Clostridium Butyricum And Thalidomide Combined To Improve Nausea And Vomiting In Cisplatin Chemotherapy For Colorectal Cancer

Background and study objectivesColorectal cancer（CRC）is a very dangerous disease with high incidence.Chemotherapy is an important adjuvant treatment for colorectal cancer;however,the targets of chemotherapy drugs are non-selective and can damage not only cancer cells but also intestinal mucosal epithelial cells,which can cause many side effects,one of which is nausea and vomiting.Chemotherapy-induced nausea and vomiting（CINV）is a form of centrally mediated vomiting in the brain,which hinders further treatment due to its high incidence.Although therapeutic agents are currently available for CINV,however,nausea and vomiting still occur in some patients after receiving these agents.Therefore,the search for drugs that can significantly reduce nausea and vomiting due to chemotherapy for colorectal cancer has become a focus of attention.Thalidomide（THD）,a derivative of glutamate,was originally used to treat pregnancy vomiting,but raised alarms among patients because of its serious side effects on the fetus.Some recent studies have shown that thalidomide has an ameliorative effect on chemotherapy-induced nausea and vomiting,however,its exact mechanism of action remains unknown.Gut microbiota are an important part of the body and influence gut function,while the gut can play an important role in chemotherapy-induced nausea and vomiting by regulating the activity of the Central Nervous System（CNS）via the gut-brain axis（GBA）.Recently our team of clinicians found that the combination of probiotics and thalidomide was nevertheless highly effective and can almost completely eliminate CINV side effects.C.butyricum is a widely-used probiotic,which regulates intestinal microorganisms after colonization in the intestine.It can secrete butyric acid,repairs damaged intestinal mucosa,delays intestinal mucosa and epithelial atrophy,thus providing protection to gastrointestinal mucosal function.In addition,it has a good protective effect on the central nervous system.However,there are few reports of probiotics combined with thalidomide for the treatment of CINV,and the mechanism is not clear.Therefore,this project is intended to construct animal models of colorectal cancer CINV,so as to explore the effect of combined administration of C.butyricum and thalidomide in the treatment of CINV by cisplatin chemotherapy for colorectal cancer,and to explore its mechanism in depth.Experimental methods1.Establishment of a mice model of nausea and vomiting caused by cisplatin chemotherapy for colorectal cancer and prevention of drug administration1)Establishment of model:Seventy-two 7-week-old C57BL/6 male mice were studied and colorectal cancer was induced by two intraperitoneal injections of azoxymethane oxide（AOM,10 mg/kg）,along with 1%dextran sodium sulfate（DSS）in mice.After the colorectal cancer was confirmed by MRI,the mice were given drugs for prevention,followed by intraperitoneal injection of cisplatin（2.5 mg/kg）for three consecutive days,and the model was considered successfully established when the mice showed significant kaolin ingestion behavior.Experimental groups:（1）normal control group（group C）,（2）colorectal cancer model group（group M1）,（3）colorectal cancer cisplatin chemotherapy group（group M2）,（4）colorectal cancer cisplatin chemotherapy+C.butyricum prevention group（group L）,（5）colorectal cancer cisplatin chemotherapy+thalidomide prevention group（group S）,（6）colorectal cancer cisplatin chemotherapy+thalidomide+C.butyricum prevention group（group SL）,12 mice in each group.2)Administration of prophylaxis:C.butyricum was administered prophylactically by gavage in a coating solution（0.9%saline containing 0.01%gelatin）at a dose of 10⁷ CFU/100μL for 21 days;thalidomide was administered intraperitoneally at a dose of 25 mg/kg/d for 14 days.The rest were given the same bacterial coating solution as control treatment.2.Efficacy test and mechanism study of C.butyricum and thalidomide combination against nausea and vomiting caused by cisplatin chemotherapy in colorectal cancer1)The combination of C.butyricum and thalidomide enhanced the antitumor effect of cisplatin by upregulating apoptotic proteins:mice were dissected to obtain colorectal tumors,and the pathological heterogeneity of colorectal cancer was evaluated by HE staining of tumors;the number of colorectal tumors in the above-mentioned groups was observed and recorded to evaluate the tumor-suppressive effect of C.butyricum and thalidomide;the apoptosis-related proteins were detected by Western blot on tumors COX2,survivin,caspase-3,cleaved-caspase-3,poly ADP-ribose polymerase PARP,cleaved-PARP were detected by western blot on tumors to investigate their tumor suppression mechanism.2)C.butyricum and thalidomide combination prevented nausea and vomiting by reducing neurotransmitters and receptors:the inhibition of nausea and vomiting caused by chemotherapy for colorectal cancer by thalidomide and C.butyricum was evaluated by relative ingestion of kaolin in mice;the activation of Fos-like immunoreactive neurons closely related to vomiting was evaluated by immunohistochemistry of Fos protein in the last region of the brain;the activation of Fos-like immunoreactive neurons closely related to vomiting was evaluated by q-PCR and The expression of SP（NK-1）and 5-HT in the brain medulla and intestine were detected by q-PCR and ELISA,respectively;the expression of neurotransmitter receptors NK-1R and 5-HT₃R on the brain and colon were detected by immunohistochemical staining.3)C.butyricum and thalidomide combination improves impaired intestinal function by regulating intestinal microbiota and reducing inflammation:fecal DNA was extracted from mice,and the number and species of intestinal microbiota were examined by high-throughput sequencing technology;pro-inflammatory cytokines:tumor necrosis factor-α（TNF-α）,interleukin-6（IL-6）,interleukin-1β（IL-1β）were detected in the intestine by HE staining of the colon and q-PCR.（IL-1β）to evaluate the inhibition of intestinal inflammation by C.butyricum and thalidomide;probe the inflammatory mechanism by detecting the inflammatory pathways:TLR4,My D88,NF-κB,p-NF-κB,and HDAC1（histone deacetylase-1）by Western blot of the colon;detect the permeability proteins ocludin,twik-related potassium channel 1（Trek1）to evaluate the protective effects of C.butyricum and thalidomide on the intestinal barrier.Experimental results1.compared with M1 and M2 groups,the number of tumors in SL group was significantly reduced（p<0.05）,which were 3.64,2.91 and 1.81,respectively;the expression of COX2 in tumor tissues was down-regulated and apoptosis execution protein cleaved-caspase-3 was up-regulated,indicating that cisplatin could inhibit tumorigenesis,and the combined administration of thalidomide and C.butyricum enhanced this inhibitory effect.2.Compared with groups C and M1,mice in group M2 significantly ingested kaolin,with an ingestion ratio of 10.17%,while the ratio of kaolin ingestion in mice in group SL was significantly decreased to 1.35%（p<0.05）.Compared with the M2group,the Fos-positive neurons,neurotransmitters 5-HT and SP in brain and colon,and receptors 5-HT3R and NK-1R in the final region of the brain were significantly reduced in the SL group mice,indicating that thalidomide and C.butyricum prevented the occurrence of nausea and vomiting caused by cisplatin chemotherapy through the above mentioned mechanisms.3.Compared with the M2 group,TNF-α,IL-6 and IL-1βwere significantly reduced in the colon of mice in the SL group（M2:7.3,11.2,9.3;SL:1.1,2.9,2.3）,colonic crypt was restored,inflammatory cell infiltration was reduced,p-p65 protein expression was decreased;intestinal barrier proteins ocludin and Trek1 expression were upregulated;compared with the M2 group,the SL group had increased intestinal microbiotaαandβdiversity,increased abundance of beneficial bacteria such as Bifidobacteria and Lactobacilli,and increased abundance of Ruminococcaceae producing cancer-resistant short-chain fatty acid（SCFA）,indicating that C.butyricum and thalidomide improved intestinal function by ameliorating inflammation and regulating intestinal microbiota.Conclusions（1）The combination of thalidomide and C.butyricum enhanced the pharmacological effect of cisplatin anticancer drugs on tumors,with a reduction in the number and size of tumors compared to the control group.The combination of the two simultaneously reduced the occurrence of CINV conditions caused by cisplatin and decreased neuronal reactivity.It also had an improvement effect on intestinal bacteria,increasing bacteria abundance and decreasing intestinal inflammatory response.（2）The mechanism of thalidomide prevention of delayed nausea and vomiting may be related to the inhibition of the expression levels of neurotransmitters 5-HT and SP in the colon and brain,and the inhibition of the activation of the receptors5-HT₃R and NK-1 receptors in the final region of the brain.（3）C.butyricum corrected intestinal dysbiosis caused by chemotherapeutic agents,maintained the functional integrity of the intestinal mucosal barrier,reduced intestinal bacterial and endotoxin ectopia,and inhibited the secretion of inflammatory cytokines.