The Roles Of Gamma-aminobutyric Acid A Receptor In Intestinal Epithelial Cells In Chemoradiotherapy-induced Intestinal Injury And Its Underlying Mechanisms

Chemoradiotherapy is a widely used cancer treatment today,which kills rapidly proliferating cancer cells by causing DNA damage.However,the normal tissues with rapid self-renewal capacity,such as the intestinal mucosa,are also easily damaged by chemoradiotherapy.Lethal intestinal tissue toxicity is a common side effect and a dose-limiting factor in chemoradiotherapy.The dose of radiation and chemotherapy drug which can effectively kill tumor cells are usually not used in the treatment of abdominal tumors due to severe intestinal tissue toxicity,which ultimately leads to a decline in patient survival and early tumor metastasis.It is quite critical to find a chemo-radioprotective agent that can effectively reduce chemoradiotherapy-induced intestinal tissue toxicity,and have no other harmful effects on the human body.However,there are currently no the United States Food and Drug Administration(FDA)-approved drugs for reducing intestinal injury or complications induced by chemoradiotherapy.This study intends to find a chemo-radioprotective agent that can selectively protect intestine from chemoradiotherapy without affecting its antitumor activity in cancer treatment.It will have extremely important clinical significance for cancer treatment.Chemoradiotherapy-induced intestinal tissue toxicity is characterized by the rapid loss of proliferating cells in the crypt of the small intestine,severe destruction of the intestinal epithelial barrier,and intestinal inflammation during or shortly after treatment.Intestinal stem cells(ISCs),located at the base of crypts,can differentiate into various mature intestinal epithelial cells(IECs)to maintain the rapid turnover of intestinal epithelium under physiological conditions.However,ISCs are very sensitive to chemoradiotherapy.Leucine rich repeat containing G protein-coupled receptor 5(LGR5)-positive ISCs disappear rapidly after chemoradiotherapy,which is the key reason for intestinal toxicity caused by chemoradiotherapy.Chemoradiotherapy mainly kills cells through DNA damage.The members of the phosphatidylinositol 3’ kinase-related kinases(PIKKs)family,including ataxia telangiectasia mutated(ATM),ATM and Rad3-related(ATR),and DNA-dependent protein kinase,catalytic subunit(DNA-PKcs),are the first kinases to initiate the DNA damage response.The activation of PIKKs can rapidly transduce DNA damage signals,leading to the phosphorylation of downstream molecules,e.g.,histone H2AX and P53.Phosphorylation of P53 following DNA damage can lead to cell apoptosis,and is responsible for most of chemoradiotherapy-induced ISC loss.Therefore,inhibition of PIKKs or P53-dependent cell apoptosis after DNA damage is an effective way to reduce chemoradiotherapy-induced ISC loss.Gamma-aminobutyric acid(GABA)is a classical inhibitory neurotransmitter,which is mainly synthesized from glutamate and catalyzed by glutamate decarboxylase(GAD).GABA performs its action by binding to its ionotropic GABA A receptor(GABAAR)or metabotropic GABA B receptor(GABABR).Extracellular GABA can be rapidly absorbed by GABA transporter(GAT)and finally degraded by GABA transaminase(GABA-T).GABA,its receptors and metabolism-related proteins are referred to as the GABAergic system.Apart from participating in the signal transduction in the central nervous system,the GABAergic system is also present in various peripheral tissues,such as the gastrointestinal tract and liver.GABA plays an important role in the innate immunity of intestinal mucosa through GABAAR.Moreover,GABA is involved in the regulation of intestinal movement or secretion.Our previous studies found that the entire GABAergic system was present in IECs,however,the function of the GABAergic system in IECs in chemoradiotherapy-induced intestinal injury is still unclear.Based on the background,we explored the expression of GABAAR subunits in IECs after chemoradiotherapy,and found that GABAAR α1(GABRA1)levels increased in the mouse intestinal crypts after chemoradiotherapy.Subsequently,we found that the conditional deletion of Gahra1 in IECs had no effect on the homeostasis of IECs in mice,but reduced chemoradiotherapy-induced intestinal injury and prolonged animal survival.Moreover,our results showed that GABRA1 was expressed in LGR5+ISCs.The deletion of GABRA1 in LGR5+ISCs protected against chemoradiotherapy-induced ISC apoptosis and intestinal organoid toxicity in mice.Notably,through multiple tumor-related mouse models,we demonstrated that the administration of bicuculline,a GABAAR antagonist,prevented chemoradiotherapy-induced P53-dependent IEC apoptosis without reducing the chemo-radiosensitivity of tumors.Mechanistically,it was found that inhibition of GABAAR reduced chemoradiotherapy-induced reactive oxygen species(ROS)-dependent DNA damage,and further reduced P53-dependent IEC apoptosis through the ATM-P53 pathway.Importantly,we found that flumazenil,a GABAAR antagonist approved by the United States FDA,attenuated chemoradiotherapy-induced mouse intestinal injury and human intestinal organoid toxicity effectively.Taken together,it was found that the activation of GABAAR in LGR5+ISCs was involved in the process of intestinal damage induced by chemoradiotherapy.Gene knock-out or pharmacological blocking of GABAAR can effectively reduce chemoradiotherapy-induced intestinal injury.This study provided a theoretical basis for the development of drugs to alleviate the intestinal toxicity caused by chemoradiotherapy.