Toll-like Receptor 4 Protects Against Stress-Induced Ulcers Via Regulation Of Glucocorticoid Production

BACKGROUNDStress is considered to be an adaptive response to various intra-or inter-stressors.Although mild stress stimulating can be benefit,severe stress can induce dysfunctions of many organs or systems in human body.Stomach is considered to be one of the most susceptible organs to stress,which can induce many gastric dysfunctions known as ‘stress related mucosa disease,SRMD'.Stress induced gastric ulcer(stress ulcer,SU),one of the most severe SRMDs,is a disease of life-threatening condition in intensive care units(ICU),may induce gastric hemorrhage and even deaths of patients.Stress ulcer is considered to be induced by the gastric ischemia related to the neuroendocrine reaction during the stress.Injury of gastric mucosal defence system and impact of further damage factors(e.g.acid,pepsin)result in gastric ischemia,making the ulceration aggravated.However,the molecular basis of stress ulcer remains largely elucidated.As many researches reported,local proinflammatory factors activation and inflammation in gastric mucosa can make the ulceration aggravated.Toll like receptor 4(TLR4)is known as a classic pattern recognition receptor(PRR),its activation induces the release of proinflammatory factors and mediates the inflammation in various cells or organs.Hence TLR4 signaling may promote stress ulceration by activating the gastric local inflammation.However,previous researches reported that TLR4 activation can stimulate the HPA axis and induce the upregulation of CRF gene expression in paraventricular nudeus neurons in the hypothalamus.The release of glucocorticoid,which can protect against the stress ulcer,is considered to be the protective mechanism of TLR4 in stress induced gastric ulceration.So far,the relationship between the TLR4 and stress ulcer remains unknown.Endogenous glucocorticoid released during acute stress is an important hormone to help body to adapt to the stress.Many researchers found the endogenous glucocorticoid can alleviate the stress induced ulcer and promote the gastric mucosa restoration.So,we hypothesized that TLR4 may play a protective role in stress ulceration.This study aimed to investigate the effect of TLR4 signaling in gastric injury during stress,to explore its role in regulating glucocorticoid production and underlie the mechanisms.OBJECTIVE1.To investigate the effect of TLR4 signaling to stress ulcer.2.To verify whether the action of TLR4 signaling to SU is mediated by the regulation of glucocorticoid production.3.To underlie mechanisms of regulation of glucocorticoid production by TLR4 signalling.METHODPart 1.To verify the effect of TLR4 signaling to SU,wild-type(WT)and TLR4 mutant(TLR4-/-)male mice were randomly divided into five groups(5 per group)and exposed to the immersion and restraint stress for 0,0.5,1,2,4 hours.The gross view,HE stained mucosa sections and ulcer index were detected for measurement of the gastric injury.The corticosterone and cortisol concentrations in different mice-types and time points were tested by Elisa.To verify whether the protective role of TLR4 signaling is mediated via the regulation of glucocorticoid,the ulcer index were measured after 10mg/ml metyrapone injected subcutaneous,or 0.4mg/ml cortisol pretreated subcutaneous in 5 mice respectively.The corticotropin tests were further applied to access the synthetic functions of adrenal gland further.Part 2.To verify the mechanism of TLR4 activation mediating the glucocorticoid synthesis,the main ligand of TLR4,LPS and its binding protein(LBP)concentrations were detected with Elisa respectively after 4 hours stress.The corticosterone and cortisol concentrations were also tested in 5 mice respectively with LPS/saline intraperitoneal injection for 4 hours.The corticosterone concentrations of LPS treated Y1 cell supernatant with TLR4 over expression lentivirus infection(LV-TLR4),negative control lentivirus infection(LV-NC)and none infection(Mock)were detected further to verified whether LPS/TLR4 signaling in cortex cells of adrenal gland mediated the glucocorticoid synthesis.Part 3.To access the glucocorticoid synthesis function of adrenal gland,Adrenal gland frozen sections were prepared and stained with oil red O for neutral lipid visualization.Protein expression of key enzymes to glucocorticoid synthesis were detected in both type mice,especially in LV-TLR4,LV-NC and Mock Y1 cells through western blot.Furthermore,cells in adrenal cortex zona fasciculata of both type mice without any treatment were detected with electron microscopy.RESULTSPart 1.TLR4 deficiency exacerbates stress ulceration by modulate serum glucocorticoid concentrations.The gross view,HE stained sections and ulcer index showed the gastric injury in TLR4 mutant were more severe than WT mice.Concentrations of corticosterone and cortisol increased after stress stimulation in WT,but not in TLR4-/-mice.The ulcer index showed increase in WT mice with metyrapone injection during stress.And cortisol pretreatment can significant reverse the gastric injury increased by TLR4 deficiency.Corticotrophin test showed the reaction to corticotrophin stimulation in TLR4-/-mice were impaired.Part 2.LPS/TLR4 signaling mediated the glucocorticoid synthesis of adrenal gland.LPS and LBP concentrations were increased during stress in both type mice.The corticosterone and cortisol concentrations increased with LPS treatment in WT only.Corticosterone concentrations in Y1 supernatant also increased after 100ng/ml LPS treated for 48 hours.And LPS induced corticosterone increase in Y1 supernatant were further enhanced with TLR4 over expression.Part 3.TLR4 deficiency impaired the glucocorticoid synthesis function of adrenal cortex.Oil red O stained frozen sections of adrenal gland showed cholesterol decrease in TLR4-/-mice.The expression of key enzyme in glucocorticoid synthesis CYP11A1 protein,decreased in TLR4-/-mice through western blot detection.And CYP11A1 increase was detected in TLR4 over-expression-cells.Furthermore,compared with WT,the zona fasciculata cells' mitochondria inner membrane which are necessary for the first limited synthesis reaction of glucocorticoid,were impaired in TLR4-/-mice.CONCLUSION1.TLR4 plays a protective role in stress ulcer via mediating glucocorticoid production.2.The increase of LPS during stress stimulate TLR4 in adrenal cortex to mediate glucocorticoid production.3.Function of glucocorticoid synthesis in adrenal cortex were impaired by TLR4 deficiency.