Anti-inflammatory Effect And Mechanism Of Huangqin Tang

Huangqin Tang is one of famous Chinese medicine formulas in "Shanghan Lun".It includes four herbal medicines, Radix Scutellariae, Radix Paeoniae Alba, Radix Rhizoma Glycyrrhizae and Jujubae Fructus. Huangqin Tang is used for the treatment of ulcerative colitis、acute gastroenteritis and bacillary dysentery diseases in clinical. It has been proved to have effects of anti-inflammatory, antipyretic and analgesic in pharmacological researches.But the anti-inflammatory mechanism of Huangqin Tang is not clearly.Now,the researches about it almost fous on macro level.There is little reach on molecular biology level.This study is guided by the theory of traditional Chinese medicine. Modern science and technology are used to research anti-inflammatory mechanism of Huangqin Tang. Rats with Ulcerative Colitis (UC) and RAW264.7inflammatory cell are studied to provide the scientific basis for Huangqin Tang in this research.Objective:1、Build UC rats and macrophage inflammatory modes, investigate the effect of Huangqin Tang and prove its anti-inflammatory effect.2、Investigate the influence of Huangqin Tang on inflammatory signaling pathways to research its anti-inflammatory mechanism.Methods:1、The treatment of ulcerative colitis rats and the anti-inflammatory mechanism of Huangqin Tang.The mode of UC rats with cell immunoreactivity were made using compound method(Trinitrobenzene sulfonic acid and ethanol). Rats were randomly divided into control group,model group, SASP group, high dose,middle dose and low dose of Huangqin Tang. The food intake,body weight and microscopic damage of rats in each group were evaluated after being treated for five days. The blood and colon tissue were also collected. Production of NO was detected by Griess assay, the expression levels of IL-6,TNF-α,PGE2were detected by ELISA. ICH method was undertaken to determine the expression of NF-κB p65protein in colon tissue.2、The treatment of LPS RAW264.7inflammatory cell caused by LPS and the anti-inflammatory mechanism of Huangqin Tang.2.1The cytotoxic effect of Huangqin Tang on cells.RAW264.7cells were maintained in DMEM containing10%FBS,100U/mL of penicillin and100μg/mL of streptomycin at37℃in a5%CO2incubator. RAW264.7cells (1×105cells/mL) were plated in96-well microtiter plates.Using the MTT-based colorimetric to evaluate the cytotoxic effect of Huangqin Tang.2.2The influence of Huangqin Tang on producting of NO.RAW264.7cells (5×105cells/mL) were plated in24-well microtiter plates.Every well was plated in0.5mL cells.The cells were pretreated with Huangqin Tang for6h.After that, the supernatant liquor was removed and putted into LPS to stimulate cells.The model group cells were only stimulated by LPS. The control group cells were putted nothing. After cultivating18h, supernatant liquor was collected and used nitrate reductase to measure NO.2.3The influence of Huangqin Tang on producting of IL-6,TNF-α and PGE2.RAW264.7cells (5×105cells/mL) were plated in24-well microtiter plates.Every well was plated in0.5mL cells.The cells were pretreated with Huangqin Tang for6h. After that, the supernatant liquor was removed and putted into LPS to stimulate cells.The model group cells were only stimulated by LPS. The control group cells were putted nothing. After cultivating18h, supernatant liquor was collected and used ELISA to measured IL-6,TNF-α and PGE2.2.4The influence of Huangqin Tang on NF-κB p65mRNA expressing.RAW264.7cells (1×106cells/mL) were plated in6-well microtiter plates.Every well was plated in2mL cells.The cells were pretreated with Huangqin Tang for6h and PDTC for2h.After that, the supernatant liquor was removed and putted into LPS to stimulate cells.The model group cells were only stimulated by LPS. The control group cells were putted nothing. After cultivating1h, all cells were collected and used Trizol to extract RNA.The expression of NF-κB p65mRNA was measured by RT-PCR.2.5The influence of Huangqin Tang on NF-κB p65protein expressing.RAW264.7cellswere plated in culture dishes. The cells were pretreated with Huangqin Tang for6h and PDTC for2h.After that, the supernatant liquor was removed and putted into LPS to stimulate cells.The model group cells were only stimulated by LPS. The control group cells were putted nothing. After cultivating1h, all cells were collected and used cell lysis buffer to extract total protein and cell nucleus and cytoplasm protein. The expression of NF-κB p65protein was measured by Western blot.2.6The influence of Huangqin Tang on MAPK signaling pathway.The cells were treated the same to above. Inhibitor groups cells were putted into p38、 ERK、JNK signaling pathway inhibitor. After been stimulated by LPS for1h, extracted RNA and used RT-PCR to measure ERK mRNA expression. After been stimulated by LPS for30min, extracted RNA and used RT-PCR to measure p38and JNK mRNA expression.After been stimulated by LPS for1h, extracted total protein and used Western blot to measure p38、ERK and JNK protein expression.2.7The influence of Huangqin Tang on JAK-STAT signaling pathway.The cells were treated the same to above. Inhibitor group cells were putted into JAK signaling pathway inhibitor. After been stimulated by LPS for6h, extracted RNA and used RT-PCR to measure JAK mRNA expression. After been stimulated by LPS for6h, extracted total protein and used Western blot to measure JAK protein expression.2.8The influence of MAPK and JAK-STAT signaling pathway on NF-κB signaling pathway.Inhibitor groups cells were putted into p38,ERK,JNK and JAK signaling pathway inhibitor. After been stimulated by LPS for1h, extracted RNA and total protein, used RT-PCR to measure NF-kB p65mRNA and used Western blot to measure NF-κB p65protein expression.Results: 1、The treatment of ulcerative colitis rats and the anti-inflammatory mechanism of Huangqin Tang.The food intake and body weight of model group rats were lower than that of control group. The expression levels of NO,IL-6,TNF-α,PGE2in serum and NF-κB p65protein of colon tissue in model group were higher than that of control group. The above indexes were improved in high and middle dose of Huangqin Tang groups.2、The treatment of LPS RAW264.7inflammatory cell caused by LPS and the anti-inflammatory mechanism of Huangqin Tang.2.1RAW264.7cells released a lot of NO,IL-6,TNF-α,PGE2after been stimulated by LPS.Following pretreatment with Huangqin Tang, LPS-induced production of NO,IL-6,TNF-α,PGE2were significantly inhibited.2.2The expression of NF-κB p65mRNA and protein in RAW264.7cells were increased after LPS stimulation. And cell nucleus protein was more than cytoplasm protein. Following pretreatment with Huangqin Tang, the expression of NF-κB p65mRNA and protein were significantly inhibited, the nuclear translocation of NF-κB p65was also inhibited.2.3The expression of p38,ERK and JNK mRNA and protein in RAW264.7cells were increased after LPS stimulation. Following pretreatment with Huangqin Tang, the expression of p38,ERK and JNK mRNA and protein were significantly inhibited.2.4The expression of JAK mRNA and protein in RAW264.7cells was increased after LPS stimulation. Following pretreatment with Huangqin Tang, the expression of JAK mRNA and protein was significantly inhibited.2.5Compared with model group, the expression of NF-κB p65mRNA and protein in p38inhibitor group were significantly inhibited. The expression of NF-κB p65protein in ERK and JNK inhibitor groups were significantly inhibited.There is a trend of decrease about expression of NF-κB p65mRNA in JAK-STAT inhibitor group.Conclusion:1、Huangqin Tang can decrease the expression levels of NO,IL-6,TNF-α and PGE2. 2、Huangqin Tang can inhibit the relative activity of NF-κB p65,then reduce the expression levels of NO,IL-6,TNF-α and PGE2.3、Huangqin Tang can inhibit the relative activity of MAPK and JAK-STAT signaling pathway to play a role of anti-inflammatory.4、There is a strong regulatory of p38signaling pathway on NF-κB signaling pathway. There is also a regulatory of ERK,JNK and JAK-STAT signaling pathway on NF-κB signaling pathway,but the regulatory is less than p38signaling pathway.