Analysis Of Chemical Constituents In Moxa Smoke And Its Acute Toxicity In SD Rats

Objective:The chemical constituents in moxa smoke were enriched and analyzed,and the toxic and side effects of moxa smoke were preliminarily discussed combined with network toxicology.Through the study of acute toxicity in moxa smoke to SD rats,the median lethal concentration was determined,and the target prediction of network toxicology was verified.Transcriptomics was used to compare the changes of lung related gene expression between all dead group and all undead group,and the target genes were further screened for qRT-PCR verification,so as to preliminarily explore the toxic and side effect mechanism of high concentration moxa smoke on rats,lung.To provide scientific data for the follow-up safety evaluation of moxa smoke,and further inherit and develop moxibustion technology.Method1.HS-GC-MS was used to analyze the chemical components of poaitang six kinds of moxa sticks,including Ordinary,Qing,Ming,Yuan,Song and Tang moxa sticks.After burning 3g moxa sticks respectively,20 m L moxa smoke were collected in a headspace bottle and detected by NIST11.L.2.Cyclohexane,ethyl acetate,n-butanol,anhydrous ethanol and water were used as absorption solvents to enrich and analyze the chemical components of Qing moxa smoke.The components were identified by GC-MS.The toxic chemical components were inquired through the CTD database,and the molecular network of target proteins and their biological pathways were constructed by Network Toxicology.3.36 SPF SD rats were randomly divided into 6 groups.The acute toxicity test was carried out in the self-made exposure cabinet（Patent No.:ZL 2020 2 0634748.8）.According to the results of the pre-experiment,the group in which all rats survived was set as the minimum dose group E,the group in which all rats died was set as the maximum dose group A,and three dose groups were set in the middle.Each group was exposed for 2 hours and observed for 14 days.Using H&E staining to observe the pathological changes of lung,liver,heart and kidney.4.Total RNA was extracted from lung tissues of all dead group and all living group,and transcriptome sequencing was performed.The sequencing data were filtered and assembled to analyze.Unigenes were annotated by GO,KEGG,COG,NR,Swiss prot and Pfam databases.All unigenes were screened by P-value<0.05&|log2FC|≥1based on DESeq2 software.5.According to the target gene design qRT-PCR primers,using the dissolution curve to carry out the specificity of them.For the remaining RNA in the library construction,firstly we removed the genomic DNA,and then took reverse transcription.7500 system software was used to analyze the stability of gene expression.GAPDH as endogenous reference,and the 2^{^（-ΔΔCt）}method was used for calculation and standardization.Result1.A total of 109 chemical components including N-methyl pyrrole,3-methyl pyrrole,toluene,1-octene,furfural and ethylbenzene were identified by HS-GC-MS with 6different processing techniques.Among them,there were 32 kinds of chemical components.The main active chemical components（+）-limonene were the highest,while camphor and cinnamonitrile were low.2.A total of 294 chemical components,including 139 in cyclohexane,145 in ethyl acetate,60 in n-butanol,89 in anhydrous ethanol and 77 in water,were identified in Qings,moxa smoke.A total of 112 toxic chemical components were found,and 25molecular networks were constructed,whose functions were mainly related to cell signal transduction,nucleic acid metabolism,inflammatory response,organ damage and apoptosis,etc.54 biological pathways of action were identified,mainly those related to cardiotoxicity,hepatotoxicity,and nephrotoxicity.3.In acute toxicity test,the maximum tolerable dose of moxa in group E was 290.036g/m³,and the LC₅₀ was 537.65 g/m³.Compared with the data of control group,there were significant differences in liver index among group A（P<0.01）,group B（P<0.01）,group C（P<0.01）and group D（P<0.01）,but there was no statistical difference in group E（P>0.05）.There was significant difference in kidney index between group A（P<0.05）and group B（P<0.05）,but there was no statistical difference among the other three groups.There was significant difference in lung index between group A（P<0.01）and group B（P<0.01）,but there was no statistical difference among the other three groups.The results of heart index in each group had no statistical significance.And the liver index and lung index were significantly higher than the control group.The results of pathological sections showed that compared with the control group,the arrangement of myocardial cells in the dead rats was disordered,and with the increase of moxa smoke concentration,inflammatory cells appeared between myocardial cells,occasionally vacuolar degeneration;hepatocyte degeneration,necrosis,nuclear fragmentation,cytoplasmic red staining,cytoplasmic vacuolization,obvious inflammatory cell infiltration appeared;the renal vesicle cavity increased,the renal vesicle increased.There was dilation of the mesentery,swelling of the epithelium,occlusion of the lumen,incomplete alveolar structure and increased inflammatory cell infiltration.Compared with the control group,there was no significant change in the pathological section in group E.4.For RNA samples used for library construction,OD260/280 1.80–2.20,RIN value greater than or equal to 8.0,RNA purity was good and qualified.Transcriptomic sequencing of 24 samples was completed,and a total of 181.14 Gb Clean Data was obtained.Most transcripts and genes were annotated using the COG database（94.79%and 94.90%,respectively）,followed by the GO database（87.59%and 87.06%,respectively）and the KEGG database（67.54%and 64.86%,respectively）.Compared with the control group,286,400 and 108 genes were up-regulated and 200,477 and 78genes were down-regulated in group A,B and E,respectively.5.Compared with the data of control group,in group A and group B the expression levels of MAFF,HSPA1B and HSPA1A were increased.There was significant difference of MAFF gene expression in group A.And in group B there were statistical differences in gene expressions among MAFF,HSPA1B and HSPA1A.In group E,MAFF,HSPA1B and HSPA1A gene expressions were decreased,AOC1 and MX2 gene expressions were increased（P<0.05）.The results of qRT-PCR showed that the expression trend of the target gene was consistent with that of RNA-seq,which indicated that RNA-seq data could reflect the change of gene expression reliably.Conclusion1.The chemical component of moxa smoke in moxa sticks with different processing technology is different,which contains both beneficial and harmful components.2.The main biological pathways of toxic chemical components of moxa smoke show that high concentration of it will cause toxicity to the heart,liver and kidney.However,there have been no clinical report of toxic and side effects.Therefore,it is not necessary to"discuss toxicity"in isolation,but to comprehensively consider the clinical features of Traditional Chinese Medicine.3.High concentration of moxa smoke can produce certain pathological changes in the tissue,which compared with the control group.But there is no significant change of undead group in the heart,liver,kidney and lung,suggesting that the dose is closely related to the toxicity.4.The results of pulmonary transcriptome sequencing showed that exposure to moxa smoke had a significant effect on the expression of lung related genes in rats,which was the result of multiple pathways and signal molecules.5.Compared with the control group,the variation trend of the expression level in MAFF,HSPA1B,HSPA1A,AOC1 and MX2 genes showed that high concentration of moxa smoke may produce cellular stress responses and cause tumor and inflammatory injury.It is suggested that it is necessary to keep ventilation during moxibustion,ensuring the safety of moxibustion treatment environment.