Chengxiang Yang

The roles of T-type calcium channel in the development of neuropathic pain following chronic compression of rat dorsal root ganglia.

This study aimed to elucidate the role of T-type calcium channels in the nociceptive signal transmission at the spinal level. The chronic compression of dorsal root ganglion (CCD) rat model was adopted. Three doses (50, 100 and 200 µg in groups Mib50, Mib100 and Mib200, respectively) of specific T-type Ca2+ channel inhibitors mibefradil (Mib) or normal saline (NS) were intrathecally administered on the 5th day after the CCD model had been established. The paw withdrawal latency from a noxious thermal stimulus and paw withdrawal mechanical threshold of von Frey filament was used to measure the thermal hyperalgesia and tactile allodynia, respectively. Lumbar spinal cords of the rats isolated on the 5th day after the operation were prepared to measure the mRNA expression of T-type (Cav3.1, Cav3.2 and Cav3.3) calcium channel with RT-PCR methods. The results demonstrated that CCD rats produced reliable thermal hyperalgesia and tactile allodynia after surgery. The intrathecal administration of Mib significantly suppressed thermal hyperalgesia and allodynia in CCD rats (p< 0.01), and the inhibitory effect lasted for 2 h. However, only Cav3.2 and Cav3.3 T-type calcium channel mRNA were detected in the lumbar spinal cord of rats, and there were no Cav3.1 calcium channels. Compared with native and sham groups, the Cav3.2 and Cav3.3 calcium channel mRNA expression increased significantly (p < 0.05). These data support the view that spinal T-type calcium (Cav3.2 and Cav3.3 but not Cav3.1) channels may play an important role in the pathogenesis of neuropathic pain.

Curcumin ameliorated diabetic neuropathy partially by inhibition of NADPH oxidase mediating oxidative stress in the spinal cord

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are the main enzymes that produce oxidative stress, which plays an important role in painful diabetic neuropathy. Curcumin has been reported to exert an antinociceptive effect in a rat model of diabetic neuropathy by suppressing oxidative stress in the spinal cord. However, it remains unknown whether the mechanism by which curcumin ameliorates diabetic neuropathy can be attributed to spinal NADPH oxidases. This study was designed to determine the effect of curcumin on diabetic neuropathy and to investigate its precise mechanism in relation to NADPH oxidase-mediating oxidative stress in the spinal cord. Diabetic neuropathy was induced in Sprague-Dawley rats by intraperitoneal injection with 1% streptozotocin (STZ; 60 mg/kg). After the onset of diabetic neuropathy, a subset of the diabetic rats received daily intragastric administrations of curcumin (200mg/kg) or intraperitoneal injections of apocynin (2.5mg/kg) for 14 consecutive days, whereas other diabetic rats received equivalent volumes of normal saline (NS). STZ resulted in diabetic neuropathy with hyperglycemia and a lower paw withdrawal threshold (PWT), accompanied by elevations in the expression of the NADPH oxidase subunits p47(phox) and gp91(phox) and in the levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA) and a reduction in superoxide dismutase (SOD) activity (P<0.05) in the spinal cord. Both curcumin and apocynin ameliorated diabetic neuropathy. In conclusion, curcumin attenuated neuropathic pain in diabetic rats, at least partly by inhibiting NADPH oxidase-mediating oxidative stress in the spinal cord.

Cardioprotection of sevoflurane postconditioning by activating extracellular signal-regulated kinase 1/2 in isolated rat hearts

AbstractAim:The activation of extracellular signal-regulated kinase (ERK)1/2 protects against ischemic–reperfusion injury. Whether ERK1/2 mediates the cardioprotection of sevoflurane postconditioning is unknown. We tested whether sevoflurane postconditioning produces cardioprotection via an ERK1/2-dependent mechanism.Methods:In protocol 1, Langendorff-perfused Sprague–Dawley rat hearts (n=84, 12 per group), with the exception of the Sham group, were subjected to 30 min ischemia followed by 90 min reperfusion and were assigned to the untreated (control) group, followed by 4 cycles of ischemic postconditioning (25 s of each), 3% (v/v) sevoflurane postconditioning (for 5 min and 10 min of washout), and the PD98059 solvent DMSO (<0.2%), ERK1/2 inhibitor PD98059 (20 μmol/L), and Sevo+PD administration. Left ventricular hemodynamics and coronary flow at 30 min of equilibrium were recorded at 30, 60, and 90 min of reperfusion, respectively. Acute infarct size was measured by triphenyltetrazolium chloride staining. The configuration of mitochondria was observed by an electron microscope. Western blot analysis was used to determine the contents of cytosolic and mitochondrial cytochrome c at the end of reperfusion. In protocol 2, after 15 min of reperfusion, the expression of total and phosphorylated forms of ERK1/2 and its downstream target p70S6K was determined by Western blotting.Results:No differences in baseline hemodynamics were observed among the experimental groups (P>0.05). After reperfusion, compared with the control group, sevoflurane postconditioning and ischemic postconditioning significantly (P<0.05) improved functional recovery and largely (P<0.05) decreased myocardial infarct size (22.9%±4.6% and 21.2%±3.8%, vs 39.4%±5.7%, both P<0.05). Sevoflurane-mediated protection was abolished by PD98059.Conclusion:Anesthetic postconditioning by sevoflurane effectively protects against reperfusion damage by activating ERK1/2 in vitro.

SEVOFLURANE POST‐CONDITIONING PROTECTS AGAINST MYOCARDIAL REPERFUSION INJURY BY ACTIVATION OF PHOSPHATIDYLINOSITOL‐3‐KINASE SIGNAL TRANSDUCTION

1 The mechanisms underlying myocardial protection by sevoflurane post‐conditioning are unclear. In the present study, we tested two hypotheses: (i) that sevoflurane post‐conditioning produces cardioprotection via a phosphatidylinositol‐3‐kinase (PI3‐K)‐dependent pathway; and (ii) combining sevoflurane and ischaemic post‐conditioning offers an additional benefit against reperfusion injury. 2 Rat isolated perfused hearts were exposed to 25 min ischaemia followed by 90 min reperfusion. Sevoflurane post‐conditioning was induced by administration of sevoflurane (3.0 vol%) for 15 min from the onset of reperfusion. In some groups, 15 µmol/L LY294002, a selective PI3‐K inhibitor, was coadministrated with sevoflurane. Other groups of hearts were exposed to ischaemic post‐conditioning or combined sevoflurane plus ischaemic post‐conditioning in the presence and absence of LY294002. After 15 min reperfusion, phosphorylation of Akt and glycogen synthase kinase 3β (GSK3β) was determined by Western blot analysis. Infarct size was determined by 2,3,5‐triphenyltetrazolium chloride staining and subsarcolemmal mitochondrial lesions were assessed by electron microscopy after 90 min reperfusion. 3 Sevoflurane post‐conditioning significantly decreased infarct size compared with control hearts (31 ± 2 vs 42 ± 3%, respectively; P < 0.05), diminished mitochondrial lesions and increased phosphorylation of Akt and GSK3β, as did ischaemic post‐conditioning. However, combined sevoflurane plus ischaemic post‐conditioning did not further improve the cardiaoprotective effects compared with either intervention alone. Sevoflurane‐mediated cardioprotection was abolished or inhibited by 15 µmol/L LY294002. 4 In conclusion, sevoflurane acts during early reperfusion after ischaemia to salvage the myocardium by activating PI3‐K. The combination of sevoflurane plus ischaemic post‐conditioning does not offer any additional benefit over either intervention alone.

Intrathecal TRESK gene recombinant adenovirus attenuates spared nerve injury-induced neuropathic pain in rats.

TRESK gene recombinant adenovirus (109 IU/ml), which has been constructed successfully in our previous study, was implemented through an intrathecal injection. The fact that the method can effectively upregulate the expression of TRESK mRNA in the dorsal root ganglia of spared nerve injury in rats was verified. We also investigated the role of TRESK gene recombinant adenovirus in attenuating tactile allodynia and thermal hyperalgesia in spared nerve injury rats. Spared nerve injury to the sciatic nerve induced persistent tactile allodynia, but had no effect on thermal hyperalgesia. Intrathecal injection of TRESK gene recombinant adenovirus (25 µl) into the region of lumbar enlargement in advance reduced tactile allodynia. Moreover, intrathecal injection of TRESK gene recombinant adenovirus (25 µl) significantly alleviated the activation of astrocytes in spinal cord induced by spared nerve injury. The current study shows that an intrathecal injection of the TRESK gene recombinant adenovirus attenuated the activity of astrocytes in spinal cord, which contributed to relieving neuropathic pain in spared nerve injury rats. According to the result reported in our previous study, attenuating the expression of TRESK in dorsal root ganglia was involved in the development of neuropathic pain. On the basis of these results, we theorized that the therapeutic utility of upregulation of TRESK in dorsal root ganglia was effective in relieving neuropathic pain syndromes induced by peripheral nerve injury.

Sevoflurane inhibits invasion and migration of lung cancer cells by inactivating the p38 MAPK signaling pathway.

PurposeSevoflurane is used widely during lung cancer surgery. However, the effect of sevoflurane on the invasion and migration of lung carcinoma cells remains unclear. The aims of this study were to explore the role of matrix metalloproteinase (MMP)-2 and MMP-9 in the effect of sevofluane on the invasion and the role of fascin and ezrin on the effect of sevofluane on the migration of human lung adenocarcinoma A549 cells. We also investigated whether sevoflurane regulates the expression of these molecules through the p38 mitogen-activated protein kinase (MAPK) signaling pathway.MethodsThe invasion of cells was evaluated using the Transwell invasion assay, and the migration of cells was determined using the wound healing assay. The expression of MMP-2, MMP-9, ezrin, fascin, and phospho-p38 MAPK in cells was determined by western blotting.ResultsA significant inhibition of cell invasion and migration was found in A549 cells which had been treated with sevoflurane. The data also revealed that sevoflurane could decrease the phosphorylation level of p38 MAPK, which is involved in the downregulation of MMP-2, MMP-9, fascin, and ezrin expression, accompanied by a concomitant inhibition of the invasion and migration of A549 cells. SB203580, a p38 MAPK inhibitor, augmented the downregulation of the expression of these proteins.ConclusionThe anti-invasion effect of sevoflurane on A549 cells was associated with a downregulation of both MMP-2 and MMP-9 expression, while the anti-migration effect was associated with a downregulation of both fascin and ezrin expression. These effects could occur partly as a result of inactivation of the p38 MAPK signaling pathway.

Identification of the Spinal Expression Profile of Non-coding RNAs Involved in Neuropathic Pain Following Spared Nerve Injury by Sequence Analysis

Neuropathic pain (NP) is caused by damage to the nervous system, resulting in aberrant pain, which is associated with gene expression changes in the sensory pathway. However, the molecular mechanisms are not fully understood. A non-coding Ribose Nucleic Acid (ncRNA) is an RNA molecule that is not translated into a protein. NcRNAs are involved in many cellular processes, and mutations or imbalances of the repertoire within the body can cause a variety of diseases. Although ncRNAs have recently been shown to play a role in NP pathogenesis, the specific effects of ncRNAs in NP remain largely unknown. In this study, sequencing analysis was performed to investigated the expression patterns of ncRNAs in the spinal cord following spared nerve injury-induced NP. A total of 134 long non-coding RNAs (lncRNAs), 12 microRNAs (miRNAs), 188 circular RNAs (circRNAs) and 1066 mRNAs were significantly regulated at 14 days after spared nerve injury (SNI) surgery. Next, quantitative real-time polymerase chain reaction (PCR) was performed to validate the expression of selected lncRNAs, miRNAs, circRNAs, and mRNAs. Bioinformatics tools and databases were employed to explore the potential ncRNA functions and relationships. Our data showed that the most significantly involved pathways in SNI pathogenesis were ribosome, PI3K-Akt signaling pathway, focal adhesion, ECM-receptor interaction, amoebiasis and protein digestion and absorption. In addition, the lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA network of NP was constructed. This is the first study to comprehensively identify regulated ncRNAs of the spinal cord and to demonstrate the involvement of different ncRNA expression patterns in the spinal cord of NP pathogenesis by sequence analysis. This information will enable further research on the pathogenesis of NP and facilitate the development of novel NP therapeutics targeting ncRNAs.

TRESK gene recombinant adenovirus vector inhibits capsaicin-mediated substance P release from cultured rat dorsal root ganglion neurons

The present study was conducted to determine whether the activation of TRESK in the dorsal root ganglion (DRG) by the TRESK gene recombinant adenovirus vector inhibits the capsaicin-evoked substance P (SP) release using a radioimmunoassay. TRESK is an outwardly rectifying K+ current channel that contributes to the resting potential and is the most important background potassium channel in DRG. Previous studies have shown that neuropathic pain (NP) is closely related to the regulation of certain potassium channels in DRG neurons, while DRG-released SP is important in the peripheral mechanism of NP. In the present study, the TRESK gene adenovirus vector significantly enhanced the TRESK mRNA and protein of the cultured rat DRG neurons. Radioimmunoassay analysis revealed that the capsaicin-mediated SP release was significantly inhibited by the TRESK gene recombinant adenovirus vector in rat DRG neurons. These findings suggest that TRESK plays a role in adjusting the release of SP in DRG, which is related to NP.

Interleukin-33 signaling contributes to renal fibrosis following ischemia reperfusion

&NA; Acute kidney injury caused by ischemia‐reperfusion injury (IRI) is a major risk factor for chronic kidney disease, which is characterized by renal interstitial fibrosis. However, the molecular mechanisms underlying renal fibrosis induced by IRI are not fully understood. Our results showed that interleukin (IL)−33 was induced markedly after IRI insult, and the kidneys of mice following IRI plus IL‐33 treatment presented more severe renal fibrosis compared with mice treated with IRI alone. Therefore, we investigated whether inhibition of IL‐33 protects against IRI‐induced renal fibrosis. Mice were administrated with soluble ST2 (sST2), a decoy receptor that neutralizes IL‐33 activity, or vehicle by intraperitoneal injection for 14 days after IRI challenge. We revealed that mice treated with sST2 exhibited less severe renal dysfunction and fibrosis in response to IRI compared with vehicle‐treated mice. Inhibition of IL‐33 suppressed bone marrow–derived fibroblast accumulation and myofibroblast formation in the kidneys after IRI stress, which was associated with less expression of extracellular matrix proteins. Furthermore, inhibition of IL‐33 also showed a significant reduction of F4/80+ macrophages and CD3+ T cells in the kidneys of mice after IRI treatment. Finally, Treatment with IL‐33 inhibitor reduced proinflammatory cytokine and chemokine levels in the kidneys of mice following IRI insult. Taken together, our findings indicate that IL‐33 signaling plays a critical role in the pathogenesis of IRI–induced renal fibrosis through regulating myeloid fibroblast accumulation, inflammation cell infiltration, and the expression of proinflammatory cytokines and chemokines.

Inhibition of PTEN Activity Aggravates Post Renal Fibrosis in Mice with Ischemia Reperfusion-Induced Acute Kidney Injury

Background: Renal fibrosis is a common pathophysiological feature of chronic kidney disease. Acute kidney injury (AKI) is defined as an independent causal factor of chronic kidney disease, with a pathological representation of post renal fibrosis. However, the etiopathogenesis underlying post renal fibrosis induced by AKI is not completely understood. Methods: BALB/c mice were treated with bpv or vehicle controls and were, respectively, the ischemia reperfusion (IR) model group and control group. All of the animals had blood taken from the orbital venous plexus at 24 hours after IR. Six mice in each group were randomly chosen and euthanized 7 days after IR treatment, and the remaining six mice in each group were euthanized 14 days after IR treatment. We examined the effect on post kidney fibrosis of inhibiting PTEN activity in mice in an IR induced AKI experimental model. Results: Compared with vehicle mice, bpv-(PTEN specific inhibitor) treated mice accumulated more bone marrow-derived fibroblasts and myofibroblasts in the kidneys. Inhibition of PTEN activity increased the expression of α-smooth muscle actin and extracellular matrix proteins and post kidney fibrosis. Furthermore, inhibition of PTEN activity resulted in more inflammatory cytokines in the kidneys of mice subjected to IR-induced renal fibrosis. Moreover, inhibition of PTEN activity up-regulated PI3K protein expression and Akt phosphorylation. Conclusions: Our study demonstrated that PTEN played an important role in post renal fibrosis in mice with ischemia reperfusion-induced AKI. These results indicated that the PTEN/PI3K/Akt signaling pathway may serve as a novel therapeutic target for AKI-induced chronic kidney disease.