Cisplatin-induced premature senescence with concomitant reduction of gap junctions in human fibroblasts

To examine the role of gap junctions in cell senescence,the changes of gap junctions in cisplatin-induced premature senescence of primary cultured fibroblasts were studied and compared with the replicative senescent human fibroblasts.Dye transfer assay for gap junction function and immunofluorescent staining for connexin 43 protein distribution were done respectively. Furthermore,cytofluorimetry and DAPI fluorescence staining were performed for cell cycle and apoptosis analysis. p53 gene expression level was detected with indirect immunofluorescence. We found that cisplatin (10 mM) treatment could block cell growth cycle at G1 and induced premature senescence. The premature senescence changes included high frequency of apoptosis,elevation of p53 expression,loss of membranous gap junctions and reduction of dye-transfer capacity. These changes were comparable to the changes of replicative senescence of human fibroblasts. It was also concluded that cisplatin could induce premature senescence concomitant with inhibition of gap junctions in the fibroblasts. Loss of functional gap junctions from the cell membrane may account for the reduced intercellular communication in the premature senescent fibroblasts. The cell system we used may provide a model useful for the study of the gap junction thus promoting agents against premature senescence.

Connexin therapeutics:blocking connexin hemichannel pores is distinct from blocking pannexin channels or gap junctions

Compounds that block the function of connexin and pannexin protein channels have been suggested to be valuable therapeutics for a range of diseases.Some of these compounds are now in clinical trials,but for many of them,the literature is inconclusive about the molecular effect on the tissue,despite evidence of functional recovery.Blocking the different channel types has distinct physiological and pathological implications and this review describes current knowledge of connexin and pannexin protein channels,their function as channels and possible mechanisms of the channel block effect for the latest therapeutic compounds.We summarize the evidence implicating pannexins and connexins in disease,considering their homeostatic versus pathological roles,their contribution to excesive ATP release linked to disease onset and progression.

The role of gap junctions in cell death and neuromodulation in the retina

Vision altering diseases,such as glaucoma,diabetic retinopathy,age-related macular degeneration,myopia,retinal vascular disease,traumatic brain injuries and others cripple many lives and are projected to continue to cause anguish in the foreseeable future.Gap junctions serve as an emerging target for neuromodulation and possible regeneration as they directly connect healthy and/or diseased cells,thereby playing a crucial role in pathophysiology.Since they are permeable for macromolecules,able to cross the cellular barriers,they show duality in illness as a cause and as a therapeutic target.In this review,we take recent advancements in gap junction neuromodulation(pharmacological blockade,gene therapy,electrical and light stimulation)into account,to show the gap junction’s role in neuronal cell death and the possible routes of rescuing neuronal and glial cells in the retina succeeding illness or injury.

Gap junctions enhance the antiproliferative effect by transfer of microRNAs in glioma cells

OBJECTIVE To investigate the permeability of gap junction composed of connexin 43(Cx43)for micro RNAs(mi RNAs)and the impact of gap junction-mediated transfer of mi RNAs in glioma U87 cells.METHODS Co-culture assay demonstrated the transmission of miR NAs through gap junction channel into adjacent cells.U87 cells were labeled with green fluorescein protein(GFP)as receivers and cells were transfected mi RNAs as donors.Receiver cells and donor cells were mixed together in a ratio of 1∶1.After 12 h co-culture,cells were separated using a BD influx flow cytometer based on the GFP labeled.Quantitative real-time polymerase chain reaction(q RT-PCR)was applied detect to the expressions of miR NAs and Cx43 mR NA.Western blotting was performed to detect the protein expressions of Cx43 and GFP in U87 cells.CCK-8 assay is used to detect cell growth.RESULTS Co-culture assays demonstrated mi R-34a could transfer between U87 cells.The role of the contact independent could also transfer of miR-34a.Gap junctions inhibitor(CBX and 18-α-GA)showed lower miR-34a expression than co-culture group,whereas gap junctions enhancer(RA and Galanglin)enhanced miR-34a expression.Knockdown of Cx43 could significantly decrease the transferring of miR-34a between U87 cells.Different length of miR NAs(miR-1827,miR-144,miR-203a and miR-1183)were similar to the expression of miR-34a between U87 cells.Additionally,we demonstrated that gap junctions mediate the effect of antiproliferation mediated by mi R-34a in U87 cells.The functional inhibition of gap junctions using either si RNA or inhibition eliminated the miR-34a mediated antiproliferation,whereas the enhancement of gap junctions treatment augmented this mi R34a-mediated antiproliferation.CONCLUSION Our study demonstrates that gap junction composed of Cx43-mediated transfer mi RNAs in different length of nucleotides and gap junction-mediated transfer of mi R-34a enhance the antiproliferative effect in glioma U87 cells.

Gap junctions enhance the antiproliferative effect of microrna-34a in glioma cells

OBJECTIVE To investigate the effect of gap junctions on the anti-tumor function induced by mi R-34a in glioma U87 cells.METHODS 1.Transfection(miR-34a mimics were transfected into glioma cells to upregulate their expression);2.Co-culture assay(U87cells were transfected with mi R-34a co-cultured with U87 cells that was transfected PCMV-eG FP plasmid);3.Flow cytometry analysis(select the e GFP labed U87 cells);4.RNA isolation and real-time PCR;5.CCK-8 assay;6.Western blotting.RESULTS Mi R-34a mimics transfered between the U87 cells.Parachute assay showed that GJ inhibition(CBX and 18-α-GA)can decrease mi R-34a expression than co-culture group.RA and galanglin enhanced mi R-34a expression than co-culture group.Mi R-34a relative expression reduced after co-culture,while gap junctions composed of Cx43 were down-regulated by sh RNA.Transfected with mi R-34a mimics reduced the survival of U87 cells in a dose-dependent manner.To more specifically establish the role of GJIC in mi R-34a induced growth inhibition of U87 cells,si RNA was used to knockdown the expression of Cx43,the dominant connexin expressed in U87 cells.CCK-8 assay showed that siR NAs have no effect on cell growth,but they could aggravate the growth inhibition of miR-34a to U87 cels.CONCLUSION Gap junctions enhance the antiproliferative effect of miR NA-34a in glioma cells.

The spike timing precision of FitzHugh-Nagumo neuron network coupled by gap junctions

It has been proved recently that the spike timing can play an important role in information transmission, so in this paper we develop a network with N-unlt FitzHugh-Nagumo neurons coupled by gap junctions and discuss the dependence of the spike timing precision on synaptic coupling strength, the noise intensity and the size of the neuron ensemble. The calculated results show that the spike timing precision decreases as the noise intensity increases; and the ensemble spike timing precision increases with coupling strength increasing. The electric synapse coupling has a more important effect on the spike timing precision than the chemical synapse coupling.

Berberine potentizes apoptosis induced by X-rays irradiation probably through modulation of gap junctions

Background Clinical combination of some traditional Chinese medical herbs, including berberine, with irradiation is demonstrated to improve efficacy of tumor radiotherapy, yet the mechanisms for such effect remain largely unknown. The present study investigated the effect of berberine on apoptosis induced by X-rays irradiation and the relation between this effect and gap junction intercellular communication （GJIC）.Methods The role of gap junctions in the modulation of X-rays irradiation-induced apoptosis was explored by manipulation of connexin （Cx） expression, and gap junction function, using oleamide, a GJIC inhibitor, and berberine.Results In transfected HeLa cells, Cx32 expression increased apoptosis induced by X-rays irradiation, while inhibition of gap junction by oleamide reduced the irradiation responses, indicating the dependence of X-rays irradiation-induced apoptosis on GJIC. Berberine, at the concentrations without cytotoxicity, enhanced apoptosis induced by irradiation only in the presence of functional gap junctions.Conclusions These results suggest that berberine potentizes cell apoptosis induced by X-rays irradiation, probably through enhancement of gap junction activity.

Role of gap junctions between keratinocyte and melanocyte in melanogenesis

The process ofmelanogenesis in melanocytes and the transport of melanin in the form ofmelanosomes to the neighboring keratinocytes are the key steps in human skin pigmentation. Keratinocytes and melanocytes interact in intricate manner to maintain the homeostasis. The present study was designed to understand the role of cell-cell interaction through the gap junctions between melanocytes and keratinocytes on melanogenesis. We show that, inhibition of the gap junctional activity between human keratinocytes and melanocytes in a coculture system using gap junction blocker lowers the expression of key regulatory genes of melanogenesis such as tyrosinase and microphthalmia- associated transcription factor （MITF）. This was followed by concurrent decrease in tyrosinase protein levels and activity. Our results show the preliminary evidence for the regulation of melanogenesis in melanocytes through direct gap junctional communication by keratinocytes. Deciphering the mechanism and factors involved in the process would uncover the significance of gap junctions in melanogenesis.

Role of gap junctions in epilepsy

Epilepsy is a common neurological disorder characterized by periodic and unpredictable seizures.Gap junctions have recently been proposed to be involved in the generation,synchronization and maintenance of seizure events.The present review mainly summarizes recent reports concerning the contribution of gap junctions to the pathophysiology of epilepsy,together with the regulation of connexin after clinical and experimental seizure activity.The anticonvulsant effects of gap junction blockers both in vitro and in vivo suggest that the gap junction is a candidate target for the development of antiepileptic drugs.It is also of interest that the roles of neuronal and astrocytic gap junctions in epilepsy have been investigated independently,based on evidence from pharmacological manipulations and connexin-knockout mice.Further studies using more specific manipulations of gap junctions in different cell types and in human epileptic tissue are needed to fully uncover the role of gap junctions in epilepsy.

Activating Connexin43 gap junctions primes adipose tissue for therapeutic intervention

Adipose tissue is a promising target for treating obesity and metabolic diseases.However,pharmacological agents usually fail to effectively engage adipocytes due to their extraordinarily large size and insufficient vascularization,especially in obese subjects.We have previously shown that during cold exposure,connexin43(Cx43)gap junctions are induced and activated to connect neighboring adipocytes to share limited sympathetic neuronal input amongst multiple cells.We reason the same mechanism may be leveraged to improve the efficacy of various pharmacological agents that target adipose tissue.Using an adipose tissue-specific Cx43 overexpression mouse model,we demonstrate effectiveness in connecting adipocytes to augment metabolic efficacy of theβ_(3)-adrenergic receptor agonist Mirabegron and FGF21.Additionally,combing those molecules with the Cx43 gap junction channel activator danegaptide shows a similar enhanced efficacy.In light of these findings,we propose a model in which connecting adipocytes via Cx43 gap junction channels primes adipose tissue to pharmacological agents designed to engage it.Thus,Cx43 gap junction activators hold great potential for combination with additional agents targeting adipose tissue.

Astrocytic Gap Junctions Contribute to Aberrant Neuronal Synchronization in a Mouse Model of MeCP2 Duplication Syndrome

Abnormal synchronous neuronal activity has been widely detected by brain imaging of autistic patients,but its underlying neural mechanism remains unclear.Compared with wild-type mice,our in vivo two-photon imaging showed that transgenic(Tgl)mice over-expressing human autism risk gene MeCP2 exhibited higher neuronal synchrony in the young but lower synchrony in the adult stage.Whole-cell recording of neuronal pairs in brain slices revealed that higher neuronal synchrony in young postnatal Tgl mice was atributed mainly to more prevalent giant slow inward currents(SICs).Both in vivo and slice imaging further demonstrated more dynamic activity and higher synchrony in astrocytes from young Tgl mice.Blocking astrocytic gap junctions markedly decreased the generation of SICs and overall cell synchrony in the Tgl brain.Furthermore,the expression level of Cx43 protein and the coupling efficiency of astrocyte gap junctions remained unchanged in Tgi mice.Thus,astrocytic gap junctions facilitate but do not act as a direct trigger for the abnormal neuronal synchrony in young Tgl mice,revealing the potential role of the astrocyte network in the pathogenesis of MeCP2 duplication syndrome.

Do the expressions of gap junction gene connexin messenger RNA in noncancerous liver remnants of patients with hepatocellular carcinoma correlate with postoperative recurrences?

AIM: To investigate whether the changes of gap junction gene connexin messenger RNA in the noncancerous liver tissue of patients with hepatocellular carcinoma (HCC) could play a significant role in its postresection recurrence.METHODS: Seventy-nine consecutive patients having undergone curative resection for HCC entered this study.Using a reverse-transcription polymerase chain reaction (RT-PCR)-based assay, connexin (Cx) 26, connexin (Cx)32 and connexin (Cx) 43 mRNAs were determined prospectively in noncancerous liver tissues from these 79 patients and in the liver tissues from 15 controls. The correlations between connexin mRNA expression and the clinicopathological variables and outcomes (tumor recurrence and recurrence related mortality) were studied.RESULTS: Compared with liver tissues of control patients,the expression of Cx 32 mRNA in noncancerous liver tissues was significantly lower (mean: 0.715 vscontrol 1.225,P＜0.01), whereas the decreased Cx 26 mRNA (mean:0.700 vs of control 1.205,P＞0.05) and increased Cx 43 mRNA (mean: 0.241 vscontrol 0.100, P＞0.05) had no statistical significance. We defined the value of Cx 32 mRNA or Cx 26mRNA below 0.800 as a lower value. By multivariate analysis for noncancerous livers, a lower value of Cx 32 mRNA correlated significantly with a risk of HCC recurrence and recurrence-related mortality. The lower value of Cx 26 mRNA did not correlate with recurrence and mortality. The increased value of Cx43 mRNA also did not correlate with postoperative recurrence and recurrence-related mortality. By multivariate analysis, other significant predictors of HCC recurrence included vascular permeation, cellular dedifferentiation, and less encaps-ulation. The other significant parameter of recurrence related mortality was vascular permeation.CONCLUSION: The decreased expression of Cx 32 mRNA in noncancerous liver tissues plays a significant role in the prediction of postoperative recurrence of HCC.

Characteristic changes in astrocyte properties during astrocyte-to-neuron conversion induced by NeuroD1/Ascl1/Dlx2

Direct in vivo conversion of astrocytes into functional new neurons induced by neural transcription factors has been recognized as a potential new therapeutic intervention for neural injury and degenerative disorders. However, a few recent studies have claimed that neural transcription factors cannot convert astrocytes into neurons, attributing the converted neurons to pre-existing neurons mis-expressing transgenes. In this study, we overexpressed three distinct neural transcription factors––NeuroD1, Ascl1, and Dlx2––in reactive astrocytes in mouse cortices subjected to stab injury, resulting in a series of significant changes in astrocyte properties. Initially, the three neural transcription factors were exclusively expressed in the nuclei of astrocytes. Over time, however, these astrocytes gradually adopted neuronal morphology, and the neural transcription factors was gradually observed in the nuclei of neuron-like cells instead of astrocytes. Furthermore,we noted that transcription factor-infected astrocytes showed a progressive decrease in the expression of astrocytic markers AQP4(astrocyte endfeet signal), CX43(gap junction signal), and S100β. Importantly, none of these changes could be attributed to transgene leakage into preexisting neurons. Therefore, our findings suggest that neural transcription factors such as NeuroD1, Ascl1, and Dlx2 can effectively convert reactive astrocytes into neurons in the adult mammalian brain.

Improvement of cardiac function and reversal of gap junction remodeling by Neuregulin-1β in volume-overloaded rats with heart failure

Objective We performed experiments using Neuregulin-1β （NRG-1β） treatment to determine a mechanism for the protective role derived from its beneficial effects by remodeling gap junctions （GJs） during heart failure （HF）. Methods Rat models of I-IF were established by aortocaval fistula. Forty-eight rats were divided randomly into the HF （HF, n = 16）, NRG-1β trealanent （NRG, n = 16）, and sham operation （S, n = 16） group. The rats in the NRG group were administered NRG-1β （10 μg/kg per day） for 7 days via the tail vein, whereas the other groups were injected with the same doses of saline, Twelve weeks after operation, Connexin 43 （Cx43） expression in single myocytes obtained from the left ventricle was determined by immunocytochemistry. Total protein was extracted from frozen left ventricular tissues for immunoblotting assay, and the ultrastmcture of myocytes was observed by transmission electron microscopy. Results Compared with the HF group, the cardiac fimction of rats in the NRG group was markedly improved, irregular distribution and deceased Cx43 expression were relieved. The ultrastmcture of myocytes was seriously damaged in HF rats, and NRG-1β reduced these pathological damages. Conclusions Short-term NRG-1β treatment can rescue pump failure in experimental models of volume overload-induced HF, which is related to the recovery of GJs structure and the improvement of Cx43 expression.

Altered Expression of Connexin-43 and Impaired Capacity of Gap Junctional Intercellular Communication in Prostate Cancer Cells

Connexin-43 (Cx43) expression in prostate cancer (PCa) cells and the potency of gap junctional intercellular communication (GJIC) in the cells were investigated, with an attempt to elu- cidate the reason why the so-called 'bystander effect' mediated by thymidine kinase (TK) suicide gene therapy on PCa cells is not of significance and to explore the role of GJIC in PCa carcinogenesis. mRNA and protein expression of Cx43 in a PCa cell line PC-3m was detected by re- verse-transcription polymerase chain reaction (RT-PCR) and strapt-avidin-biotin-enzyme complex (SABC) immunohistochemical staining, and inherent GJIC of PC-3m cells was assayed by scrape-loading and dye transfer (SLDT) assay. The expression of Cx43 in human normal and malig- nant prostate tissues was determined by SABC immunohistochemistry as well. It was found that Cx43 mRNA and protein expression in PC-3m cells was slightly reduced as compared with positive controls and the location of Cx43 protein was aberrant in cytoplasm rather than on membrane. As- sessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was ab- normally located and markedly diminished as compared with normal prostatic epithelial ones, dis- playing a negative correlation to the pathological grade (χ2=4.025, P<0.05). Additionally, capacity of inherent GJIC in PC-3m cells was disrupted, which was semi-quantified as (+) or (-). It was indi- cated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein par- ticipated in the mechanisms leading to deficient GJIC in PC-3m cells. Lack of efficient GJIC is a molecular event, which may contribute not only to limited extent of 'bystander effect', but also to initiation and progression of prostatic neoplasm.

THE EFFECT OF ALL-TRANS RETINOIC ACID ON GAP JUNCTIONAL INTERCELLULARCOMMUNICATION AND CONNEXIN 43 GENE EXPRESSION IN GLIOMA CELLS

To illuminate the regulating effect of all trans retinoic acid (ATRA ) on gap junctional intercellular communication (GJIC) and connexin 43 (Cx43) ge ne expression in glioma cells, which is tissue and organ specific. Method. Rat C6 glioma cells were exposed to ATRA at a concentration of 1, 10, 10 0 μmol/L respectively, and the GJIC function of the cells was examined with scr ape loading dye transfer assay 24 hours, 48 hours and 72 hours after ATRA treat ment. The effect of ATRA on Cx43 gene expression was measured with semiquantitat ive reverse transcription polymerase chain reaction (RT PCR) 24 hours after ATR A exposure. Results. The GJIC function of C6 glioma cells was significantly increased by ATR A at each concentration applied. The dye passed 4 to 5 rows of cells from the sc raping edge in ATRA treated cells, but only 1 or 2 rows in the control. The augm ent effect was observed 24 hours after each concentration ATRA treatment, and la sted till 72 hours after treatment with 1μmol/L and 10μmol/L ATRA. Forty eigh t hours after exposed to 100μmol/L ATRA, the enhancement of GJIC was less obvi ous. There was no significant increase induced by ATRA on the transcription of C x43 gene, as demonstrated by semiquantitative RT PCR. Conclusion. ATRA turned out to be a potent enhancer on GJIC function in C6 gliom a cells, and the enhancement effect was most probable at post transcriptional l evel.

The gap junction blocker carbenoxolone enhances propofol and sevoflurane-induced loss of consciousness

General anesthetics induce loss of consciousness by inhibiting ascending arousal pathways, and they interfere with gap junction electrical coupling. The present study aimed to determine whether inhibition of gap junction-mediated signaling could influence general anesthetic-induced loss of consciousness. The general anesthetics sevoflurane and propofol were used. Intracerebroventricular administration of carbenoxolone, a gap junction blocker, significantly decreased the time to loss of the righting reflex （P 0.05）, but prolonged the time to recovery of the reflex （P 0.05）. Moreover, intracerebroventricular administration of carbenoxolone increased the sensitivity to sevoflurane, with a leftward shift of the loss of righting reflex dose-response curve, and decreased the 50% effective concentration of sevoflurane. These results suggest that the gap junction blocker carbenoxolone enhances propofol and sevoflurane-mediated general anesthesia.

Quantitative Structure-activity Relationship Studies on the Antioxidant Activity and Gap Junctional Communication of Carotenoids

The antioxidant and gap junctional communication（GJC） activities of carotenoids are known to be the two main anticancer mechanisms.Quantitative structure-activity relationship（QSAR） models of the two activities were developed using stepwise regression and multilayer perceptron neural network based on the calculated descriptors of quantum chemistry.The results showed that the significant molecular descriptor related to the antioxidant activity of carotenoids was the HOMO-LUMO energy gap（EHL） and the molecular descriptor related to the GJC was the lowest unoccupied molecular orbital energy（ELUMO）.The two models of antioxidant activity both showed good predictive power,but the predictive power of the neural network QSAR model of antioxidant activity was better.In addition,the two GJC models have similar,moderate predictive power.The possible mechanisms of antioxidant activity and GJC of carotenoids were discussed.

Gap junction communication involved in brain protection following focal ischemia and reperfusion in rats

BACKGROUND： Studies have suggested that gap junctions not only modulate the fate of the neocortex, but are also involved in maintaining homeostasis in the mature brain. However, the neuroprotective effects of gap junction communication following brain ischemic injury remain poorly understood. OBJECTIVE： To investigate the neuroprotective effects and possible mechanisms of gap junction communication following focal ischemia and reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the School of Basic Medical Sciences of Lanzhou University between June 2007 and May 2008. MATERIALS： Rabbit polyclonal anti-connexin 43 （Cx43） and gap junction blocking agent octanol were purchased from Sigma, USA; mouse monoclonal anti-rat glial fibrillary acidic protein （GFAP） was provided by Santa Cruz, USA; mouse monoclonal anti-rat CD11 b was produced by Abcam, England. METHODS： A total of 52 adult, male, Sprague Dawley rats were randomly assigned to three groups： sham-operated （n = 12）, vehicle control （n = 20）, and octanol-treated （n = 20）. Brain ischemia and reperfusion were induced by transient middle cerebral artery occlusion （MCAO） in vehicle control and octanol-treated groups, while no MCAO was administered to the sham-operated group. In the octanol-treated group, 5 mmol/kg octanol was dissolved in dimethyl sulfoxide （0.005% v/v） and was intraperitoneally injected 30 minutes prior to ischemic onset. Sham-operated and vehicle groups received equivalent volumes of dimethyl sulfoxide. MAIN OUTCOME MEASURES： Infarct volumes in ipsilateral striatum after MCAO were measured using cresyl violet dye; GFAP, CD11 b, and Cx43 expression in the ipsilateral striatum following MCAO were detected by immunohistochemistry; Western blot analysis was employed to determine Cx43 and GFAP expression. RESULTS： At 1 and 3 days following MCAO and reperfusion, ipsilateral striatum infarct volumes in the octanol group were significantly greater than in the vehicle group （P 〈 0.05）. There was no infarction in the sham-operated group. Cx43 and GFAP expression in the ipsilateral striatum of the octanol group was remarkably decreased compared with the vehicle group （P 〈 0.05）, and expression in the sham-operated group was less than in the other two groups （P 〈 0.05）. In the octanol-treated group, CD11 b expression was significantly increased compared with the vehicle group （P 〈 0.05）, and there were less CD11 b-immunoreactive cells in the sham-operated group compared with the other two groups （P 〈 0.05）. CONCLUSION： The pretreatment of blocking gap junction aggravated brain injury following MCAO. These results were possibly due to reduced astrocyte proliferation and activation, as well as reduced inflammatory response via activated microglia.

Mechanical allodynia and affective behavior are improved by INI-0602,a gap junction hemichannel inhibitor,in a rat model of neuropathic pain induced by sciatic nerve injury

OBJECTIVE To investigated the effects of INI-0602 on nociceptive reflex,depression-associated andanxiety-related behaviors caused by neuropathic pain in sciatic nerve injury rats.METHODS Male rat were subjected to sciatic nerve injury(SNI)or sham surgery.Rat received daily treatment with INI-0602 intrathecally,at a dose of 0.25μg/10μL.The response frequency to mechanical allodynia in animals was measured with von Frey hairs on day 1,3,5,7,14,21.Rats were evaluated in the forced swimming test(FST)test,tail suspension test(TST),sucrose preference test(SPT)for depression-like behavior.We performed open field test(OFT)and elevated plus-maze test(EPM)to evaluate anxiety-associated behaviors.Besides,we investigated the alterations of NMDA receptor and the brain-derived neurotrophic factor(BDNF)and also the expression of connexin43 and connexin32,structure protein of gap junction channel,on the protein level and the number of activated astrocyte showed by immunohistochemical.RESULTS The SNI procedure produced mechanical allodynia and accompanied with depressive-like and anxiety-like behavior.Treatment with INI-0602 produced a significant analgesic effect in SNI rats at day 7(model+NS:11.017±1.506 g;model+INI-0602:31.157±1.532 g,P<0.01),and still obviously on the 21th day(31.067±1.787,P<0.01).INI-0602 could also improve the performance of sciatic nerve injury rats among program behavior tests related to depression and anxiety.In parallel with relief of pain,the alterations of NMDA receptor and the brain-derived neurotrophic factor(BDNF),involved in central sensitization and synaptic plasticity,were investigated.INI-0602 not only could inhibited spared nerve injury induced up-regulated of NR2B and phosphorylation NR2B in early and late neuropathic pain(early phase:Nr2b:2.897±0.228,P<0.01;p-Nr2b:2.984±0.236,P<0.01;late phase:Nr2b:2.594±0.187,P<0.01;p-Nr2b:3.124±0.330,P<0.01),but also could inhibit the increased of BDNF in the early(model+NS:3.637±0.381,model+INI-0602:1.148±0.372,P<0.01)and upregulate the BDNF in late stage(model+NS:0.438±0.103,model+INI-0602:1.222±0.092,P<0.01).Meanwhile,INI-0602 significantly decreased the expression of connexin43 and connexin32,structure protein of gap junction channel,on the protein level and the number of activated astrocyte showed by immunohistochemical.CONCLUSION INI-0602 blocked behavioral changes induced by neuropathic pain,suggesting that it might be a promising pharmacological approach of painemotion diseases.