| Objective: the purpose of this study was to investigate whether the activation of NLRP3 inflammasome is related to the regulation mechanism of mitophagy in the pathogenesis of depression,whether Xiaojianzhong Decoction has antidepressant effect,whether the mechanism of Xiaojianzhong Decoction is related to up-regulating the level of mitophagy,improving mitochondrial function,inhibiting TLR4/NF-?B pathway and NLRP3 inflammasome activation in the hippocampus of CUMS rats,and whether the inhibitory effect of Xiaojianzhong Decoction on NLRP3 inflammasome depends on mitophagy mediated by PINK-1/Parkin pathway.Methods: Experiment 1: 40 SD rats were used to establish the model with CUMS except the control group(n = 8).After 3 weeks,the body weight and sucrose preference rate of rats were measured to determine whether the model was successful or not.The modeled rats were randomly divided into CUMS group(n = 8),fluoxetine group(n = 9)and rapamycin group(n = 9).Rats were treated with drugs for 3 weeks respectively,and the CUMS model was continued at the same time.After treatment,the behavior tests of rats were detected,and the hippocampal tissue and serum were separated.The copy number of Mt DNA,the changes of mitophagy related m RNA(PINK-1,Parkin,Beclin-1,LC3,P62)and NLRP3 inflammasome pathway related m RNA(NLRP3,caspase-1,ASC,IL-1?,IL-18)were detected by RT-PCR.The expressions of mitophagy related proteins(PINK-1,Parkin,Beclin-1,LC3,P62)and NLRP3 inflammasome pathway related proteins(NLRP3,caspase-1,ASC,IL-1?,IL-18)were detected by WesternBlot.The expressions of IL-1?,IL-18,DA and 5-HT in serum were tseted by ELISA.Experiment 2: 60 SD rats were used to establish the model with CUMS except the control group(n = 7).After 3 weeks,the body weight and sucrose preference rate of rats were measured to determine whether the model was successful or not.The modeled rats were randomly divided into CUMS group(n = 7),fluoxetine group(n = 7),low-dose group(n = 8),middle-dose group(n = 8),middle-dose group(n = 8)and high-dose group(n = 8).Rats were given drug intervention for 3 weeks,and CUMS model was made at the same time.After the treatment,the behavior tests were detected,and then the hippocampal tissue and serum were separated.The morphology of pyramidal cells in hippocampal CA3 region was detected by Nissl staining,and the ultrastructure of pyramidal cells and mitochondria in hippocampal CA3 region was observed by transmission electron microscope.Mt DNA copy number,mitophagy related m RNA(PINK-1,Parkin,Beclin-1,P62),NF-?B signal pathway related m RNA(TLR4,I?B-?,NF-?B1,RELA and TNF ?),NLRP3 inflammasome related m RNA(NLRP3,caspase-1,ASC,IL-1?,IL-18)were measured by RT-PCR.Western-Blot was used to detect the changes of mitophagy-related proteins(PINK-1,Parkin,Beclin-1,LC3,P62),NF-?B signaling pathwayrelated proteins(TLR4,p-i?B-?,p-p50 and p-p65).Western-Blot and immunofluorescence were used to detect the expressions of NLRP3 inflammasome pathway related proteins(NLRP3,caspase-1,ASC,IL-1?,IL-18).The changes of IL-1?,IL-18,DA,5-HT,SOD and MDA in serum were tested by ELISA.Experiment 3: 50 SD rats were used to establish the model with CUMS except the control group(n = 7).After 3 weeks,the body weight and sucrose preference rate of rats were measured to determine whether the model was successful or not.The modeled rats were randomly divided into CUMS group(n = 8),fluoxetine group(n = 8),Xiao Jian Zhong group(n = 8)and Xiao Jian Zhong + inhibitor group(n = 8).Rats were treated with drugs for 3 weeks respectively,and the CUMS model was made at the same time.After treatment,the behavior test was detected.RT-PCR was used to detect the changes of mitophagy related m RNA(PINK-1,Parkin,Beclin-1,P62),NLRP3 inflammasome pathway related m RNA(NLRP3,caspase-1,ASC,IL-1?,IL-18).Western-Blot was used to detect the changes of mitophagy related proteins(PINK-1,Parkin,Beclin-1,LC3,P62)and NLRP3 inflammasome pathway related proteins(NLRP3,caspase-1,ASC,IL-1?,IL-18).Results: 1.Changes of behavior,mitophagy and NLRP3 inflammasome in CUMS rats.1.1 Behavior: Compared with the control group,the body weight and sucrose preference rate of rats in the CUMS group decreased significantly(P < 0.01).The immobility time of rats in the CUMS group was longer than that in the control group(P < 0.01)in the forced swimming test(FST).In the open field test(OFT),the crossing score,rearing score,total exercise distance and total exercise time of rats were decreased than that in the control group(P < 0.01).1.2 Mitophagy: Compared with the control group,the m RNA relative expressions of PINK-1,Parkin and Beclin-1 in hippocampus of rats in CUMS group were significantly decreased,while the relative expression of m RNA of p62 was increased.Compared with the control group,the protein expression levels of PINK-1,Parkin,Beclin-1 and LC3B2/1 in the hippocampus of rats in CUMS group were decreased significantly,while the expression of p62 protein was increased significantly.1.3 NLRP3 inflammasome: When compared with the control group,the m RNA relative expressions of NLRP3,ASC,caspase-1,IL-1? and IL-18 in the hippocampus of rats in CUMS group were significantly increased.Compared with the control group,the protein expression levels of NLRP3,ASC,caspase-1,IL-1? and IL-18 of rats in CUMS group were significantly increased.2.Effects of rapamycin on behavior,mitophagy and NLRP3 inflammasome in CUMS rats 2.1 Behavioral test: Compared with the CUMS group,the sucrose preference rate of rapamycin group was significantly increased(P < 0.01).The immobility time of FST was significantly decreased(P < 0.01).2.2 Mitophagy: Compared with the CUMS group,the m RNA relative expressions of PINK-1(P < 0.01),Parkin(P < 0.05)and Beclin-1(P < 0.05)in hippocampus of rats in rapamycin group were increased,while the relative expression of p62(P < 0.01)m RNA were decreased.Compared with the CUMS group,the protein expressions of PINK-1,Parkin,Beclin-1 and LC3B2/1 in the hippocampus of rats in rapamycin group were increased,while the protein expression of p62 were decreased.Rapamycin up-regulated the mitophagy level in CUMS rats.2.3 NLRP3 inflammasome: Compared with the CUMS group,the relative m RNA expressions of NLRP3,caspase-1,ASC,IL-1? and IL-18 in hippocampus of rats in rapamycin group were decreased.At the same time,the protein expressions of NLRP3,caspase-1 p20,ASC,IL-1? and IL-18 were decreased.Rapamycin inhibited the activation of NLRP3 inflammasome in CUMS rats.3.Effects of Xiaojianzhong Decoction on behavior of CUMS rats.Compared with the CUMS group,the body weights of the rats were increased(P < 0.01)and the immobility time of FST in Xiaojianzhong Decoction groups were shorter(P < 0.01).Compared with the CUMS group,the crossing score of the low and middle dose groups were increased(P < 0.05),and the rearing score,the total exercise distance and the total exercise time of the rats in Xiaojianzhong Decoction groups were increased(P < 0.01)in OFT.Xiaojianzhong Decoction significantly reversed the depression-like behavior of CUMS rats.4.Regulation of Xiaojianzhong Decoction on mitophagy in CUMS rats.Compared with the CUMS group,the m RNA relative expressions of PINK-1(P < 0.01)and Parkin(P < 0.05)were increased,while the relative expression of m RNA of p62(P < 0.01)were decreased in hippocampus of rats in low dose group.The relative expressions of m RNA of PINK-1(P < 0.01),Parkin(P < 0.05)and Beclin-1(P < 0.01,P < 0.05)were increased,while the m RNA expression of p62(P < 0.01)were decreased in the hippocampus of rats in middle dose group and high dose group.Compared with the CUMS group,the protein expressions of PINK-1,Parkin and Beclin-1 were significantly up-regulated,while the protein expression level of p62 was decreased in the hippocampus of rats in the low dose group(P < 0.01).The protein expressions of PINK-1,Parkin,Beclin-1 and LC3B2/1 were up-regulated,while the protein expression of p62 was decreased in hippocampus of rats in middle and high dose group(P < 0.01).Xiaojianzhong Decoction upregulated the level of mitophagy in CUMS rats through PINK-1/Parkin pathway.5.Effect of Xiaojianzhong Decoction on mt DNA copy number of CUMS rats.Compared with the control group,the copy number of mt DNA in the hippocampus of the CUMS group was significantly increased(P < 0.01).Compared with the CUMS group,the copy numbers of hippocampal mt DNA of rats in the low,middle and high dose groups of Xiaojianzhong Decoction were significantly increased(P < 0.01).6.Effect of Xiaojianzhong Decoction on SOD and MDA in CUMS rats.Compared with the control group,the level of serum SOD(P < 0.01)was decreased and MDA(P < 0.01)was increased in the CUMS group.When compared with the CUMS group,the expressions of serum SOD were increased(P < 0.05,P < 0.01,P < 0.01),and the expression of MDA were decreased significantly(P < 0.01,P < 0.05,P < 0.01)in the low,middle and high dose groups.Xiaojianzhong Decoction improved the level of lipid peroxidation in CUMS rats.7.Effect of Xiaojianzhong Decoction on TLR4/NF-?B signal pathway in CUMS rats.When compared with the control group,the m RNA relative expressions of TLR4,I?B-?,NF-?B1,RELA and TNF-? were increased in the hippocampus of rats in the CUMS group(P < 0.01).Compared with the CUMS group,the levels of TLR4(P < 0.01),I?B-?(P < 0.01),NF-?B1(P < 0.01),RELA(P < 0.01)and TNF-?(P < 0.01)in the Xiaojianzhong Decoction groups were decreased.Compared with the control group,the protein levels of p-p50,p-p65,p-i?B-? and TLR4 in the hippocampus of the CUMS group were significantly increased(P < 0.01).Compared with the CUMS group,the protein levels of p-p50,p-p65,I?B-? and TLR4 in the hippocampus of rats in the low,middle and high dose groups of Xiaojianzhong Decoction were significantly lower(P < 0.01).Xiaojianzhong Decoction inhibited the activation of TLR4/NF-?B signal pathway in CUMS rats.8.Effect of Xiaojianzhong Decoction on NLRP3 inflammasome in CUMS rats.Compared with the CUMS group,the relative expression of m RNA and levels of protein of NLRP3,ASC,caspase-1,IL-1? and IL-18 in the low,middle and high groups of Xiaojianzhong Decoction were significantly lower(P < 0.01).Xiaojianzhong Decoction inhibited the activation of NLRP3 inflammasome in CUMS rats.9.Effect of Xiaojianzhong Decoction on Serum 5-HT and DA in CUMS rats.Compared with the control group,the protein levels of DA and 5-HT in the serum of rats in the CUMS group were significantly lower(P < 0.01).The expressions of DA and 5-HT were up-regulated in the low,middle and high dose groups as compared with the CUMS group.Xiaojianzhong Decoction increased the levels of serum 5-HT and DA in CUMS rats(P < 0.01).10.Effect of Xiaojianzhong Decoction + mitophagy inhibitor on mitophagy in CUMS rats.Compared with Xiaojianzhong Decoction group,the relative expression of m RNA of PINK-1(P < 0.01),Parkin(P < 0.05)and Beclin-1(P < 0.01)were decreased while the relative expression of m RNA of p62(P < 0.01)were increased in Xiaojianzhong Decoction + inhibitor group.Compared with the Xiaojianzhong Decoction group,the protein expression levels of PINK-1,Parkin,Beclin-1 and LC3B2/1 in the hippocampus of the rats were decreased significantly(P < 0.01),while the expression of p62 protein increased in the Xiaojianzhong Decoction + inhibitor group(P < 0.01).Mitophagy inhibitor attenuated the enhancement effect of Xiaojianzhong Decoction on mitophagy in CUMS rats.11.Effect of Xiaojianzhong Decoction + mitophagy inhibitor on NLRP3 inflammasome in CUMS rats.Compared with the Xiaojianzhong Decoction group,the relative m RNA expressions and protein expressions of NLRP3(P< 0.01),ASC(P< 0.05),caspase-1(P< 0.01),IL-1?(P< 0.01)and IL-18(P< 0.01)in the hippocampus of rats in the Xiaojianzhong Decoction + inhibitor group were increased.Mitophagy inhibitor attenuated the inhibitory effect of Xiaojianzhong Decoction on the NLRP3 inflammasome in CUMS rats.Conclusions: 1.There are both low level of mitophagy and activation of NLRP3 inflammasome pathway in the hippocampus of CUMS rats,which may one of the important mechanisms of depression.2.Rapamycin,a mitophagy agonist,improved the depression-like behavior of CUMS rats and decreased the levels of NLRP3 inflammasome and downstream inflammatory factors.It is further verified that the activation of NLRP3 inflammasome is related to the mitophagy,which is a negative regulatory mechanism in the pathogenesis of depression.3.Xiaojianzhong Decoction could effectively reverse the depression-like behavior of CUMS rats by improving the autonomous activity and reducing the immobility time of FST in CUMS rats.It has shown that Xiaojianzhong Decoction has an antidepressant effect.4.The pharmacological mechanism of Xiaojianzhong Decoction in the treatment of depression is related to its up-regulation to mitophagy and inhibition to TLR4/NF-?B pathway,NLRP3 inflammasome and the inflammatory cytokines of CUMS rats.5.The inhibitory effect of Xiaojianzhong Decoction on NLRP3 inflammasome depends on its regulation of mitophagy mediated by PINK-1/Parkin pathway. |
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