The Role Of Gut Microbiome/Bile Acids/TGR5/CCR5 Signaling Pathway In Paclitaxel-induced Peripheral Neuropathy

Introduction: Along with surgery and radiotherapy,chemotherapy is one of the main treatments for cancer.With the emergence of new therapies such as targeted-therapy and immunotherapy,which have been proposed to become more effective and have fewer side effects in recent years,the debate on whether chemotherapy can be replaced has become one of the hot topics.However,emerging research have found that targeted-therapy can only kill a subset of cancer cells that express specific proteins.Despite the current emergence of multiple anti-tumour therapies,chemotherapy continues to be a cornerstone of first-line clinical treatment.Chemotherapy-induced peripheral neuropathy is a common side effect of numerous chemotherapeutic drugs,with approximately 50% of patients suffering from sensory,motor and autonomic deficits within 24-72 h of treatment,and around 30% of the patients suffering from symptoms several years after the chemotherapy.Among which,sensory deficits are most common,with the main clinical manifestations such as numbness,nociceptive hyperalgesia,sensory loss,chronic pain and irreversible nerve damage.About 25% of the patients need to adjust or even discontinue the chemotherapy to limit the drug neurotoxicity,which seriously affects the anti-tumour process and patients’ life quality.According to the latest data issued by the World Health Organization’s International Agency for Research on Cancer(IARC)on the global burden of cancer in 2020,breast cancer has ranked the first in the world,of which China has the highest incidence.As a common first-line therapy for breast cancer,the prevention and treatment of paclitaxel-induced peripheral neuropathy(PIPN)has become a clinical challenge.In addition,the pathogenesis of PIPN is still unclear,therefore,how to find the target for treatment of PIPN and how to identify the sensitive patients,so as to find a balance between anti-tumour therapy and anti-PIPN side effect become a bottleneck problem that needs to be addressed in chemotherapy.Due to the systemic effects of chemotherapeutic drugs,the possibility of providing new ideas from the perspective of host metabolism become a current research hot topic.As one of the most important metabolites of intestinal microbiota,alterations in microbial-related bile acid profiles have been observed in human and mouse models of Parkinson’s disease,Alzheimer’s disease,multiple sclerosis and autism in recent years,but whether and how bile acids play the role in PIPN has not yet been reported.Bile acids as the main metabolic mediators of gut microbiota–host crosstalk have been involved in cholesterol and energy homeostasis.Bile acids are synthesized in hepatocytes via cytochrome P450-mediated oxidation of cholesterol.Then some bile acids escape into the colon,where gut commensal bacteria convert them into various secondary bile acids.Deoxycholic acid,as one of the secondary bile acids,is unique due to its potential roles,not only in local gastrointestinal inflammation,but also in affecting neuronal functions,in partuclar,induces visceral hypersensitivity through a bile acid receptor TGR5,also known as Gp BAR1.Recent studies have shown that intestinal microbiota play an important role in oxaliplatin-induced peripheral neuropathy,which the mechanical nociception not present in germ-free mice.In addition to the neurological damage,paclitaxel also disrupts the composition of intestinal commensal microbiota,damages the intestinal epithelium and induces dysbiosis and mucosal inflammation.It suggests that chemotherapeutic agents may cause changes in the serum bile acid profile by affecting the structure of intestinal microbiota,however whether and how both are involved in the development of CIPN has not been reported.It has been established that peripheral nociceptors not only respond to gut microbiota and microbiota-derived molecules,but also respond to immune mediators such as cytokines and chemokines.Emerging evidences suggest that the activity of CCR5,a G-protein-coupled receptor,significantly influences the promotion of cancer invasion and metastasis.CCR5-targeted therapeutics with small molecule inhibitors(maraviroc and vicriviroc)and humanized m Ab(leronlimab)are now being repositioned in clinical trials for breast cancer treatment.In addition,a previous study has demonstrated that paclitaxel-treated rats showed a significant increase of CCR5 in spinal dorsal horn.However,whether and how CCR5 regulates peripheral neuropathy and sensitization of peripheral nociceptors remains unclear.Therefore,this study aims to identify the microbiomic and metabolomic signatures in patients and rodent models with different PIPN grades,to explore the roles and mechanisms of identified gut microbiota and bile acids on the induction and development of PIPN,and to provide novel prevention or treatment strategies by modulating gut microbiota / bile acids / TGR5 / CCR5 signaling pathway.Methods:Part I.To verify the changes of intestinal flora and serum bile acid profiles of PIPN patients and rodent models.Clinical study part: A total of 80 subjects were included,including 60 breast cancer patients treated with paclitaxel(30 patients with peripheral neuropathy of grade 2+,30 patients with peripheral neuropathy of grade 1),and 20 healthy controls volunteer.Grading of sensory neuropathy was assessed using the National Cancer Institute-Common Terminology Criteria for Adverse Events(NCI-CTCAE)and the European Organization for Cancer Research and Treatment-Chemotherapy-Induced Peripheral Neuropathy Questionnaire(EORTC QLQ-CIPN20).1)16Sr RNA sequencing was used to detect the composition and abundance of intestinal flora in feces,and 2)liquid chromatography tandem mass spectrometry technology(LC-MS/MS)was used to detect serum targeted metabolomics respectively.Animal research part: The effect of PIPN model on intestinal microbiota and host metabolism was verified.1)Established a rat model of PIPN by intraperitoneal injection of paclitaxel 2mg/kg for four times;2)Behavior tests for detecting mechanical hyperalgesia and cold hypersensitivity by von frey and cold acetone test;3)Dorsal root ganglion were stained and morphologically observed by Nissle staining and transmission electron microscopy;4)16Sr RNA sequencing was used to determine the composition and abundance of intestinal microbiota,5)Liquid chromatography tandem mass spectrometry(LC-MS/MS)was applied to determine the untargeted metabolomics of serum and feces.Part II.To verify the effects of gut microbiota and bile acids on neuropathic nociception and molecular phenotypes.1)Using antibiotic gavage to mimic a model of germ-free rats;2)Using anaerobic bacteria culture technology and fecal bacterial transplantation to determine the role of specific intestinal microbiota in PIPN;3)Using deoxycholic acid gavage to clarify the direct effects of bile acids on PIPN;4)Liquid chromatography tandem mass spectrometry(LC-MS/MS)was applied to determine the changes of bile acid profiles in serum and feces;5)Whole-Cell patch clamp was applied to determine the action potential frequency,resting potential and basal current of DRG neurons in vivo;6)Real-time quantitative PCR was used to detect Nav1.3,Nav1.7,Nav1.8,Nav1.9,TRPA1,TRPV1,TRPV4,MBP,GAP43,C-Fos,C-Jun m RNA levels.Part III.To verify the role and mechanisms of bile acids receptor TGR5 and key protein CCR5 in the development of PIPN.1)Real-time quantitative PCR was used to detect the CCR5 m RNA levels in the dorsal root ganglias;2)ELISA was used to detect serum CCL5 level;3)The RNA-sequencing was applied to clarify the differentially expressed genes and important signaling pathways which CCR5 participated in;4)The cellular localization of CCR5 in dorsal root ganglia,and the immuno-activity of GFAP-positive astrocytes and IBA1-positive microglia in spinal dorsal horn cord were detected by touble-immunofluorescence;5)Von frey and cold acetone tests were used to detect the nociceptive responses to mechanical and cold stimuli;6)Western blot was used to detect the expression of important proteins in dorsal root ganglia in vivo and ND7/23 cells in vitro;7)CCR5 knockout rats were constructed by CRISPR/Cas9 technology;8)Electrophysiological indicators such as action potential frequency,resting potential,and basal current of DRG neurons were detected by whole-cell patch clamp;9)DRG intra-injection with AAV9 were used to knockdown TGR5 and CCR5 in DRG neurons of rats;10)Small interfering RNA(si RNA)was used to knockdown TGR5 and FXR in ND7/23 cells in vitro.Results:1.The composition of gut microbiota in PIPN patients was substantially different from that in healthy controls,with the observed species and the α-diversity of gut microbiota slightly decreased in high-PIPN patients.2.In serum of high-PIPN group,the level of secondary bile acid—deoxycholic acid was significantly increased,whereas the levels of primary bile acids—GHCA,GUDCA,TCA,TUDCA,and TCDCA were decreased.3.The secondary to primary bile acid ratio DCA/TCA reflecting Clostridium activity was significantly increased in patients with severe PIPN.Five parameters,DCA,GHCA,GUDCA,TUDCA,and DCA/TCA,can be used as potential markers to assess the clinical grade of PIPN.4.The composition of gut microbiota in PIPN rodent models was significantly altered,with the most significant increase in Clostridium spp.5.Serum untargeted metabolomics showed that the secondary bile acids synthesis signaling pathway was the most significantly changed metabolic pathway in PIPN rodent group,among which deoxycholic acid increased most significantly.6.The relative abundance of Clostridium was strongly associated with DCA levels in PIPN rodent models.7.The BAs profiles in serum and feces can be significantly affected by the modulation of gut microbiota,and the altered gut microbiota and SBAs in the PIPN rat model can induce peripheral neuropathy-related phenotypes(including mechanical hyperalgesia,cold hyperalgesia,Nav and TRP ion channel receptor families,transcriptional regulators c-fos/c-jun,and myelin protein MBP).8.The expression of G protein-coupled receptor 5(CCR5)was significantly up-regulated in the dorsal root ganglia of PIPN rodent models,and were modulated by gut microbiota and bile acids.9.CCR5 contributes to the induction of PIPN by regulating glial cell activity in the spinal dorsal horn,dorsal root neuron excitability,myelin protein homeostasis,and MAPK signaling pathway.10.The roles of gut microbiota and SBAs in the pathogenesis of PIPN are mediated through TGR5/CREB/CCR5 signaling pathway.Conclusion:1.There was a significant correlation between increased serum deoxycholic acid levels and increased relative abundance of Clostridium spp.in the intestinal microbiota in patients and rodent models of PIPN.2.Clostridium spp.and deoxycholic acid can significantly affect the phenotype associated with peripheral neuropathy.3.The neurotoxic effects of deoxycholic acid are partly dependent on the TGR5/CREB/CCR5 signalling pathway in the dorsal root ganglion.