| BackgroundAs a stressor,chronic pain is the primary determinant of mood disorders.Common mood disorders include anxiety and depression.The incidence of major depression is as high as 30%in neuropathic pain patients.The molecular mechanism of anxiety and depression induced by neuropathic pain is still unclear.Fat mass and obesity-associated protein(FTO),as a demethylase,is involved in the regulation of post-transcriptional mRNA m6A modification.FTO is involved in the generation and maintenance of neuropathic pain in primary sensory neurons.FTO-knockout mice exhibited anxiety-and depression-like behaviors.However,the role and specific mechanism of FTO in anxiety and depression induced by neuropathic pain are still unclear.This study intends to evaluate the role and molecular mechanism of FTO in anxiety-and depression-like behaviors induced by neuropathic pain in mice,in order to provide new ideas for the study of the mechanism of chronic pain-induced anxiety and depression.Objective1.To explore the role of FTO in anxiety-and depression-like behaviors induced by neuropathic pain.2.To explore the molecular mechanisms which FTO mediates in anxiety-and depression-like behaviors induced by neuropathic pain.Experimental methods1.Chronic sciatic nerve compression injury(CCI)induced hyperalgesia and anxiety-and depression-like behaviors in miceMale C57BL/6J mice were randomly divided into two groups(n=10):experimental group(CCI group)and control group(Sham group).The paw withdrawal threshold(PWT)and paw withdrawal latency(PWL)of mice were measured at 1 d before surgery,3 d,7 d,14 d,21 d and 28 d after surgery,respectively.After 28 days,the anxiety-like behavior of mice was observed by open field test(OFT)and elevated cross test(EPM),and the depression-like behavior of mice was observed by forced swimming test(FST)and tail suspension test(TST).2.Expression and cell distribution of FTO in ACC in neuropathic pain mice7 d,14 d,21 d and 28 d after surgery,qRT-PCR was used to detect the expression changes of FTO mRNA level in ACC of mice,and Western blot technology was used to detect the expression changes of FTO protein level in ACC of mice.Immunofluorescence technique was used to detect the expression changes and cell distribution types of FTO in ACC.3.Effects of FTO overexpression in ACC on anxiety-depression-like behavior in neuropathic pain miceMale C57BL/6J mice were randomly divided into 2 groups(n=20):the experimental group(CCI group)and the control group(Sham group).Group Mice were randomly divided into 2 groups(n=10),a total of four groups:CCI+AAV-EGFP group,CCI+AAV-FTO group,Sham+AAV-EGFP group and Sham+AAV-FTO group.Mice in each group underwent ACC microinjection of AAV-FTO or AAV-EGFP 28 days after surgery.21 days after AAV injection,qRT-PCR was used to detect the expression changes of FTO mRNA in ACC of mice,and Western blot was used to detect the expression changes of FTO protein level in ACC of mice.AAV transfection and transfection cell types were assessed by immunofluorescence.To verify whether the overexpression of FTO in the ACC can reverse the anxiety-and depression-like behaviors caused by neuropathic pain,the anxiety-like behavior of mice were observed by OFT and EPM in turn,and the depression-like behavior of mice were observed by TST and FST,respectively.4.Effects of knockdown of FTO expression in ACC on the behavior of Naive miceMale C57BL/6J mice were divided into two groups(n=10):Naive+AAV-FTO shRNA group and Na?ve+AAV-scrRNA group.Mice in each group were microinjected AAV-FTO shRNA or AAV-scrRNA into the ACC contralateral to CCI surgery by stereotaxic brain.21 days after AAV injection,the expression changes of FTO mRNA in ACC of neuropathic pain mice were detected by qRT-PCR.The expression changes of FTO protein in ACC of neuropathic pain mice were detected by Western blot.The anxiety-like behaviors of mice were observed by OFT and EPM,respectively,and the depression-like behaviors of mice were observed by TST and FST,respectively.5.Molecular mechanisms of FTO mediating anxiety-and depression-like behaviors induced by neuropathic pain in miceThe expression changes of MMP-9 mRNA in the ACC of neuropathic pain mice were detected by qRT-PCR.Western blot was used to detect the protein levels of MMP-9,proBDNF and mBDNF in the ACC of neuropathic pain mice.In situ hybridization technique combined with immunofluorescence technique was used to detect the cellular co-localization of FTO protein and MMP-9 mRNA.The co-localization of FTO protein and MMP-9 protein was detected by immunofluorescence technique.28 days after CCI,the enrichment of FTO on MMP-9 mRNA(n=4)and the enrichment of m6A on MMP-9 mRNA(n=4)were detected by RIP-PCR.The expression changes of MMP-9 mRNA were detected by qRT-PCR technology after overexpression of FTO in ACC of mice.Western blot was used to detect the expression changes of MMP-9,proBDNF and mBDNF protein levels after FTO was overexpressed in ACC of mice.The expression changes of MMP-9 mRNA were detected by qRT-PCR technology after knocked down of FTO expression in ACC of mice.Western blot was used to detect the expression changes of MMP-9,proBDNF and mBDNF protein levels after knocked down FTO expression in ACC of mice.Results1.Chronic sciatic nerve compression injury(CCI)induces chronic hyperalgesia and anxiety-depression-like behavior in miceThe results of pain behavior showed that compared with the mice in the Sham group,the PWT and PWL values of the mice in the CCI group decreased significantly on the 3 d after operation(P<0.05),and could be maintained until the 28 d after the operation,suggesting that Chronic compression sciatic nerve injury mice could induced the developed hyperalgesia.The neuropathic pain model was successfully prepared.The OFT results showed that there was no significant difference in the exercise ability of the two groups of mice during the test(P>0.05);compared with the mice in the Sham group,the percentage of time spent in the central area of the CCI group was significantly reduced(P<0.05),and the number of times entering the central area was significantly reduced(P<0.05).The results of the EPM experiment showed that there was no significant difference in the exercise ability of the two groups of mice during the test(P>0.05);compared with the mice in the Sham group,the percentage of time that the mice in the CCI group stayed in the open arm was significantly reduced(P<0.05),and the number of times they entered the central area was significantly reduced(P<0.05),indicating that neuropathic pain induced anxiety-like behaviors in mice.The results of FST showed that compared with the mice in the Sham group,the mice in the CCI group remained stationary during the test period significantly longer(P<0.05).In the TST experiment,compared with the mice in the Sham group,the still time of the mice in the CCI group was significantly longer during the test period(P<0.05),suggesting that neuropathic pain can lead to depression-like behaviors in mice.These results suggested that neuropathic pain can induce anxiety-and depression-like behaviors in mice.2.FTO expression was down-regulated in ACC of neuropathic pain miceqRT-PCR and Western blot results showed that FTO mRNA and protein expression were downregulated at 7 d after surgery in the contralateral ACC of neuropathic pain mice compared with the sham group and could persist until 28 days after surgery(P<0.05).Immunofluorescence single staining results showed that the number of FTO positive cells was decreased in the contralateral ACC of neuropathic pain mice compared with the Sham group(P<0.05).Immunofluorescence double staining results showed that FTO was highly co expressed with neuronal nuclear antigen(NeuN)and sparsely expressed with glial fibrillary acidic protein(GFAP)or microglia(OX42).3.Overexpression of FTO in ACC can reverse anxiety-and depression-like behaviors in neuropathic pain miceResults of qRT-PCR showed that FTO mRNA levels were significantly upregulated in the ACC of mice treated with Sham+AAV-FTO and CCI+AAV-FTO groups compared with Sham+AAV-EGFP and CCI+AAV-EGFP groups(P<0.05).The results of Western blot indicated that the protein levels of FTO in mice treated with Sham+AAV-FTO and CCI+AAV-FTO groups were significantly up-regulated compared with those in the Sham+AAV-EGFP and CCI+AAV-EGFP groups(P<0.05).Immunofluorescence results showed that AAV was mainly transfected into ACC neurons,and there was no obvious transfection in astrocytes and microglia.The OFT results showed that there was no significant difference in exercise capacity during the test among the four groups of mice(P>0.05);CCI+AAV-FTO mice had significantly longer percentages of their stay in the central open field area and significantly greater numbers of entries into the central area compared with the CCI+AAV-EGFP group(P<0.05).The results of the EPM experiment showed no significant differences in exercise capacity during the testing period among the four groups of mice.Compared with the CCI+AAV-EGFP group,mice in the CCI+AAV-FTO group exhibited a significantly longer percentage of exploration time in the open arm(P<0.05)and an increased number of entries into the open arm,although the differences were not statistically significant(P>0.05).The above results indicate that reversing the downregulation of FTO in the ACC of CCI mice can improve neuropathic pain induced anxiety like behaviors.FST results showed that mice in the CCI+AAV-FTO group had significantly shorter immobility time compared with the CCI+AAV-EGFP group(P<0.05).The above results indicated that depressive like behaviors induced by neuropathic pain can be ameliorated by reversing the downregulation of FTO in the ACC of CCI mice.4.Knockdown of FTO expression in ACC induces anxiety-and depression-like behaviors in Na?ve miceqRT-PCR and Western blot showed that FTO mRNA and protein expression were significantly downregulated in the ACC of mice in the Na?ve +AAV-FTO shRNA group compared with the Na?ve+AAV-scrRNA group(P<0.05).The OFT results found that there was no significant difference in exercise capacity during the test between the two groups of mice(P>0.05);Compared with the Na?ve+AAV-scrRNA group,mice in the Na?ve+AAV-FTO shRNA group spent significantly less time in the middle zone of the open field(P<0.05)and less time in the center zone(P<0.05).In the EPM experiment,there was no significant difference in exercise capacity during the test between the two groups of mice(P>0.05;Compared with the Na?ve+AAV-scrRNA group,mice in the Na?ve+AAV-FTO shRNA group spent significantly less time in the open arm(P<0.05)and less time in the center zone(P<0.05),indicating that FTO knockdown in the ACC induced anxiety like emotions in mice.Results from FST and TST showed that mice in the Na?ve+AAV-FTO shRNA group spent significantly more time immobile compared with the Na?ve+AAV-scrRNA group(P<0.05),suggesting that FTO knockdown in the ACC evoked depression like behaviors in mice.5.FTO is involved in anxiety-depression-like behavior induced by neuropathic pain by regulating MMP-9/BDNF signaling pathwayThe results of qRT-PCR indicated that,28 days after surgery,there was no significant change in the mRNA expression level of MMP-9 in the ACC of mice in both groups(P>0.05).The results of Western blot indicated that the protein level of MMP-9 in the ACC of mice in the CCI group decreased significantly(P<0.05),and the expression of proBDNF protein in the ACC was increased(P<0.05),whereas the mBDNF protein level was decreased(P<0.05)compared with the Sham group.In situ hybridization and immunofluorescence results showed that MMP-9 mRNA was highly co expressed with FTO protein in ACC neurons.Immunofluorescence double staining showed that FTO protein was highly co expressed with MMP-9 protein in ACC.FTO RIP-PCR showed that,the enrichment of FTO on MMP-9 mRNA was significantly reduced in the ACC of the CCI mice compared with the Sham group(P<0.05);m6A RIP-PCR showed that the enrichment of m6A on MMP-9 mRNA was increased compared with the Sham group(P<0.05),and the above results suggest that FTO in ACC of mice with neuropathic pain may be through decreased binding to MMP-9 mRNA and increased m6A modification on MMP-9 mRNA,leading to downregulation of MMP-9 protein expression.Compared with the CCI+AAV-EGFP group,the levels of MMP-9 mRNA in the ACC of CCI+AAV-FTO group were unchanged(P>0.05),MMP-9 protein was upregulated(P<0.05),mBDNF protein was upregulated(P<0.05),and proBDNF protein was downregulated(P<0.05).Compared with Na?ve+AAV-scrRNA,there were no significant changes in MMP-9 mRNA levels(P>0.05),downregulation of MMP-9 protein(P<0.05),and downregulation of mBDNF protein(P<0.05)but upregulation of proBDNF protein(P<0.05)in the ACC of mice treated with Na?ve+AAV-FTO shRNA.These results indicated that FTO regulates MMP-9 protein expression by regulating m6A modification on MMP-9 mRNA,affects the dynamic balance of proBDNF/mBDNF,and participates in anxiety and depression-like behaviors caused by neuropathic pain.ConclusionFTO was down-regulated in contralateral ACC of mice with Chronic compression injury of sciatic nerve,and the enrichment degree of FTO on MMP-9 mRNA was reduced.m6A modification level on MMP-9 mRNA was up-regulated,which reduced the expression of MMP-9 protein,by affecting the dynamic balance of proBDNF/mBDNF,participate in anxiety-and depression-like behaviors induced by neuropathic pain. |
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