Study On The Antitumor Effect Of Fuzi And Its Improvement On Cisplatin-induced Nontargeted Tissue Toxicity

ObjectiveAconiti Lateralis Radix Praeparata(Fuzi,FZ),a traditional Chinese medicine for warming Yang,has been used for cancer patients in clinic,but there is still a lack of systematic basic research evidence on the anti-tumor effect of FZ.Based on the current status of traditional Chinese medicine adjuvant anticancer therapy,combined with the doubts and controversies about the rationality of FZ in anti-tumor treatment,this study will clarify the anti-tumor effect of FZ at the animal and cellular levels,and whether the combination of FZ and cisplatin could alleviate the organ damage caused by cisplatin,thus improve the survival condition of chemotherapy mice.In addition,this study discussed the effects of FZ on cisplatin induced kidney injury from the perspective of DNA damage and pyroptosis,and revealed the linkage mechanism of FZ combined with cisplatin on various tissues of the body.At the same time,the content of traditional Chinese medicine as an adjunct therapy is enriched and innovated,and the research evidence is provided for the clinical application of FZ in chemotherapy patients in the future.Methods1.Research on the anti-tumor effect and the material basis of drug effect of FZ① Combined with the literature and our team’s previous research on the chemical basis of FZ,this project selected Heishunpian of FZ processed products and 19 kinds of monomer components derived from FZ as the research objects.First,CCK8 method was used to observe the effects on the proliferation of cancer cell lines(Huh-7,HepG-2)and normal tissue-derived cell lines(LX-2,THLE-3).higenamine(HG),higenamine hydrochloride(HHc),and Salsolinol(SAL)have anti-tumor effects.Furthermore,the effects of FZ and its derived compounds(HG,HHc,SAL)on the proliferation,migration and invasion of tumor cell lines were observed by Transwell and scratch experiments.②To observe the inhibitory effect of FZ and its derived compounds(HG,HHc,SAL)on tumor growth.BALB/C-Nude mice models of subcutaneous transplanted tumors were established and randomly divided into 10 groups:The Vehicle group,Cisplatin group,FZ group,FZ+Cisplatin group,HG group,HG+ Cisplatin group,HHc group,HHc+Cisplatin group,SAL group,SAL+ cisplatin group.The body weight and tumor growth curve of each group were monitored.The tumor size was recorded,and the histomorphological changes were observed by H&E staining.③ RNA-seq technology was used to analyze the effects of FZ and active compounds on differentially expressed genes in tumor cells and potential targets.The intervention measures of each group were as follows:Vehicle group(DMSO),FZ group(2 μg/μL),HG group(120 μM),HHc group(120 μM),and SAL group(120 μM).2.Effect of FZ on cisplatin-induced non-targeted tissue toxicity① CCK-8 assay was used to observe the effect of cisplatin(0-25 μM)on the cell viability of cancer cell lines(Huh-7,HepG-2)and normal tissue-derived cell lines(HT22,H9C2,BEAS-2B,293T),and to analyze the difference in the killing ability of different cell lines at the same dose of cisplatin.② To observe the effects of FZ and its derived compounds(HG,HHc,SAL)on cisplatin-induced organ damage in mice.Through the detection and analysis of organ coefficient.H&E histology of each organ,serum biomarkers(SCR,BUN,ALT,AST,CKMB,LDH)in each group of mice,combined with the body weight growth curve of mice,to determine whether the compound derived from FZ can slow down the organ damage caused by cisplatin.In order to answer the question of improving the survival condition of chemotherapy mice.3.Explore the mechanism of FZ in improving cisplatin-induced kidney injury① To observe the effect of FZ and its derived compounds(HG,HHc,SAL)on cisplatin-induced 293T cell damage.② Tumor tissues and kidney tissues of mice:Western blot and other molecular biological methods were used to detect DNA damage related proteins(P-H2A.X and P-p53)in tumor tissues and kidney tissues of tumor-bearing mice.And the expression levels of GSDMD-mediated pyroptosis related proteins(GSDMD.IL-1β,Cleaved Caspase-1,Caspase-11)in kidney tissue.Results1.Research on the anti-tumor effect and the material basis of drug effect of FZ① Anti-tumor effects of FZ and its derived compounds(HG,HHc,SAL)can significantly inhibit the proliferation,migration and invasion of tumor cells.With Huh-7,HepG-2.LX-2 and THLE-3 as the order,the IC50 of FZ water extract for 48 h was 1.683μg/μL,1.505 μg/μL,4.985 μg/μL and 3.908 μg/μL,respectively.The IC50 values of HG and HHc were 220.4 μM,152.7 μM.156.7μM,185.1 μM and 207.3μM,172.8 μM,271.1μM,223.7 μM,respectively.The IC50 of SAL was 124.5 μM,128.4 μM.87.48μM and 137.1 μM,respectively.② Compared with the Vehicle group.the tumor weight of FZ,HG,HHc,SAL andCisplatin groups was significantly reduced;Compared with the Cisplatin group.the tumors in the FZ+Cisplatin group,HG+Cisplatin group,HHc+Cisplatin group and SAL+Cisplatin group were slightly reduced,but the difference was not statistically significant.Tumor histomorphology showed that the tumor cells in the Vehicle group were closely arranged and structurally intact.In the Cisplatin group,the tumor cell space increased,the cavity increased,the cells arranged disorderly,and the broken cells could be seen.In FZ group,HG group,HHc group and SAL group,the tumor cells arranged loosely and disorderly,with vacuoles and necrosis.In the FZ+Cisplatin group,HG+Cisplatin group,HHc+Cisplatin group and SAL+Cisplatin group,the outline of tumor cells was not clear,the inflammatory infiltration cells increased,and the necrosis area expanded.③ Differentially expressed genes analysis:compared with Vehicle group,there were 946 significantly different genes after FZ intervention,including 761 up-regulated genes and 185 down-regulated genes:There were 791 significantly differentially expressed genes after HG intervention,including 343 up-regulated genes and 448 down-regulated genes.A total of 139 genes were significantly different after HHc intervention,including 61 up-regulated genes and 78 down-regulated genes.A total of 103 genes were significantly differentially expressed after SAL intervention,including 48 up-regulated genes and 55 down-regulated genes.Among them,FZ_vs_Vehicle and HG_vs_Vehicle shared 219 differential genes,FZ_vs_Vehicle and HHc_vs_Vehicle shared 49 differential genes,FZ_vs_Vehicle and SAL_vs_Vehicle shared 33 differential genes.According to GSEA gene set enrichment results,the common pathways enriched by differentially expressed genes of each intervention drug included:A subgroup of genes regulated by MYC pathway in tumor cell cycle progression,apoptosis and cell transformation,Tumor cell proliferation and immune-related Genes regulated by NF-kB in response to TNF pathway,Biological oxidations pathway,up-regulated in response to IFNG pathway,Complement and Coagulation Cascades pathway,and other Genes related to inflammation and immune response.2.Effect of FZ on cisplatin-induced non-targeted tissue toxicity① Cisplatin can cause serious damage to normal cells at low doses.The IC50 of cisplatin treatment for 48 h in Huh-7.HepG-2.HT22.H9C2,BEAS-2B and 293T cells was 21.99 μM,200.2 μM.9.067 μM.13.66 μM,11.76 μM and 10.60 μM.respectively.② FZ could significantly ameliorate cisplatin-induced organ toxicity in mice,combined with organ coefficient,H&E histomorphology,serum biomarkers(SCR,BUN.ALT,AST,CK-MB,LDH)and body weight changes.The toxicity of FZ,HG,HHc and SAL alone or in combination with Cisplatin in mice was analyzed,and it was found that compared with cisplatin group,In FZ+Cisplatin group or SAL+Cisplatin group,the abnormal increase of related indexes of kidney,spleen,liver,heart and weight loss caused by cisplatin were improved to varying degrees,especially in kidney.3.Explore the mechanism of FZ in improving cisplatin-induced kidney injury① FZ up-regulated DNA damage-related proteins in tumor tissues of tumor-bearing micep-H2A.X:Compared with Vehicle,the protein expression of p-H2A.X in the tumor tissues of the Cisplatin group and the FZ group showed an upward trend,but there was no statistically significant difference.The protein expression of p-H2A.X in the tumor tissues of the FZ+Cisplatin group was up-regulated.Compared with the Cisplatin group,the protein expression of p-H2A.X in the tumor tissues of the FZ+Cisplatin group was increased.p-P53:Compared with Vehicle,the expression of p-P53 protein in tumor tissues of Cisplatin group and FZ group was significantly increased.Compared with the Cisplatin group,the expression of p-P5 3 protein in the tumor tissues of the FZ+Cisplatin group had a certain increase trend,but there was no statistically significant difference.② FZ inhibits cisplatin-induced high expression of DNA damage related proteins in kidney tissue of tumor-bearing miceCompared with Vehicle,the protein expression levels of p-H2A.X and p-P53 in kidney tissue of Cisplatin group were not significantly different from those of FZ group.Compared with the Cisplatin group,the protein expressions of p-H2A.X and p-P53 in the kidney tissues of the FZ+Cisplatin group were significantly down-regulated.③FZ inhibits cisplatin-induced high expression of pyroptosis-related proteins in kidney tissue of tumor-bearing miceCompared with Vehicle group,the protein expressions of GSDMD and IL-1β in kidney tissue of Cisplatin group were increased,while the protein expressions of GSDMD,IL-1β,Cleaved Caspase-1 and Caspase-11 in kidney tissue of FZ group were not significantly different.Compared with the Cisplatin group,the protein expression of GSDMD,IL-1β,Cleaved Caspase-1 and Caspase-11 in the kidney tissue of tumor-bearing mice in the FZ+Cisplatin group was significantly decreased.Conclusions1.FZ and its derived compounds(HG,HHc,SAL)can inhibit the proliferation,migration and invasion of tumor cells in vitro,and inhibit the growth of tumor in tumorbearing mice.2.HG,HHc and SAL,which derived from FZ,have their own roles in tumor chemotherapy,and their effects and targets are relatively single.HG and HHc have significant anti-tumor effects,but can not effectively improve cisplatin-induced organ damage,while SAL is more effective in improving cisplatin-induced non-tissue targeted toxicity.Compared with other monomers,FZ can exert significant anti-tumor effect and improve some organ damage induced by cisplatin,especially the kidney.3.The mechanism by which FZ ameliorates cisplatin-induced kidney injury may be related to inhibiting DNA damage and GSDMD-mediated pyroptosis.