Study On The Adverse Reaction Of Gardenia Jasminoides Ellis And Mechanism Of Liver And Kidney Toxicity

[Object]In order to reasonably apply gardenia,master its relevant toxicity data and understand the toxic reaction of gardenia,this paper takes gardenia jasminoides Ellis as the research object,and carries out relevant research on its toxic reaction substances and its hepatorenal toxicity mechanism.It is hoped that through the above research,the chemical components that gardenia jasminoides Ellis can cause toxic reaction can be further clarified,the toxic effect of gardenia jasminoides Ellis on liver and kidney can be clarified,and the toxic molecular mechanism can be discussed,so that gardenia jasminoides Ellis can avoid or reduce toxic effect on the basis of exerting pharmacological activity,thus laying a foundation for the development of gardenia resources and rational clinical application.[Research methods and results]1. The toxic components of Fructus Gardeniae were screened based on in vitro cells.Water decoction method and alcohol extraction method were used to extract Fructus Gardeniae respectively.CCK-8 method was used to detect the activity of the extracts on BRL-3A cells.The inhibition rate showed that the water extract inhibited rate was higher than that of alcohol extract.LC-ESI-MS/MS method was developed for the extraction of Fructus Gardeniae.According to the peak time,mass spectrometry data and standard comparison,it was determined 9 compounds such as Shanzhiside,Geniposidic acid,Gardenoside,Genipin-1-Gentiobioside,Chlorogenic acid,Geniposide,Crocin-?,Crocin-?and Crocetin.Subsequently,CCK-8 method was used to screen 9 compounds of Fructus Gardeniae and the metabolite of geniposide is genipin for cell viability.The comparison of the cell survival rate calculations revealed that the iridoid terpenoids Shanzhiside,Gardenoside,Genipin-1-gentiobiside,Geniposide and Genipin have inhibitory effects on cells.The Crocetin,Crocin-?and Crocin-?can promote cell growth,while Chlorogenic acid and Geniposidic acid cell viability of the blank group were similar,not significant.Furthermore,Geniposide and Genipin were selected for cell cycle,apoptosis,and fluorescence color analysis.The results showed that Geniposide and Genipin can cause G₂/M arrest in the cell cycle,prolong S phase,and cells apoptotic rate increased with the increase of drug concentration,and the number of cells decreased.2. Study on hepatotoxicity and nephrotoxicity of Fructus Gardeniae in ratsAccording to the cell component screening results,the liver and kidney toxicity of Fructus Gardeniae was further evaluated.The rats were divided into blank group and different doses of water extract group,geniposide group and genipin group administration group.Observation,analysis and evaluation of the condition,liver and kidney tissue morphology and histopathology,and detection of biochemical indicators and inflammatory factors in rat serum.The results showed that:compared with the control group,the rats in the administration group lost weight,and the rats in the individual group would die,and their liver and kidney biochemical indexes AST,ALT,ALP,CRE and BUN increased.In particular,the high-dose group of the administration group was more significant,and the levels of serum inflammatory factors TNF-?,IL-6?,and NO levels have also increased to varying degrees.Pathological examination microscopic observation showed that the liver and kidney tissues had significant inflammatory cell infiltration and swelling in the high dose group.These biochemical indexes and histopathological changes all proved that the liver and kidney cells of rats were damaged under the influence of high dose gardenia toxic components.3. Transcriptome effects of hepatotoxicity of Fructus GardeniaeGene sequencing and analysis were carried out on rat liver tissues before and after gardenia administration based on transcriptomic technology.Total RNA was extracted from liver tissues by TRIzol?reagent.After the quality evaluation was qualified,the samples were prepared and sequenced.Then gene comparison was carried out to find differentially expressed genes.Gene expression analysis was carried out using GO and KEGG databases to clarify gene function and enrichment pathway.The results showed that compared with normal liver tissue genes,3122differentially expressed genes were screened in gardenia aqueous extract administration group,including 1703 genes with down-regulated expression and 1419 genes with up-regulated expression.GO analysis was mainly concentrated in the regulation process of cell migration,and KEGG was mainly concentrated in fatty acid biosynthesis,proteasome and other pathways.617 differentially expressed genes were selected from geniposide administration group,including 370 genes with down-regulated expression and 247 genes with up-regulated expression.GO analysis was mainly concentrated in redox enzyme activity process,and KEGG was mainly concentrated in circadian rhythm,steroid hormone biosynthesis and other pathways.1,451 differentially expressed genes were screened in genipin administration group,of which686 were down-regulated and 765 were up-regulated.GO analysis was mainly concentrated in bile acid and bile salt transport process,and KEGG was mainly concentrated in amino acid metabolism and other pathways.There are 176 common differentially expressed genes in the three administration groups and the gene expression trends of gardenia extract and genipin administration group are consistent.Subsequently,q-PCR was used to verify the common differentially expressed genes such as Rhoc,Akr7a3,Gpx2,Isg20,Tmed3,Cbr1,and the m RNA expression level was consistent with the transcriptome analysis data.4. Study on the molecular mechanism of hepatorenal toxicity induced by Fructus GardeniaeFructus Gardeniae studied from oxidative stress,inflammatory pathway and apoptosis.First,the levels of SOD,GSH and MDA in liver and kidney tissues of rats were detected,and the expressions of NF-?Bp65 and Caspase-3 in liver and kidney tissues were detected by immunohistochemical staining.The liver and kidney of rats were detected by Western Blot Protein expression of Nrf2,HO-1,NF-?Bp65,Cox-2,i NOS,Caspase-3 and Bax in tissues,and molecular docking simulation technology was used to further explain the mechanism of action of TNFR1 ligands and receptors.The experimental results showed that the levels of SOD and GSH in liver and kidney tissues of rats were reduced to varying degrees,and the levels of MDA were increased.The expressions of NF-?Bp65,Cox-2,i NOS,Bax and Caspase-3 were increased,and Nrf2,HO-1 protein expression decreased,and the molecular docking simulation results of Geniposide,Genipin and TNFR1 were the lowest binding energy of-7.34 kcal/mo L and-6.97kcal/mo L.[Conclusion]Through in vitro screening of 10 chemical components in Fructus Gardeniae,iridoid components are found to be the main substances causing toxic reactions in Fructus Gardeniae.Its hepatorenal toxicity mechanism is related to the fact that Fructus Gardeniae components can activate inflammatory pathways and reduce anti-oxidative stress ability,thus promoting cell apoptosis and showing differential expression of transcription genes in cell transduction,metabolism,redox and other aspects.Therefore,Fructus Gardeniae should not be taken in large doses or for a long time.This study has laid a certain foundation for toxicological research and rational application of Fructus Gardeniae.