Study On The Mechanism Of Rhubarb Promoting DEN-induced Hepatocarcinogenesis In Rats Based On Pentose Phosphate Pathway

Rhubarb is a natural herb widely used in clinical practice,with a variety of pharmacological effects including anti-cancer.In particular,free anthraquinones in Rhubarb have an inhibitory effect on the proliferation of hepatocellular carcinoma cells.However,numerous studies have shown that Rhubarb has both hepatoprotective and hepatotoxic effects.Therefore,the efficacy and safety of Rhubarb in the in vivo treatment of liver cancer should be further elucidated.Objective:The pentose phosphate pathway was used as an entry point to explore the mechanism of Rhubarb promoting DEN-induced hepatocarcinogenesis in rats,providing a basis for the correct understanding of the efficacy and safety of Rhubarb in the treatment of hepatocellular carcinoma.Methods:1 Metabolomic analysis of the effect of Rhubarb on the pentose phosphate pathway in the liver tissue of rats with DEN-induced hepatocellular carcinoma60 SPF-grade SD rats were random Ly divided into 6 groups: Control group,LDR-treated group,HDR-treated group,DEN-treating group,LDR-DEN-treated group,HDR-DEN-treated group.After treatment for 17 weeks,all the rats were additionally raised for 3 weeks without any treatment.Subsequently,they were deeply anesthetized by intraperitoneally injecting pentobarbital sodium(30 mg/kg,Merck,USA),and euthanized by decapitation.Glucose metabolism changes in rat liver tissues of each group were analyzed using metabolomics techniques,and two-to-one comparisons were made between control group vs DEN-treated group,control group vs LDR-DEN-treated group,and DEN-treated group vs LDR-DEN-treated group to screen for differential metabolites and perform KEGG metabolic pathway analysis.2 Effect of Rhubarb on the pentose phosphate pathway in the hepatocarcinogenesis stage of DEN-induced ratsHistological alterations,liver function,hepatic protein levels of alpha-fetoprotein(AFP),pyruvate kinase isozyme type M2(PKM2)and proliferating cell nuclear antigen(PCNA)were measured in animals at 20 weeks in method 1 to analyze liver injury in rats.Hepatic glucose 6 phosphate(G6P)levels and glucose 6 phosphate dehydrogenase(G6PD)activity were measured to assess the pentose phosphate pathway.3 Effect of G6PD inhibitor on the stage of hepatocarcinogenesis in Rhubarb-DEN treated rats80 SPF-rated SD rats were random Ly divided into 8 groups.Control group,LDR-treated group,Polydatin-treated group,DEN-treating group,LDR-DEN-treated group,Polydatin-DEN-treated group,Polydatin-Rhubarb-treated group,Polydatin-Rhubarb-DEN-treated group.After treatment for 17 weeks,all the rats were additionally raised for 3 weeks without any treatment.Subsequently,they were deeply anesthetized by intraperitoneally injecting pentobarbital sodium(30 mg/kg),and euthanized by decapitation.Liver injury in rats was analyzed by histological alterations,liver function,hepatic protein levels of pyruvate kinase isozyme type M2(PKM2)and proliferating cell nuclear antigen(PCNA).Hepatic glucose 6 phosphate(G6P)levels and glucose 6 phosphate dehydrogenase(G6PD)activity were measured to assess the pentose phosphate pathway.4 Extraction of active parts of RhubarbThe active parts of Rhubarb were extracted by the systematic solvent method,and the aqueous extract of Rhubarb,ethyl acetate extract of Rhubarb,n-butanol extract of Rhubarb and petroleum ether extract of Rhubarb were obtained.5 Effect of active parts of Rhubarb on DEN-induced liver injury in ratsThe yield of different solvent extracts of Rheum palmatum was calculated,and different solvent extracts of Rheum palmatum were prepared with 0.5% CMC-Na(equivalent to 4 g/kg of raw drug).50 SPF-grade SD rats were random Ly divided into 5 groups: Control group,DEN-treated group,Rhubarb aqueous extract-DEN-treated group,Rhubarb ethyl acetate extract-DEN-treated group,and Rhubarb n-butanol extract-DEN-treated group.Rats were sampled at week 14 and liver injury was analyzed by histological alterations,liver function,pyruvate kinase isoenzyme type M2(PKM2)and proliferating cell nuclear antigen(PCNA)levels of hepatic proteins.Hepatic glucose 6phosphate(G6P)levels and glucose 6 phosphate dehydrogenase(G6PD)activity were measured to assess the pentose phosphate pathway.Results:1 Analysis of non-targeted metabolomics techniques showed that:(1)A total of 63 different metabolites appeared between the DEN-treated group and the control group.Among these metabolites,3 metabolites were related to glutathione metabolism based on KEGG results.(2)A total of 97 potential biomarker metabolites were noted between the Rhubarb-DEN-treated and control groups.Among these metabolites,3 metabolites were related to glutathione metabolism based on KEGG results.(3)Moreover,44 potential biomarker metabolites were found between the Rhubarb-DEN-treated and DEN-treated groups.Among these metabolites,2 metabolites were related to glutathione metabolism based on KEGG results.These findings demonstrated that glutathione metabolism and the PPP were regulated in the liver of Rhubarb-DEN-treated rats.2 HE staining showed that: The hepatic pathological changes in the Rhubarb-DEN-treated rats were more severe than those in the DEN-treated rats,and the severity of hepatic pathological changes depended on Rhubarb dosage;The results of serum GPT and GOT levels showed that the liver function of rats in the LDR-DEN-treated and HDR-DEN-treated groups was significantly impaired;Western Blot assay results showed that,AFP: The level of AFP remarkably increased in the liver of HDR-DEN-treated rats compared to the control.And the AFP levels in the LDR-DEN-treated rats and the HDR-DEN-treated rats were similar to that in the DEN-treated rats.Similarly,there was no significant difference between the LDR-DEN-treated group and the HDR-DEN-treated group;PKM2: PKM2 levels in the LDR-treated rats were similar to those in the control group.PKM2 levels were increased in the DEN-treated,HDR-treated,LDR-DEN-treated and HDR-DEN-treated groups compared to the control and LDR-treated groups.However,PKM2 levels in the LDR-DEN-treated rats and HDR-DEN-treated rats were similar to those in the HDR-treated and DEN-treated rats,and there were no significant differences between the LDR-DEN-treated and HDR-DEN-treated.PCNA : PCNA levels in DEN-treated rats,LDR-treated rats and HDR DEN-treated rats were similar to those in control rats.However,PCNA levels were elevated in the livers of LDR-DEN-treated and HDR-DEN-treated rats compared to control and LDR-treated rats.In addition,PCNA levels in LDR-DEN-treated rats were similar to those in DEN-treated rats.However,PCNA levels were elevated in the liver of HDR-DEN-treated rats compared to DEN-treated rats.In addition,there was no significant difference between the LDR-DEN-treated and HDR-DEN-treated groups.Compared with rats in the DEN-treated group,the activity of G6PD in the liver of rats in the LDR-DEN-treated and HDR-DEN-treated groups was significantly increased,and there was no significant difference between the LDR-DEN-treated and HDR-DEN-treated groups;compared with rats in the DEN-treated group,the level of G6P in the liver of rats in the LDR-DEN-treated and HDR-DEN-treated groups was reduced,respectively.In addition,G6P levels in the liver of rats in the HDR-DEN-treated group were significantly reduced compared to those in the LDR-DEN-treated group.These findings suggest that Rhubarb activated PPP in the liver of Rhubarb-DEN-treated rats.3 Polydatin-HDR-DEN-treated rats showed decreased G6PD activity and increased hepatic G6P levels compared to the LDR-DEN-treated group.These findings confirmed that Polydatin,a specific inhibitor of G6PD,inhibited G6PD activity in the liver of Rhubarb-DEN-treated rats.HE staining results showed that Rhubarb aggravated the pathological changes in the liver of DEN-treated rats,while Polydatin attenuated such changes;serum GPT level assay results showed that both Polydatin and Rhubarb increased the DEN-induced increase in GPT levels,but there was no significant difference in GPT activity between Polydatin-DEN-treated rats and Polydatin-LDR-DEN-treated rats;serum GOT level assays showed an increase in GOT activity in LDR-DEN-treated rats.However,there was no significant difference in GOT activity between the Polydatin-treated and Polydatin-LDR-DEN-treated rats.These findings suggest that Polydatin attenuated Rhubarb-DEN-induced liver function impairment.western blot assay results showed that the levels of PKM2 and PCNA were reduced in liver tissues of Polydatin-LDR-DEN-treated rats compared with the LDR-DEN-treated group.These results indicated that Polydatin attenuated the promotional effect of low-dose Rhubarb on DEN-induced hepatocarcinogenesis.4 The yields of the active parts of Rhubarb extracted with water,ethyl acetate,n-butanol and petroleum ether were 1.5%,0.98%,0.58% and 0.029%,respectively.Since the yield of petroleum ether extract of Rheum palmatum was too low for experimental significance,the aqueous extract of Rheum palmatum,ethyl acetate extract of Rheum palmatum and n-butanol extract of Rheum palmatum were selected to further study the effects of the active parts of Rheum palmatum on liver injury caused by DEN in rats.5 HE、Masson staining results showed that rats in the Rhubarb aqueous extract-DEN-treated group showed more severe pathological changes in liver tissue compared to the DEN-treated group;The results of serum GPT and GOT levels showed that the liver function was more significantly impaired in rats in the Rhubarb aqueous extract-DEN treated group.The results of Western Blot showed that the levels of PCNA and PKM2 in the liver tissues of rats in the Rhubarb aqueous extract-DEN-treated group were significantly higher compared with the DEN-treated group.Compared with rats in the DEN-treated group,G6PD activity was significantly increased and G6P levels were significantly decreased in the Rhubarb aqueous extract-DEN-treated group.These findings suggest that aqueous extracts of Rhubarb promote the development of DEN-induced liver injury in rats via the pentose phosphate pathway.Conclusion:The toxic-volume-effect relationship of Rhubarb in a DEN-induced liver cancer model is different from that in a normal model;PPP is the mechanism in a DEN-induced liver cancer model in rats;and Rhubarb aqueous extract promotes DEN-induced liver injury.This suggests that the study of Rhubarb toxicity-volume-effect relationships in normal models has limitations,and it is more important to evaluate the efficacy and safety of Rhubarb in animal models that simulate clinical disease states.