Toxicity And Efficacy Of Water-Eluted Fraction From Siegesbeckiae Herba Against Human Embryonic Lung Fibroblasts

Objective:Molecular biological techniques were used to detect the toxic effects of Water-Eluted Fraction from Siegesbeckiae Herba（SWEF）on Human embryonic lung fibroblasts（MRC-5）and on the expression ofα7 nicotinic acetylcholine receptor（α7nAchR）and inflammatory factors in MRC-5 cells,so as to find the material basis of toxicity and efficacy of Siegesbeckiae herba,and provided a scientific basis for the rational application of Siegesbeckiae herba.Methods:1.Toxic effect of SWEF on MRC-5 cells According to the results of pre-experiment,MRC-5 cells in logarithmic growth phase were divided into blank control group（CK group）and different concentration drug group(1,6,10,20,50mg·ml^-1).The survival rate of cells was detected by CCK-8 method,and the 50%inhibitory concentration(IC₅₀)was calculated.The apoptosis and necrosis rate of cells treated with different concentrations of drugs were detected by Flow cytometry and Trypan blue staining.2.Effects of SWEF onα7nAchR gene expression and inflammatory factors in MRC-5cells MRC-5 cells in logarithmic growth phase were divided into CK group and different concentration drug group(1,6,10,20,50mg·ml^-1).The effect of SWEF on the expression ofα7nAchR m RNA in MRC-5 cells was detected by real-time fluorescence quantitative,and the levels of interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）in supernatant were detected by Enzyme-linked immunosorbent assay（ELISA）.3.Effects of silencingα7nAchR and adding 4mg·ml^-1SWEF onα7nAchR Gene,protein expression and inflammatory factors in MRC-5 cells MRC-5 cells in logarithmic growth phase were divided into CK group,negative control group（NC group）,si RNA（small interference RNA）group,CK+SWEF group,NC+SWEF group and si RNA+SWEF group.The effects of silencingα7nAchR and adding SWEF on the gene expression ofα7nAchR,IL-6 and TNF-αin MRC-5 cells were detected by real time fluorescence quantitative assay.The effects of silencingα7nAchR and adding SWEF on the expression ofα7nAchR protein in MRC-5 cells were detected by Western Blot.ELISA method was used to detect the effects of silencingα7nAchR and adding SWEF on the levels of IL-6 and TNF-αin the supernatant of MRC-5 cells.Results:1.SWEF has obvious toxic effect on MRC-5 cells CCK-8 detection showed that SWEF significantly inhibited the survival rate of MRC-5 cells.When the concentration of the drug was 6mg·ml^-1,the cell survival rate was about 70%.The cell survival rate of10mg·ml^-1and above was less than 10%,which was statistically significant compared with the blank group（p<0.01）.The IC₅₀was 6.03mg·ml^-1,and there was no significant change in the solvent control group.The results of Flow cytometry and Trypan blue staining showed that obvious apoptosis and necrosis occurred at the concentration of 6mg·ml^-1（the apoptosis rate was about 13%,and the apoptosis+necrosis rate was about 21%）.At the concentration of 50mg·ml^-1,the proportion of apoptosis and necrosis was more than 80%,which was consistent with the results of CCK-8 detection（p<0.01）.The results showed that the safe dose concentration of the drug was within 6mg·ml^-1.2.SWEF can up-regulate the expression ofα7nAchR m RNA and decrease the level of inflammatory factors in MRC-5 cells The results of PCR and ELISA showed that SWEF at different concentrations(1mg·ml^-1to 50mg·ml^-1)could up-regulate the expression ofα7nAchR m RNA in MRC-5 cells by tens to hundreds of times（P<0.05）,while the solvent control group at the corresponding concentration only increased the expression ofα7n ACh R m RNA by about 7 times at the concentration of 50mg·ml^-1（the highest concentration）.The levels of IL-6 and TNF-αdecreased at the concentration of 1mg·ml^-1to 50mg·ml^-1（P<0.05,P<0.01）.Correlation analysis showed that there was a negative correlation between the expression ofα7nAchR m RNA and the level of pro-inflammatory factors,indicating that the down-regulation of pro-inflammatory factors by SWEF is related to the up-regulation ofα7nAchR m RNA expression.3.α7nAchR gene silencing test confirms that SWEF containsα7nAchR agonist-like substance The safe drug concentration of 4mg·ml^-1was used in the experiment ofα7nAchR gene silencing（small interference RNA）.After silencingα7nAchR gene,the expression ofα7nAchR m RNA and protein decreased（P<0.05 P<0.01）,the level of inflammatory factors did not change significantly,but after adding SWEF,the expression ofα7nAchR m RNA and protein increased（P<0.05 P<0.01）,and the level of inflammatory factors decreased significantly（P<0.05 P<0.01）.It is suggested that SWEF containsα7nAchR agonist-like substance,which can increase the expression ofα7nAchR and decrease the level of inflammatory factors.4.Excessiveα7nAchR agonist-like substances may be one of the toxic components of Siegesbeckiae herba In the previous acute toxicity test,it was found that after intragastric administration of SWEF,mice showed rest prone,bluish blue,curled up,intermittent convulsion,tremor and other symptoms,which were similar to the nicotinic symptoms of acetylcholine（ACh）accumulation in the body,whileα7nAchR agonists have ACh-like effects.MRC-5 cell gene silencing test confirmed that SWEF containsα7nAchR agonist-like substances,so we speculate that excessiveα7nAchR agonist-like substances may be one of the toxic components of Siegesbeckiae herba and need to be further studied.Conclusion:In this study,molecular biological methods were used for the first time to observe the toxic effect of SWEF on MRC-5 cells and its effect onα7nAchR expression and inflammatory factors in MRC-5 cells before and after silencing gene,so as to explore the material basis of toxicity and efficacy of Rabdosia humilis.The results showed that SWEF(≥6mg·ml^-1)had significant toxic effect on MRC-5 cells.The pharmacodynamic study confirmed that SWEF containedα7nAchR agonist-like substance,which could increase the expression ofα7nAchR and decrease the level of inflammatory factors.There was a significant negative correlation between the expression ofα7nAchR and the level of inflammatory cytokines.Combined with the results of previous animal acute toxicity experiments,we speculated that the excessiveα-7nAchR agonist-like substance may be one of the toxic components of Siegesbeckiae herba.