Study on difference ofanti-inflammatory activity and non-targeted metabolomics of 16HBE cells between a single decoction formulation of Hanchuan Zupa and its combination
Objective To examined the anti-inflammatory activity of Hanchuan Zupa according to the tradi-tional combination of decoction and single decoction formulations,and to studied the non-targeted metabo-lomics differences by UPLC-Q-TOF/MS.Methods 16HBE inflammatory cells were induced by lipopo-lysaccharide in vitro,and the dosages of single decoction formula group and the combined decoction group were screened using CCK-8 method;the mRNA expression levels of inflammatory factors IL-6,IL-8 and TNF-α in the cells were determined using Reverse(reverse transcription-polymerase chain reaction,RT-PCR)was used to determine the mRNA expression levels of the inflammatory factors IL-6,IL-8 and TNF-αin the cells,and UPLC-TOF/MS was used to analyze the cells for non-targeted metabolomics.They were divided into the normal group(16HBE cells+medium),the model group(16HBE cells+medium)and the model group(16HBE cells+medium).The cells were divided into normal group(16HBE cells+culture medium),model group(16HBE cells+culture medium+0.1 μg/mL LPS),Hanchuan Zupa single decoction formula group(16HBE cells+culture medium+0.1 μg/mL LPS+50 μg/mL single decoction formula),and Hanchuan Zupa combined decoction group(16HBE cells+culture medium+0.1 μg/mL LPS+50 μg/mL combined decoction).To investigate the metabolic pathways involved in the anti-inflam-matory activity of Hanchuan Zupa granules,and to search for the differential metabolites associated with the anti-inflammatory activity of Hanchuan Zupa granules.Results Compared with the normal group,the cell growth and survival rate of the model group was not restricted at a concentration of 0.1 μg/mL,and the difference was not statistically significant(P＞0.05).In the combined decoction group,the cell growth and survival rate was not affected at a concentration of 0～400 μg/mL,and the cell growth and survival rate wasdecreased at a concentration of ≥800 μg/mL,and the difference was statistically signifi-cant(P＜0.01);while in the single decoction group,the cell growth and survival rate was not restricted at a concentration＜50 μg/mL,and it wasdecreased at a concentration ≥100 μg/mL,and the difference was statistically significant(P＜0.01).Whereas in the single decoction formula group,cell growth and surviv-al were not restricted at concentrations＜50 μg/mL,and it was decreased at concentrations ≥100 μg/mL,with statistically significant differences(P＜0.01).The results of the RT-PCR experiments showed that,com-pared with the normal group,the expression of IL-6,IL-8 and TNF-α mRNAs in the model group was in-creased,with statistically significant differences(P＜0.00).The results of the RT-PCR experiments showed that,compared with normal group,the model group's IL-6,IL-8 and TNF-α mRNA expression was increased,with statistically significant differences(P＜0.001),and 50 μg/mL single decoction for-mulation was decreased the expression of IL-6,IL-8 and TNF-α mRNA compared with 100 μg/mL,and the difference was statistically significant(P＜0.001).Under the intervention of the same drug concentra-tion,the expression of IL-6,IL-8 and TNF-α mRNA wasincreased in the model group compared with that in the normal group,and the difference was was statistically significant(P＜0.001).Compared with the model group,the single-decoction formulation group and the combination-decoction group could simulta-neously reduce the expression of IL-6,IL-8 and TNF-α mRNA,and the difference was statistically signifi-cant(P＜0.001).When combination-decoction group was compared with the single-decoction formulation group,the expression of IL-6 and IL-8 in the combination-decoction group was lower than that in the sin-gle-decoction formulation,and the difference was statistically significant(P＜0.05).Metabolomics analy-sis identified 68 endogenous metabolites that were mainly related to tryptophan metabolism,glycerophos-pholipid metabolism and pyrimidine metabolism between the model group and the combined decoction group,while 62 endogenous metabolites were identified between the model group and the single decoction formula group,which were mainly related to glycerophospholipid metabolism,arginine and proline metab-olism,and so on.Conclusion The anti-inflammatory effects of the single decoction formula of Hanchuan Zupa and the combined decoction of the formula on the LPS-induced inflammatory cell model differed,and the results of the non-targeted metabolomics analysis indicated that the 2 modalities exerted their anti-in-flammatory activities through different metabolic pathways.
万方数据
维普
