC for 24 h: CCK-8 assay results showed that,compared with the LPS treated group,RC in 0.175 mg / ml can enable BRL cell survival rate from 73.79% increase to 81.62%,0.1 mg/mL can increase the survival rate of BRL cells to 91.23%.that is,the concentration of 0.1 mg/mL,the protective effect is better.(3)Apoptosis rate detection: BRL cells in normal control group,the LPS model control group,0.175 mg/mL RC + LPS group,0.1 mg/mL RC + LPS group,0.1 mg/mL Dex + LPS group total apoptosis rate(%)were: 0.32 ± 0.04,19.26 ± 0.65,16.43 ± 0.73,9.33 ± 0.47 and 6.34 ± 0.43,total cell apoptosis rate in drug treatment group was significantly lower than that of LPS control group(p < 0.01),Among them,RC was 0.1 mg/mL,the apoptosis rate of BRL was reduced from 19.26% to 9.33%.RC can effectively inhibit the LPS induced apoptosis of BRL cells,and RC in 0.1 mg/mL when the effect is better.(4)Fluorescence quantitative PCR detection: Compared with normal control group,LPS can enhance the TNF alpha,IL-6,IL-1 beta,TLR4,NF-kappa B and IRF3 m RNA expression significantly(p < 0.05),which indicated that the LPS induced inflammatory injury relates to TLR4/NF-kappa-B pathway and TLR4/IRF3 pathways and lead to inflammatory mediators such as IL-1 beta,IL-6,TNF alpha release,resulting in liver cells damage eventually.Compared with LPS treatment group,RC intervention group ould inhibit the expression of gene above(p < 0.05),and play a protective role in liver injury,its mechanism are related with the TLR4/NF-kappa-B pathway and TLR4/IRF3 pathways.(5)Result of immunofluorescence detection.BRL cells in the normal control group,NF kappa B p65 protein labeled by FITC mainly distributed in the cytoplasmshow green fluorescence,and the DAPI staining nuclei were blue fluorescence.After treatment of RC,the BRL cells nuclear color showed blue green at different degrees.The results show that NF kappa B p65 protein transfer from the cytoplasm to the nucleus under the action of LPS.and the drug treatment can inhibit NF kappa B p65 nuclear transfer induced by LPS at different degree,BRL nuclear color changed to varying degrees of blue-green.The RC intervention group can inhibit LPS induced B nuclear transfer of NF-nuclear p65,and the effect was obvious when RC was 0.1 mg/mL.Conclusion: Rhizoma Coptis has significant protective effect on rat liver cell injury induced by LPS,and it can significantly improve the cell growth and inhibit cytotoxicity induced by LPS,decline the apoptosis rate.The mechanism was that Rhizoma Coptis can influence TLR4 signaling pathway,hindering activation of NF kappa B and IRF3 pathway,inhibit NF kappa B p65 transfer into the nucleus,reduce the inflammatory factors such as TNF alpha,IL-1 beta and IL-6 release.