Objective:To study whether cisplatin may induce apoptosis in the pericytes of cochlear stria vascularis and underlying mechanisms.Methods:Twenty male C57BL/6J mice aged 6-8 weeks were divided into control group and a cisplatin group. Primary cultured mouse cochlear vascular peristriatal cells were identified and divided into control group, cisplatin group, and N-acetylcysteine+cisplatin group. Auditory brainstem response (ABR) was used to detect hearing in mice. Hematoxylin eosin (HE) staining was used to observe morphological changes in the stria vascularis of the cochlea. The total superoxide dismutase (SOD) activity test kit (WST-1 method) and thiobarbituric acid (TBA) method were used to detect SOD activity and Malondialdehyde (MDA) content, respectively. DCFH-DA fluorescence probe was used to detect the content of reactive oxygen species in peripheral cells. Hoechst 33 342 and flow cytometry were used to detect the apoptosis rate of pericytes. Immunofluorescence technology was used to detect the distribution and expression of apoptosis related proteins in pericytes of cochlear stria vascularis. Immunohistochemistry and Western blotting (WB) were used to detect the expression of apoptosis-and mitochondrial-related proteins. Mito SOX TM-red and JC-1 were used to detect the mitochondrial function of pericytes. Evans blue staining was used to observe the permeability of the blood labyrinth barrier (BLB). Statistical analysis was conducted using SPSS 18.0 software. Results:Compared with the control group, the cisplatin group significantly increased in the hearing threshold ( t=4.72, P<0.01), Ⅰ-wave latency ( t=12.25, P<0.05), and the levels of oxidative stress in the cochlea and pericytes ( t=38.34, P<0.01), and also cisplatin caused disorder and contraction of the cochlear stria vascularis structure, increased BLB permeability [Evans blue leakage (1.08±0.42) AU vs (0.55±0.23) AU, t=4.64, P<0.05], with a statistically significant difference, enhanced the expressions of apoptotic proteins c-Caspase-3 ( t=5.01, P<0.01) and Bax ( t=6.33, P<0.01) in the peristriatal cells of cochlear blood vessels in mice treated with cisplatin increased. And cisplatin can induce apoptosis of primary cultured pericytes and up-regulate the expression of c-Caspase-3 and Bax ( P<0.05). The NAC+cisplatin group partially reversed cisplatin-induced pericyte apoptosis ( P<0.05). Cisplatin caused damage to the mitochondrial function of peripheral cells, and induced the release of apoptosis-inducing factor (AIF) and cytochrome C (cyt-c) into the cytoplasm ( P<0.05). The NAC+cisplatin group partially reversed cisplatin induced pericyte apoptosis ( P<0.05) and mitochondrial damage ( P<0.05). Conclusion:Cisplatin can increase the level of oxidative stress in the cochlea and cause mitochondrial pathway apoptosis in C57BL/6J mouse cochlear vascular peristriatal cells.

Objective: To investigate the role of connexin 43 ( Cx43) in acute sympathetic stress induced by phe- nylephrine (PE) overactivation of α1-adrenergic receptor ( α1-AR) in cardiomyocytes． Methods: Cardiomyocytes of H9C2 rats were randomly divided into control group，PE alone treatment group，Gap26 ( Cx43 specific inhibitor) intervention group and Gap26 alone treatment group．PE alone treatment group was treated with 50 μmol / L PE for 15 min．The Gap26 intervention group was pretreated with 0. 5 μmol / L Gap26 for 30 min，and then treated with 50 μmol / L PE for 15 min．The protein and mRNA expression levels of Cx43，NLRP3 inflammasome，interleukin-1 β (IL-1 β) ，Caspase-1，interleukin-18 (IL-18 ) were detected by Western blot and qRT-PCR. The expression and co-location of Cx43 in cardiomyocytes were observed by immunofluorescence assay，and the expression of inflamma- tory cytokines IL-1 β and IL-18 in cardiomyocytes was detected by ELISA. Results: Compared with control group， the protein and mRNA levels of Cx43，NLRP3，Caspase-1 and IL-18 in PE group increased．Compared with PE a- lone treatment group，the protein and mRNA levels of Cx43，NLRP3，Caspase-1 and IL-18 decreased after Gap26 intervention，but they were still higher than those of control group． Similarly ，immunofluorescence showed that Cx43 protein expression increased in PE alone group，while Cx43 expression was down-regulated in Gap26 inter- vention group compared with PE alone group．ELISA results showed that the expression of IL-1 β and IL-18 was sig- nificantly up-regulated in PE alone group，but down-regulated in Gap26 intervention group． Conclusion Cx43 is involved in α1-AR activation induced cardiac acute sympathetic stress by regulating NLRP3 inflammasome.

Objective:To evaluate the relationship between transforming growth factor beta-3 (TGF-β 3)/mammalian homologs of the drosophila mad gene 3 (Smad3) signaling pathway and neuronal apoptosis during reduction of focal cerebral ischemia-reperfusion (I/R) injury by isoflurane postconditioning (ISO) in rats. Methods:Sixty clean-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 210-230 g, were randomly divided into 5 groups ( n=12 each): sham operation group (group S), cerebral I/R group (group I/R), cerebral I/R plus isoflurane postconditioning group (group I/R+ ISO), cerebral I/R plus pirfenidone group (group I/R+ P), and cerebral I/R plus isoflurane postconditioning plus pirfenidone group (group I/R+ ISO+ P). Local cerebral I/R was produced by middle cerebral artery occlusion for 1.5 h followed by 24-h reperfusion in anesthetized rats.Pirfenidone 5 μg/kg was injected into the lateral ventricle at 30 min before ischemia in I/R+ P and I/R+ ISO+ P groups.In I/R+ ISO and I/R+ ISO+ P groups, 1.5% isoflurane was inhaled for 1 h starting from the time point immediately after onset of reperfusion.Neuro-functional deficit was assessed using neurologic deficit scores (NDS) at the end of reperfusion.Then the animals were sacrificed, and brain tissues were removed for determination of the neuronal damage rate (by Nissl staining), neuronal apoptosis rate (by TUNEL), expression of TGF-β 3 (using immunofluorescence), and expression of TGF-β 3, phosphorylated Smad3 (p-Smad3), caspase-3, Bax and Bcl-2 (by Western blot). Results:Compared with group S, the NDS, neuronal damage rate and apoptosis rate of neurons were significantly increased, the expression of TGF-β 3, caspase-3 and Bax was up-regulated, and the expression of p-Smad3 and Bcl-2 was down-regulated in group I/R ( P<0.05). Compared with group I/R, the NDS, neuronal damage rate and apoptosis rate of neurons were significantly decreased, the expression of TGF-β 3, p-Smad3 and Bcl-2 was up-regulated, and the expression of caspase-3 and Bax was down-regulated in group I/R+ ISO, and the NDS, neuronal damage rate and apoptosis rate of neurons were significantly increased, the expression of TGF-β 3 and p-Smad3 was down-regulated, and the expression of caspase-3 was up-regulated in group I/R+ P ( P<0.05). Compared with group I/R+ ISO, the NDS, neuronal damage rate and apoptosis rate of neurons were significantly increased, the expression of TGF-β 3, p-Smad3 and Bcl-2 was down-regulated, and the expression of caspase-3 and Bax was up-regulated in group I/R+ ISO+ P ( P<0.05). Conclusion:Isoflurane postconditioning can inhibit neuronal apoptosis by activating the TGF-β 3/Smad3 signaling pathway, thus reducing focal I/R injury in rats.

Objective To investigate whether Wnt/β-catenin signaling pathway mediating the neuroprotection of isoflurane post-conditioning in hippocampal neurons damage induced by ischemia/reperfusion injury in rats.Methods According to the randomized principle, 60 male Sprague-Dawley rats were randomly divided into five groups (12 rats in each group):sham group (group S), model group (group M), ISO+model group (group MI), ISO+model+DKK-1 group (group MDI) and model+DKK-1 group (group MD).A rat model of middle cerebral artery occlusion (MCAO) was established with 90 min ischemia followed by 24 hreperfusion.Group S was only exposed to one side of the internal carotid artery without fishing line.Isoflurane post-conditioning groups (group MI, MDI) were immediately treated with 1.5％isoflurane for 60 min at the onset of reperfusion.DKK-1 (5μg/kg) was injected intracerebroventricularly 30 min before the model established in group MDI and group MD.After reperfusion for 24 h, Longa score method was used for neurological deficit score.HE staining and Tunel fluorescence was employed to observe the morphological changes of neurons.Immunohistochemistry and Western Blot were applied to detect the expression of target protein in CA1 region.Results Compared with group S, the neurobehavioral score, the number of apoptosis and the expression of Bax and GSK-3βprotein in group M all increased (P<0.05), while the expression ofβ-catenin and Bcl-2/Bax ratio decreased (P<0.05) ;Compared with group M, the neurobehavioral score, the number of apoptosis and the expression of Bax protein were significantly decreased (P<0.05), while the expression of Bcl-2, β-catenin protein and the Bcl-2/Bax ratio were significantly increased (P<0.05) in group MI.Compared with group MI, the neurobehavioral score, the number of apoptosis, Bax and GSK-3βprotein in group MDI were significantly increased (P<0.05), while the Bcl-2, β-catenin protein expression, and Bcl-2/Bax ratio were significantly decreased (P<0.05).Conclusion Isoflurane post-conditioning may protect the hippocampus neurons against cerebral ischemic reperfusion-induced damage via the way that the Wnt/β-catenin signaling pathway regulates the expression levels of Bcl-2 and Bax proteins in rats.

Objective: The aging model of guinea pigs induced by D-galactose was set up to investigate the changes of BK_Ca expression and function on cochlear pericytes and their relationship with age-related hearing loss. Methods: Thirty healthy 8-week-old guinea pigs were randomly divided into three groups, with 10 in each group: D-galactose aging model group, subcutaneous injection of D-galactose (500 mg/kg) daily for 6 weeks; saline control group, the same amount of saline was injected into the neck of the aging model group for 6 weeks; the blank control group, no treatment was performed. The threshold of auditory brainstem response (ABR) was detected. The content of BK_Ca in the perivascular cells of the guinea pig cochlear cells was detected by immunofluorescence technique. The changes of peripheral current density and BK_Ca current were detected by patch clamp technique. The data were analyzed by GraphPad Prism software. Results: Compared with the saline group and the control group, the ABR threshold and the amplitude of the wave I were significantly decreased in the aging model group, and the difference was statistically significant (P<0.01). Compared with the control group, the expression of BK_Ca in the vascular pericytes of guinea pigs in the aging model group was significantly reduced (1.00±0.08 vs 0.27±0.03,the difference was statistically significant P<0.01), and the cell current density and BK_Ca net current value were also significantly reduced with statistically significant (P<0.01). Conclusions D-galactose can successfully induce guinea pig aging model, in which BK_Ca expression decreases and net current value decreases in pericytes of cochlear striavascularis, and changes in BK_Ca expression and function may be related to age-related hearing loss.

Objective To evaluate the effects of remifentanil (RMF)on large conductance cal-cium-activated potassium channel (BKCa)and voltage-gated potassium channel (KV)activition currents in basilar arterial smooth muscle cells (BASMCs)of normotensive and hypertensive rats. Methods Spontaneously hypertensive rats (SHR)and homologous normotensive wistar-kyoto (WKY)rats,were used in this study.BASMCs were obtained freshly by the method of enzymolysis. Six basilar artery smooth muscle cells of each rat were chosen and analyzed.Outward current ampli-tude was recorded by the whole-cell patch clamp technique.The outward current amplitude under all stimulation voltage in set of step stimulation protocol before (basal level)and after administration of RMF (3×10-7mol/L)were recorded respectively and net current was calculated (net current=cur-rent amplitude after administration-basic value).With administration by concentrations cumulative method,the current amplitude under +60 mV stimulation voltage was separately recorded before (basic value)and after application of different concentrations of RMF (10-10,10-9,10-8,10-7, 10-6,10-5mol/L),then calculated current increasing rate and the half effective concentration (EC50)of RMF increasing current amplitude in BASMCs.Another six basilar artery smooth muscle cells of each rat were chosen and given RMF (3×10-7mol/L),and separately treated with BKCa channel blocker (tetraethylammonium,TEA)and Kv channel blocker (4-aminopyridine,4-AP),and then administrated the corresponding RMF mixture.The current amplitude was recorded after each dose.Results At 0,+20,+40 and +60 mV,the net current generated by RMF on both BASMCs of rats was successively and significantly increased (P <0.05).The increment rate of outward currents in BASMCs generated by 10-10,10-9,10-8,10-7RMF successively and significantly went upward (P<0.05).Compared to WKY rats,the half-effective concentration(EC50)of RMF increas-ing the current amplitude in BASMCs of SHR significantly rose(P<0.05).Compared with the base-line,the current amplitude in BASMCs of the two kind rats was significantly increased after adminis-tration of RMF,and decreased after administration of TEA or 4-AP (P<0.05);Compared to ad-ministration of TEA or 4-AP,the current amplitude in BASMCs of the two kind rats was significantly in-creased after administration of TEA+RMF or 4-AP+RMF (P<0.05).Conclusion Bkcaand Kv currents in both BASMCs of SHR and WKY rats were activated by RMF in a voltage-dependent and dose-dependent manner,and the effect of RMF on BKCaand Kvactivition currents in BASMCs of SHR was weaker than WKY rats.

Objective To investigate the effect of G protein?coupled estrogen receptor (GPER) on the diastolic function of renal interlobular artery and reduce renal ischemia?reperfusion injury in rats. Methods Female ovariectomized rats were divided into control group; ischemia?reperfusion injury (IRI) group;GPER?specific agonist (G1) intervention group;GPER?specific blocker+GPER?specific agonist (G15+G1) intervention group. Histopathological examination (HE staining), renal function test and Paller score were used to identify the success of the model and the degree of kidney damage. In vitro microvascular pressure diameter measuring instrument was used to detect the relaxation and contraction activity of renal interlobular artery in each group. Immunofluorescence technique was used to observe the expression of GPER on the renal interlobular artery. Westernblotting was used to detect the expression of GPER protein in renal interlobular artery of rats in each group. The NO content was determined by a nitrate reductase method. Results Compared with IRI group, serum BUN, Scr level and Paller score in G1 intervention group were significantly decreased (all P<0.05). The systolic rate of renal interlobar artery was significantly increased [(40.76 ± 1.57)％ vs (29.78 ± 1.87)％, P<0.05]. The results of immunofluorescence showed that GPER was expressed in renal interlobular artery smooth muscle cells and endothelial cells, and the expression of IRI group was higher than that of the control group. The expression of G15+G1 intervention group was lower than that of G1 intervention group (all P<0.05). Compared with the IRI group, the NO content in the G1 intervention group increased significantly (all P<0.05). Conclusions During renal ischemia ?reperfusion injury, GPER may regulate the systolic and diastolic activity of the renal interlobar artery by increasing the content of NO, so as to alleviate the renal ischemia?reperfusion injury.

Objective To evaluate the effect of oxycodone on function of GABAA receptors in dor-sal root ganglion ( DRG ) neurons of rats with neuropathic pain ( NP ) . Methods Thirty-six adult male Sprague-Dawley rats, weighing 180-220 g, aged 10 weeks, were allocated into 3 groups ( n=12 each) u-sing a random number table method: sham operation group ( group S ) , group NP and oxycodone group ( group O) . The sciatic nerve was only isolated but not ligated in group S. NP was induced by chronic con-striction injury. The sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1 mm intervals with 4-0 chromic catgut. Oxycodone 15μg∕kg was intraperitoneally injected once a day for 14 con-secutive days from ligating the sciatic nerve to satisfaction in group O. The thermal paw withdrawal latency( TWL) was measured at 1 day before establishing the model ( T0 ) and 3, 5, 7, 10 and 14 days after es-tablishing the model ( T1-5 ) . The rats were sacrificed after measurement of pain threshold at T5 , and DRG neurons were acutely isolated for recording the amplitude of GABAA receptors-activated currents using whole-cell patch-clamp technique. Results Compared with group S, the TWL was significantly shortened at T1-5, and the amplitude of GABAA receptors-activated currents in DRG neurons was decreased in NP and O groups (P<0. 05). Compared with group NP, the TWL was significantly prolonged at T1-5, and the ampli-tude of GABAA receptors-activated currents in DRG neurons was increased in group O ( P<0. 05) . Conclu-sion Oxycodone can enhance the function of GABAA receptors-activated currents in DRG neurons and thus enhance GABAA receptors-mediated presynaptic inhibition, which may be related to the mechanism of oxyc-odone-induced reduction of NP in rats.

Objective To observe the effects of propofol intervention on renal ischemia-reperfusion injury on the expression of Cx43 in rat renal interlobar artery.Methods Male SD rats were randomly divided into control 4h and 24h groups (control), sham operation 4h and 24h groups (sham), ischemia reperfusion 4h and 24h groups (I/R), propofol 4h and 24h groups (propofol), and fat emulsion 4h and 24h groups (intralipid).Ischemia/reperfusion model was prepared by resection of right kidney and noninvasive arterial occlusion of left kidney, with renal ischemia for 45min and reperfusion for 4h or 24h depending on different group.Serum urea nitrogen (BUN) and creatinine (Cr) were detected by automatic biochemical analyzer.HE staining was used to observe the pathological changes of renal tissue.The changes of renal artery systolic and diastolic lobes were examined by pressure myographic technique.The expression of Cx43 protein in renal interlobar artery was analyzed by Western blot.Results The concentrations of serum BUN and Cr in sham group did not differ significantly from those in the blank control group.Renal HE staining showed no significant lesions;the pressure myogram of motor renal interlobar artery contraction rate showed no significant difference.The expression of Cx43 protein did not change significantly.Compared with sham operation group, the concentrations of serum BUN and Cr in ischemia-reperfusiongroup were significantly increased.HE slices kidney showed that the pathological changes of renal tissue became obvious;pressure motor indicated renal interlobar artery contraction rate was decreased;the expression of Cx43 protein was increased significantly (P<0.05).Compared with ischemia-reperfusion group, the concentrations of serum BUN and Cr in propofol group were decreased;renal HE slices showed reduced renal tissue lesions, increased renal interlobar artery contraction rate, and decreased expression of Cx43 protein (P<0.05).Conclusion Propofol can change renal ischemia-reperfusion injury by reducing the expression of Cx43 protein in vasomotor in renal interlobar artery.

Objective To investigate the effect of estrogen (E2) on the connexin43 (Cx43) expression of renal interlobar arteries after renal ischemia-reperfusion injury (I/R).Methods The experiment was carried out in vivo using an SD rat I/R model.SD rats were randomly divided into normal group,sham-operation group,I/R group,and estrogen-intervention group.The functional changes of the kidney were analyzed after 24 hours of I/R;nephridial tissue section was stained by hematoxylin-eosin (HE),and Paller scores were used to evaluate the degree of kidney damage.Pressure myography was utilized to detect the vasomotor function of renal interlobar arteries.Immunofluorescence technique,qRT-PCR and Western blot were applied to determine the expression of Cx43 in renal interlobar arteries in different groups.Results Estrogen markedly decreased the levels of Cr and BUN in the serum of I/R rats (P＜0.05),and the damage of the kidney tissue could be improved noticeably.The vasomotor rate of renal interlobar arteries was (24.80 ± 3.70)％ after I/R and (41.60 ± 3.50)％ after treatment with estrogen,which was higher than that of I/R group (P＜0.05).The expression of Cx43 was lower in renal interlobar arteries of estrogen-intervention group than that in I/R group (P＜0.01).Conclusion Estrogen may reduce vascular tension and boost dilation of the artery by inhibiting Cx43 expression and GJ function.Therefore,estrogen may attenuate the damage of I/R and improve renal function.