| Background:Safety is the fundamental guarantee for promoting the high-quality development of traditional Chinese medicine(TCM),and herbal compatibility is the key link to the rational,safe,and effective use of herbal medicine in clinical practice.In the clinical practice of TCM,the toxic and side effects of herbal medicine can be effectively reduced through the compatibility theory of“mutual restraint" and "mutual detoxication",which still affect the understanding of drug interactions in modern medicine.Therefore,it is valuable to perform research on the combined detoxification of herbal medicines based on the theory of TCM.Radix Bupleuri(RB)is commonly used to treat depression,but it can also lead to hepatotoxicity after long-term use.Currently,although RB has antidepressant effects,the hepatotoxicity exhibited by RB also limits its clinical application.In many anti-depression TCM prescriptions,RB is often used in combination with Radix Paeoniae Alba(RPA)as herb pair.In TCM theory,RPA has the effect of "nourishing blood and smoothing the liver",which can alleviate the“RB decreasing liver’s vin essence" by mutual-detoxication compatibility rules.It achieves the effect of detoxification through the compatibility theory of "mutual detoxication".However,whether RPA could alleviate RBinduced toxic and side effects and the mechanism remains unclear.Aims:(1)To clarify the "dose-effect/toxicity" relationship of RB,and to comprehensively analyze the effect of the combination of RB and RPA attenuates RB-induced toxic and side effects.(2)To systematically elucidate the key effect mechanism of the combination of RB and RPA attenuates RB-induced toxic and side effects.(3)To clarify the material basis of the combination of RB and RPA attenuates RBinduced hepatotoxicity,and to reveal the key molecular targets of the combination of RB and RPA to reduce the toxic and side effects of RB.Methods:(1)The chronic unpredictable stress(CUMS)depression rat model was used to evaluate the antidepressant effect of RB at 1.7,3.4,6.8,and 13.6 times the dosage specified in the pharmacopeia,using the sucrose preference test,forced swim test,and open-field test as indicators.At the same time,the "dose effect/toxicity" relationship of RB was comprehensively evaluated using diagnostic indicators of "liver yin deficiency"and hepatotoxicity indicators.Furthermore,the effect of compatibility of RB and RPA attenuates RB-induced toxic and side effects was evaluated through "liver yin deficiency"and hepatotoxicity indicators,to comprehensively characterize the efficacy of compatibility of RB and RPA on "reducing toxicity and retaining efficacy".(2)In the research on the key effect mechanism of the combination of RB and RPA attenuates RB-induced toxic and side effects,a non-targeted metabonomics study was conducted on liver samples.Combining with multivariate statistical analysis methods to screen out the differential metabolites associated with the toxicity and side effects of RB.On this basis,the callback ability of compatibility of RB and RPA to these differential metabolites was analyzed to preliminarily clarify which metabolic pathways are regulated by RB-RPA herb pair to alleviate RB-induced toxicity and side effects.Further,apply the"bottom-up" oriented target-finding strategy of metabolomics to preliminarily identify the potential targets of compatibility of RB and RPA for detoxification.Secondly,targeted metabolomics technology was applied to qualitatively and quantitatively confirm the key metabolic pathways identified by non-targeted metabolomics technology.Further,western blot,enzyme-linked immunosorbent assay,and biochemical enzyme activity assay techniques were used to verify the relevant targets and signal pathways in the key metabolic pathways.(3)In the study of the material basis of the compatibility of RB and RPA attenuates RB-induced toxic and side effects.Firstly,the difference in serum chemical profiles and liver chemical profiles of RB and RPA before and after the combination was studied by integrating UPLC-MS/MS analysis,mass spectrometry background subtraction technology,and multivariate statistical analysis methods.On this basis,the key differential components were quantitatively confirmed through target quantitative analysis.Secondly,a correlation analysis was conducted between the exogenous chemical component profiles and the endogenous metabolite profiles to preliminarily identify the potential chemical components that affect the key targets related to the RB-RPA herb pair RB-induced toxic and side effects.Next,molecular docking technology,isothermal titration calorimetry,and atomic force microscopy were used to determine the binding mode and binding power of key molecular targets.Further,the in vitro enzyme activity half maximal inhibitory concentrations(IC50)were measured to verify the regulatory effect of key chemical components on key enzyme targets,in order to reveal the potential material basis for the compatibility of RB and RPA to restrict the toxic side effects of RB.(4)In the study of the RB-RPA herb pair influencing the transformation of the RB toxic side effect components by modulating gut microbiota.Firstly,the target quantitative analysis method was used to quantitatively analyze the key chemical components related to the mutual-detoxication compatibility of RB and RPA in the intestine.Combined with relevant literature reports,it was preliminarily determined whether the RB-RPA herb pair affected the transformation of the RB toxic side effect components by modulating gut microbiota.Secondly,the effects of RB and RPA before and after the combination on the composition of the gut microbiome were analyzed using the16S rRNA third-generation full-length sequencing of the intestinal microflora.Further,use the PICRUSt2 software performs functional prediction analysis based on the obtained 16S rRNA full-length sequencing data,and combined with functional unit principal coordinate analysis(PCoA)analysis to screen the significantly altered bacterial functions before and after compatibility of RB and RPA.So as to screen the key differential functional units that affect the transformation of the RB toxic side effect components,and to validate the key differential functional units using enzyme-linked immunosorbent assay.Finally,we further screened the key gut microbiota that regulates the metabolic transformation of the RB toxic side effect components by constructing "significantly altered gut microbiotadifferential components" association pairs and "significantly altered gut microbiota-drug metabolizing enzymes" association pairs.To clarify the key mechanism of the compatibility of RB and RPA influencing the transformation of the RB toxic side effect components by modulating gut microbiota.Results:(1)The research results of the "dose-effect/toxicity" relationship of RB and the effect of the combination of RB and RPA attenuates RB-induced toxic and side effects showed that:After 49 days of RB administration,RB(1.8-14.4 g-herb/kg)improved the behavioral indicators that reflected the depression status in a concentration-dependent manner,among them,14.4 g-herb/kg RB had the best antidepressant effect.Additionally,the "liver yin deficiency" side effects and the hepatotoxicity of RB were assessed.The results showed that 7.2 and 14.4 g-herb/kg RB caused the side effects of "liver Yin deficiency "-like behavior.Moreover,14.4 g-herb/kg RB has the potential to cause hepatotoxicity.These results indicated that RB has a dose-dependent antidepressant effect,but the optimal antidepressant dose of RB causes "liver yin deficiency"-like behavior and hepatotoxicity.Besides,the combined use of RPA and RB reversed the "liver Yin deficiency "-like behavior induced by 7.2 and 14.4 g-herb/kg RB in CUMS depression model rats.It has been confirmed that in the compatibility environment of the TCM prescription,the RB-RPA "Sanshou" herb pair can reduce the toxic and side effects of RB,and it has been verified that herbal compatibility is the key link to the rational,safe,and effective use of RB in clinical practice.(2)The research results on the key effect mechanism of the combination of RB and RPA attenuates RB-induced toxic and side effects showed that:Integrated non-targeted metabolomics,targeted metabolomics,and bioinformatics network analysis confirm that glutathione metabolism was a key metabolic pathway for RB-RPA herb pair to reverse RB-induced toxic and side effects,and glutathione synthase(GSS)in the glutathione metabolism pathway was a potential target for RB-RPA herb pair to reverse RB-induced toxic and side effects.Afterward,molecular biological techniques have found that RBinduced hepatotoxicity was related to inhibiting GSS enzyme activity but does not regulate the protein level,and RB-RPA herb pair can reverse the inhibitory effect of RB on GSS enzymatic activity.Then,protein immunoblotting and enzyme-linked immunosorbent assay results further confirm that the RB-RPA herb pair suppresses RB-induced hepatotoxicity via oxidative stress and the NF-κB/NLRP3 pathway due to inhibition of GSS.(3)The research results on the material basis of the compatibility of RB and RPA attenuates RB-induced toxic and side effects showed that:The results of the difference in serum chemical profiles and liver chemical profiles of RB and RPA before and after the combination,as well as the results of the target quantitative analysis of the key differential components,confirmed that the combined use of RB and RPA promotes the conversion of saikosaponins to saikogenins in vivo compared with RB alone.Furthermore,based on the investigation of chemical component profiles and endogenous metabolite profiles,correlation analysis was used to find the potential link between significantly altered chemical components and regulated endogenous metabolites between the RB group and RB-RPA group.Furthermore,molecular docking technology,isothermal titration calorimetry,atomic force microscopy,and in vitro GSS inhibitory activity experiment indicated that saikosaponin A and saikosaponin D exhibit relatively strong binding with GSS protein and inhibit GSS enzyme activity at the present concentrations,while their deglycosylated conversion products saikogenin F and saikogenin G do not.This result confirmed that promoting the conversion of saikosaponins to saikogenins in vivo is the key material basis for the RB-RPA herb pair attenuating RB-induced toxic and side effects.(4)The research results on the RB-RPA herb pair influencing the transformation of the RB toxic side effect components by modulating gut microbiota showed that:the quantitative analysis of saikosaponins and saikogenins in colon contents revealed that the main site of RPA-promoted conversion of saikosaponins to saikogenins occurs in the intestine.In addition,full-length 16S rRNA sequencing revealed the composition of the gut microbiome in each group.The results showed that there were significant differences in the composition and abundance of the gut microbiome among the RB,RPA,and RBRPA herb pair groups.Next,bacterial functions were predicted via PICRUSt2 based on 16S marker sequences,and the principal coordinate analysis(PCoA)plot of the prediction of metagenome functional profiles depicted that the relative abundances of β-Dglucosidase and β-D-fucosidase were significantly increased in the RB-RPA herb pair group compared to the RB group.Following this lead,the levels of β-D-glucosidase and βD-fucosidase in the colon tissues were tested.Consistently,the results also showed that the levels of β-D-glucosidase and β-D-fucosidase in the colon tissues were significantly increased in the RB-RPA herb pair group compared to the RB group.These results indicated that RPA increases the abundance of intestinal bacteria with β-Dglucosidase and β-D-fucosidase activity in the intestine,thereby promoting the conversion of saikosaponins to saikogenins.Moreover,the analysis results of the "significantly altered gut microbiota-differential components" association pairs and "significantly altered gut microbiota-drug metabolizing enzymes" association pairs showed that RPA promoted the conversion of saikosaponins to saikogenins in the colon by increasing the abundance of Bacteroides,Escherichia_coli,and Lactobacillus_koreensis.Conclusions:In this work,our study aims to investigate the mechanisms by which RB-RPA herb pair attenuates RB-induced toxicity and side effects.The results showed that RB-RPA herb pair increased the abundance of intestinal bacteria with glycosidase activity in the intestine,thereby promoting the conversion of saikosaponins to saikogenins.Different from saikosaponin A and saikosaponin D,which are directly combined with GSS as an inhibitor,their deglycosylation conversion products saikogenin F and saikogenin G exhibited no GSS binding activity.Based on this,RB-RPA herb pair can effectively alleviate the inhibitory effect of saikosaponins on GSS activity and reshape the liver redox balance by increasing the GSH-GSSG ratio.Meanwhile,RB-RPA herb pair inhibited RB-induced liver oxidative injury and alleviated the liver inflammatory response mediated by the NFκB/NLRP3 pathway.In conclusion,the present study suggests that promoting the conversion of saikosaponins by modulating gut microbiota composition to attenuate the inhibitory effect on GSS activity is a potential mechanism by which RB-RPA herb pair prevents RB-induced toxicity and side effects. |
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