| Part I Experimental Research on Toxicological Pathology of Acute Cantharidin Poisoning in Sprague Dawley RatsObjective:We aim to enrich the contents of toxicological pathology of cantharidin poisoning,which is of great significance for assessing the safety of drug use and monitoring adverse drug reactions.Methods:Nine dose groups（1,1.3,1.69,2.19,2.82,3.67,4.73,6.15,8 mg/kg,respectively）of cantharidin and a control group,with the method of intragastric administration,were formed to establish a model of acute cantharidin poisoning using ninety-six healthy Sprague-Dawley（SD）rats（forty-eight male and forty-eight female）.The toxic manifestations,death time,death cases,temperature changes before and after exposure,score of visceral pain and neurological deficit score were recorded in detail.Improved Karber method and BLISS weighted regression method were used to calculate the lethal dose interval of acute cantharidin poisoning.The pathological changes of organs were counted and scored from the aspects of degeneration and necrosis,hemorrhage,edema and inflammatory cells infiltration by the method of hematoxylin-eosin staining.Results:We observed that reduced activity,cold clammy skin,hematuria,respiratory abnormality,visceral pain,penile erection/vaginal bleeding,and neurological deficit were the toxic manifestations of cantharidin poisoning.The temperature changes of rats in experiment groups were higher compared with that in the control and no significant gender difference was found in each group.Both of the scores of visceral pain and neurological deficit were increased with the dose of cantharidin and no significant gender difference was found in each group.The median lethal dose(LD50)of the cantharidin was 2.68 mg/kg and 2.67 mg/kg respectively analyzed by the improved Karber method and BLISS weighted regression method.The target organ damages caused by cantharidin in different organs were judged by histopathologic score and the results indicated that the damages including hypoxia,inflammation and edema,the highest of score was derived from the heart and histopathologic scores of other organs（from high to low）were lung,kidney,liver,intestine,brain,stomach,bladder,sciatic nerve,testis,spleen,facial nerve,and esophagus.Conclusions:The main toxic manifestations of acute cantharidin poisoning were reduced activity,temperature changes,visceral pain and neurological deficit.And the scores of visceral pain and neurological deficit were positively related to the dose of cantharidin.We adapted the LD50（2.67 mg/kg）of cantharidin in acute oral toxicity for further study.The pathological changes of multiple organs were ischemic and hypoxic,inflammation,hemorrhage and so on,and the heart was the main target organ damage of cantharidin exposure.Part II Experimental Research on Molecular Biology of Cantharidin-induced Cardiotoxicity in Sprague Dawley RatsObjectives:To explore the biomarkers which may be used to assess the severity of myocardial injury induced by cantharidin.To explore the biomarkers which may be used for the pre-and postmortem diagnosis of cantharidin-induced myocardial injury and PMI estimation.To screen and verify the differentially expressed genes（mRNA and miRNA）in heart of rats that were exposed to cantharidin,especially the mRNAs and miRNAs were associated with circulatory system diseases.Methods:Twenty-five SD rats were randomly divided into 5 groups according to the administration dose of cantharidin(0,0.5 LD50,LD50,1.5 LD50and 2 LD50,n=5 per group).The rats were killed and their hearts were removed 6 h after cantharidin administration.Immunofluorescence staining and western blotting were performed to investigate the effects of cantharidin on VEGF,HIF-1αand caspase9 expression.Forty male SD rats were randomly divided into control group（n=4）and cantharidin exposure（2.67 mg/kg）group（n=36）,the rats in cantharidin exposure group were randomly divided into nine groups according to the administration time（1,2,4,6,8,12,24,48,and 72 h,n=4 per group）.And fifty-four male SD rats were randomly divided into control group（n=24）and cantharidin（4 mg/kg）exposure group（n=30）,rats in each group were randomly divided into six groups（6,12,24,48,72,and 168?h after death,n=4 per time interval in control and n=5 per time interval in cantharidin exposure group）.The cardiac blood were collected in all rats at the given time and the determination of TN-T,VEGF and HIF-1αconcentrations in rat serum were followed by ELISA method.Thirteen male SD rats were randomly divided into control group（n=3）,low level(0.5LD50)and high level(LD50)of cantharidin exposure group（n=5 per group）.The rat hearts were removed 24 h after cantharidin administration and were stored at-80℃.TotalRNA was extracted andRNA-sequencing was performed to detect the mRNA expression in rat hearts.DEGseq method and MA-plot method were performed to detect and screen the differentially expressed mRNAs.Gene ontology（GO）analysis,kyoto encyclopedia of genes and genomes（KEGG）pathway enrichment analysis and gene set enrichment analysis（GSEA）were performed to explore the potential roles of the differentially expressed mRNAs.Bioinformatics analyses methods were used to establish the mRNA-miRNA interaction network and to predict the target miRNAs of the differentially expressed mRNAs.The real time polymerase chain reaction（RT-q PCR）was used to validate the differentially expressed mRNAs and miRNAsResults:Co-expression of VEGF,HIF-1αand caspase9 in heart of the cantharidin-treated rats was observed by immunofluorescence staining.The results of western blotting showed that the expression of VEGF in the groups of LD50,1.5 LD50and 2 LD50were significantly increased compared with that in the control group.The levels of HIF-1αfollowing exposure to cantharidin for different doses were higher than that in the control group.The expression of caspase9 in the groups of 1.5 LD50and 2 LD50were significantly increased compared with that in the control while it presented a descending trend in 0.5 LD50and LD50.The plasma levels of TN-T,VEGF and HIF-1αwere significantly increased in cantharidin exposure group compared with that in the control.The trends of TN-T,VEGF and HIF-1αexpression in each time interval after cantharidin administration indicated that the TN-T expression in each time interval was significantly increased compared with that in control and the peak value of TN-T expression was 1 h after administration.Besides the group of 12 h,the levels of VEGF in other time intervals were significantly increased compared with that in control and the peak value of VEGF expression was 2 h after administration.The change trend of HIF-1αin each time interval was smooth and the statistical difference was found in the groups of 6 h,8 h,and 24 h.The linear relationship between the expression of VEGF and TN-T was analyzed using Pearson correlation analysis.Statistically significant differences in the postmortem plasma expression of TN-T and the ratio of HIF-1α/TN-T were found between the cantharidin exposure group and control group,while we did not observe any significant differences in the expression of VEGF and HIF-1α,the ratio of VEGF/TN-T and VEGF/HIF-1α.between the two groups.The tendency of postmortem changes of all above biomarkers in control group and postmortem changes of TN-T and HIF-1α/TN-T in cantharidin exposure group were stable.In cantharidin exposure group,postmortem plasma VEGF levels in the group of48 h and 72 h were all significantly decreased compared with that in the group of 12 h and 168 h,respectively.And statistically significant differences in the postmortem plasma HIF-1αexpression were also found between the group of 168 h and 12 h,24 h,48 h;the ratios of VEGF/TN-T and VEGF/HIF-1αwere stable within 72 h but the ratios were significantly increased in group of 168 h.ROC curve showed that TN-T and HIF-1α/TN-T with higher postmortem diagnostic adequacy,and the area under the ROC curve of TN-T and HIF-1α/TN-T were 0.7809,0.7037 respectively.The linear regression equations between postmortem interval（PMI）and all above biomarkers suggested that VEGF/HIF-1αwith potential value in the estimation of the PMI.171 differentially expressed mRNAs were found in the three groups.GO analysis showed that the differentially expressed mRNAs were mainly enriched in extracellular matrix.KEGG pathway analysis indicated that the differentially expressed mRNAs were associated with organismal systems,environmental information processing,metabolism,cellular processes and genetic information processing pathway.KEGG pathway enrichment showed that the differentially expressed mRNAs between control and low dosage group,control and high dosage group,low dosage group and high dosage group were mainly enriched in 18,9,5 pathway respectively.Results of GSEA indicated that the significantly associated gene sets among the 3 groups were focal adhesion,ECM-receptor interaction,protein digestion and absorption pathway,and so on.We screened 5 differentially expressed mRNAs（Myh7,Tnc,Ldlr,11β-HSD2,Scd）with higher expression which were closely related to the circulatory system for further verification.The results of RT-q PCR showed that the expression of Myh7 and11β-HSD2 in cantharidin exposure groups were significantly increased while the expression of Tnc,Ldlr and Scd were decreased compared with that in control.Based on the mRNA-miRNA interaction network,1386 target miRNAs of the 171differentially expressed mRNAs were obtained by bioinformatics analyses methods.Among of them,216 target miRNAs were associated with Myh7,Tnc,Ldlr,11β-HSD2and Scd.Combined with the results of literature review,function and structural stability of target miRNAs,we finally screened mi R-133a-3p,mi R-150-3p,mi R-214-3p and mi R-146a-5p for verification.The results of RT-q PCR showed that the expression of mi R-133-3p in LD50group was significantly increased compared with that in control and 0.5 LD50group,the expression of mi R-146a-5p in cantharidin exposure groups were decreased compared with that in control.Compared with the control,the expression of mi R-150-3p and mi R-214-3p in cantharidin exposure groups were lower without statistical difference.Conclusions:Co-expression of VEGF,HIF-1αand caspase9 was presented in heart samples that were exposed to cantharidin,and the expression of VEGF,HIF-1αand caspase9 in cantharidin exposure groups were increased as the dosage increased.Plasma expression of TN-T and VEGF might be used for prediction and diagnosis of cantharidin-induced myocardial injury.Plasma expression of TN-T and HIF-1α/TN-T are cardiac biomarkers with higher diagnostic accuracy for postmortem diagnosis of cantharidin-induced myocardial injury.Plasma VEGF/HIF-1αexpression can be used for PMI estimation in cantharidin poisoning cases and it promises to be a biomarker for PMI estimation in other causes of death.Cantharidin poisoning can cause the changes of the mRNAs and miRNAs expression profiles in rat hearts.Both the differentially expressed mRNAs and predicted target miRNAs may be involved in the pathophysiological process of cantharidin-induced cardiotoxicity.Myh7,Tnc,Ldlr,11β-HSD2,Scd,mi R-133a-3p and mi R-146a-5p are molecular biomarkers which could be used for prediction and diagnosis of cantharidin-induced myocardial injury.To sum up,our results provide experimental basis on toxicological pathology changes of cantharidin poisoning and suggest that cardiotoxicity of cantharidin is more serious.Preliminary results serve to illustrate that TN-T,VEGF,HIF-1α,Myh7,Tnc,Ldlr,11β-HSD2,Scd,mi R-133a-3p,and mi R-146a-5p might be valuable molecular markers in cantharidin-induced myocardial injury and the diagnostic accuracy needs to be further studied.And our study demonstrated cantharidin-induced myocardial injury by multiple modulatory mechanisms,which provide new clues for further study on the pathophysiological mechanism of cantharidin-induced myocardial injury. |
