Study On The Protective Effect Of Cinnamaldehyde Against Corticosterone-induced Mouse Kidney Injury And Its Underlying Molecular Mechanism

A large number of clinical and experimental results show that the disorder in sleep cycle,hormone rhythm and metabolic function leads to dysregulation of hypothalamus pituitary adrenal(HPA)axis,resulting in unbalance of cortisol physiological rhythm.High level of cortisol damages the renal function directly or indirectly,while cortisol promotes the expression of serum and glucocorticoid-regulated kinase 1(SGK1),increases the phosphorylation level of murine double minute 2(MDM2),inhibits the phosphorylation level of p53(p-p53)and promotes the phosphorylation of nuclear factor-?B(NF-?B),suggesting the involvement of SGK1/MDM2/p53/NF-?B signaling pathway.Cortisol can promote reactive oxygen species(ROS)production and then induce thioredoxin-interacting protein(TXNIP)to activate the NOD like receptor protein 3(NLRP3)inflammasome and produce interleukin-1?(IL-1?).Therefore,whether cortisol mediates ROS/TXNIP/NLRP3 inflammasome signaling pathway and SGK1/MDM2/p53/NF-?B signaling pathway to injure kidney needs to be further explored.Cinnamaldehyde is the major ingredient of Cinnamomum cassia with multiple activities,including antioxidant,anti-inflammatory and renal protective effects.However,the mechanism of cinnamaldehyde on improving renal injury induced by cortisol is still unclear.Therefore,C57BL6/J mouse model of renal injury induced by corticosterone and human renal tubular epithelial cell(HK-2)model stimulated by cortisol were used to explore pathological pathway,as well as the protective mechanism of cinnamaldehyde.A corticosterone-induced model of renal injury in C57BL6/J mice was established according to the method reported by other researchers.Cinnamaldehyde(10,20,40 mg/kg)and fluoxetine(15 mg/kg,positive control)were given for 3 weeks respectively.The behavioral changes were evaluated.Compared to the normal group,the immobility time in forced swimming test and tail suspension test was increased significantly in the model group.Meanwhile,corticosterone increased the time spent in center during the open field test,decreased the percentage of sucrose preference significantly,suggesting the depressive behavior.The results of tissue immunofluorescence showed that corticosterone significantly increased the fluorescence intensity of NLRP3 and IL-1? in the kidney of mice,exhibiting the inflammation occurrence.At the same time,serum levels of urea nitrogen and creatinine were significantly increased,and urine levels of creatinine were obviously decreased in this animal model,further confirming corticosterone-induced renal function damage.Cinnamaldehyde significantly reduced the increased immobility time in forced swimming test and tail suspension test,increased the time spent in center in the open field test and the consumption of sucrose water in corticosterone-stimulated mice.Furthermore,cinnamaldehyde reduced fluorescence intensity of kidney NLRP3 and IL-1?,decreased serum urea nitrogen and creatinine levels,and increased urea creatinine levels in this animal model.These findings indicate that cinnamaldehyde may effectively improve depressive behavior,kidney inflammatory response and function impairment in corticosterone-stimulated mice.Next,whether corticosterone or cortisol targets the ROS/TXNIP/NLRP3 inflammasome signaling pathway and the possible protective role of cinnamaldehyde in mice and in vitro HK-2 cells were investigated.Compared with the normal group,the renal ROS levels and renal protein levels of TXNIP,NLRP3,caspase-1,ASC and IL-1? were significantly increased in corticosterone-stimulated mice.Similar results were observed in cortisol-exposed HK-2 cells,suggesting the activation of ROS/TXNIP/NLRP3 inflammasome signaling.Cinnamaldehyde obviously reduced renal ROS,TXNIP,NLRP3,caspase-1,ASC and IL-1? protein levels induced by corticosterone in the kidney.Similar results were also obtained in HK-2 cells incubated with cortisol.Therefore,cinnamaldehyde inhibits the activation of ROS/TXNIP/NLRP3 inflammasome signaling,leading to ameloriation of inflammatory responses in rats and HK-2 cells.The effects of corticosterone or cortisol on the SGK1/MDM2/p53/NF-?B signaling and the effects of cinnamaldehyde in mice kidney and HK-2 cells were studied further.Compared with the normal group,the fluorescence intensity of renal SGK1,protein levels of SGK1,p-MDM2 and p-NF-?B,as well as the phosphorylation protein level of p53 increased significantly in corticosterone-treated group.Similar results were also obtained in HK-2 cells incubated with cortisol.Therefore,the SGK1/MDM2/p53/NF-?B signaling activated by corticosterone to promote the renal inflammatory responses.Cinnamaldehyde reduced the fluorescence intensity of SGK1,and the protein levels of SGK1,p-MDM2,p-NF-?B protein,and phosphorylated protein levels of p53 remarkably,suggesting that cinnamaldehyde may inhibit the activation of SGK1/MDM2/p53/NF-?B signaling and alleviate the renal inflammation triggered by corticosterone.In order to determine the relationship between ROS/TXNIP/NLRP3 inflammasome signaling and SGK1/MDM2/p53/NF-?B signaling induced by cortisol,ROS inhibitor NAC co-incubation and SGK1 inhibitor GSK650394 pre-incubation were adopted in cells experiments.Compared with the cortisol-treated group,the protein levels of SGK1,TXNIP and IL-1? decreased significantly with cortisol and NAC coincubation.However,the protein expression level of SGK1 induced by cortisol and NAC coincubation was significantly higher than that of the NAC-treated group,suggesting that ROS mediates SGK1/MDM2/p53/NF-?B signaling with cortisol exposure.Compared with the cortisol-treated group,cortisol and GSK650394 co-incubation significantly decreased the protein levels of TXNIP and NLRP3 in HK-2 cells,but had no significant effect on the fluorescence intensity of ROS,the protein levels of caspase-1,ASC and IL-1?,suggesting that SGK1 promotes the expressions of NLRP3 and TXNIP,but has no obvious effect on ROS/TXNIP/NLRP3 inflammasome signaling with cortisol treatment.Compared with cortisol and NAC co-incubation group,treatment with cinnamaldehyde,NAC and cortisol had no significant effect on the protein levels of TXNIP and IL-1?,but significantly reduced the protein elevations of SGK1.It was supposed that cinnamaldehyde may regulate the SGK1/MDM2/p53/NF-?B signaling in HK-2 treated with cortisol and NAC,exhibting certain superimposed effect with NAC.Compared with the cortisol and GSK650394 co-incuabtion group,cinnamaldehyde had no significant effect on the protein levels of TXNIP and NLRP3,but could reduces the fluorescence intensity of ROS,the protein levels of caspase-1,ASC and IL-1? significantly.It was suggested that cinnamaldehyde may regulate the ROS/TXNIP/NLRP3 inflammasome signaling with SGK1 inhibitor exposure.Therefore,cinnamaldehyde can alleviate renal inflammatory responses induced by cortisol via inhibiting ROS/TXNIP/NLRP3 inflammasome signaling and SGK1/MDM2/p53/NF-?B signaling.In summary,the present work confirmed that cortisol or corticosterone could activate ROS/TXNIP/NLRP3 inflammasome signaling and SGK1/MDM2/p53/NF-?B signaling,and ROS may target the SGK1/MDM2/p53/NF-?B signaling to trigger renal injury and inflammatory responses in mice.Cinnamaldehyde could ameliorate renal damage and inflammatory responses by inhibiting the ROS/TXNIP/NLRP3 inflammasome signaling and SGK1/MDM2/p53/NF-?B signaling.In this dissertation.a novel mechanism of corticosterone induced renal injury in mice was discovered.And the preliminary findings about the protective effect of cinnamaldehyde on corticosterone-induced renal injury was reported,which provided an experimental basis for cortisol rhythm disorder and renal injury caused by diverse pathological factors.