Expression and significance of non-canonical pyroptosis pathway-related proteins in muscle tissues of patients with dermatomyositis/polymyositis / 中华微生物学和免疫学杂志

Objective:To detect the expression of cysteinyl aspartate specific proteinase 4 (caspase-4), caspase-5, gasdermin D (GSDMD), nucleotide-binding oligomerization domain-like receptor protein-3 (NLRP3), Pannexin-1 and P2X7 involved in non-canonical pyroptosis pathway in muscle tissues of patients with dermatomyositis (DM)/polymyositis (PM) and to investigate the roles and significance of them in the pathogenesis of DM and PM.Methods:Altogether 13 DM patients, nine PM patients and 20 volunteers (control group) treated in the Affiliated Hospital of Qinghai University from January 2019 to September 2020 were enrolled in the present study. The 20 volunteers with no additional concomitant diseases underwent debridement due to simple orthopedic trauma. Pathological changes in muscle tissues were detected by hematoxylin & eosin (HE) staining. Expression of caspase-4, caspase-5, GSDMD, NLRP3, Pannexin-1 and P2X7 in muscle tissues was measured using immunohistochemistry (IHC).Results:(1) HE staining results showed that the muscle fibers in the control group had basically normal morphology and structure with no obvious inflammatory cell infiltration, atrophy, degeneration or necrosis. However, the size and thickness of muscle fibers in DM and PM groups were different with excessive inflammatory cell infiltration, atrophy, degeneration and necrosis to varying degrees. Moreover, the pathological scores of HE staining in muscle tissues of DM and PM groups were significantly higher than that of the control group and the differences were of statistical significance ( P<0.05). (2) IHC staining results suggested that the expression of caspase-4, caspase-5, GSDMD, NLRP3, Pannexin-1 and P2X7 in muscle tissues was higher in DM and PM groups than in the control group ( P<0.05). (3) As indicated by Pearson correlation analysis, the pathological scores of HE staining in muscle tissues of DM and PM groups were positively correlated with the IHC scores of caspase-4, caspase-5, GSDMD, NLRP3, PAnnexin-1 and P2X7 ( P<0.05). Furthermore, the IHC scores of caspase-4 and caspase-5 were positively correlated with the IHC scores of GSDMD and Pannexin-1 ( P<0.05); the IHC score of GSDMD was positively correlated with the IHC score of NLRP3 ( P<0.05); the IHC score of Pannexin-1 was positively correlated with the IHC score of P2X7 in muscle tissues ( P<0.05). Conclusions:The non-canonical pyroptosis pathway might be involved in the pathogenesis of DM and PM, which was possibly achieved by promoting inflammatory response. These results suggested that the non-canonical pyroptosis pathway played crucial roles in the immune pathogenesis of DM and PM.

Down-regulation of pannexin 2 channel enhances cisplatin-induced apoptosis in testicular cancer I-10 cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of down-regulation of pannexin 2 (Panx-2) channels on cisplatin-induced apoptosis in I-10 cells.@*METHODS@#The expression of Panx-2 protein in testicular cancer cells was detected with Western blotting. The testicular cancer cell line I-10 was transfected with two short hairpin RNA (shRNA1 and shRNA2) Lipofectamine, the empty vector (NC group) or Lipofectamine2000 (blank control group), and the changes in the expression of Panx-2 was detected with Western blotting. The effects of transfection with a Panx-2 inhibitor on surviving fraction of the cells treated with cisplatin (16 μmol/L) for 24 h, 48 h and 72 h was assessed with MTT assay, and the clonogenic capacity of the cells was evaluated with colony-forming assay. At 8 h after incubation with 16 μmol/L cisplatin, AnnexinV/PI double staining was used to detect the early apoptosis of the cells. After 24 h of treatment with 16 μmol/L cisplatin, the cells were examined for expressions of caspase-3, Bcl-2 and Bax using Western blotting.@*RESULTS@#The expression of Panx-2 was significantly increased in cisplatin-resistant I-10/DDP ( < 0.001) cells and Tcam-2/DDP ( < 0.01) cells as compared with I-10 cells and Tcam-2 cells. Transfection of I-10 cells with shRNA1 and shRNA2 resulted in significantly decreased Panx-2 expression ( < 0.05) and significantly reduced cell surviving fraction ( < 0.001). In the presence of cisplatin, the cells in NC group showed a higher clonogenic efficiency than those in shRNA1 and shRNA2 groups ( < 0.001). The early-stage apoptosis rate of the cells in shRNA1 and shRNA2 groups were significantly higher than that in NC group ( < 0.01). Panx-2 knockdown in I-10 cells significantly increased caspase-3 and Bax expressions ( < 0.05) and significantly decreased the expression of Bcl-2 ( < 0.01).@*CONCLUSIONS@#Down-regulation of Panx-2 channel enhances cisplatin-induced apoptosis in cultured testicular cancer cells.

Blocking pannexin-1 alleviates cisplatin-induced acute kidney injury in mice by reducing renal inflammatory cell infiltration / 南方医科大学学报

OBJECTIVE@#To investigate the effect of blocking pannexin-1 against acute kidney injury induced by cisplatin.@*METHODS@#Twenty-six male C57BL/6 mice aged 6-8 weeks were randomly divided into control group, cisplatin model (Cis) group and cisplatin + carbenoxolone treatment group (Cis + CBX). In Cis group and Cis + CBX group, the mice were injected intraperitoneally with 20 mg/kg of cisplatin and with CBX (20 mg/kg) at 30 min before and 24 and 48 h after cisplatin inhjection, respectively. All the mice were sacrificed at 72 h after cisplatin injection, and plasma and kidney samples were collected for testing mRNA and protein expression levels of pannexin-1 in the renal tissue using RT-qPCR and Western blotting and for detecting plasma creatinine and BUN levels; the pathological changes in the renal tissues were observed using Periodic Acid-Schiff staining. The expression of kidney injury molecule 1 (KIM-1) was examined using immunohistochemistry and the mRNA expressions of KIM-1 and neutrophil gelatinase- related lipid transport protein (NGAL) were detected by RT-qPCR to evaluate the injuries of the renal tubules. The infiltration of F4/80-positive macrophages and CD4-positive T cells were observed by immunofluorescence. In the experiment, human proximal tubule epithelial cell line HK-2 was stimulated with 50 μmol/L cisplatin to establish a cell model of acute kidney injury, and the mRNA and protein expressions of pannexin-1 were detected by RT-qPCR and Western blotting at 4, 6, 12, 18 and 24 h after the stimulation.@*RESULTS@#Compared with the control mice, the cisplatin-treated mice showed significantly up-regulated protein levels ( < 0.05) and mRNA levels ( < 0.005) of pannexin-1 in the kidney tissue. Cisplatin stimulation also caused significant increases in the protein levels ( < 0.005) and mRNA levels ( < 0.005) of pannexin-1 in cultured HK-2 cells. Compared with cisplatin-treated mice, the mice treated with both cisplatin and the pannexin-1 inhibitor CBX showed obviously lessened kidney pathologies and milder renal tubular injuries with significantly reduced plasma BUN and Scr levels ( < 0.01), expressions of KIM-1 and NGAL in the kidney ( < 0.05), and infiltration of F4/80-positive macrophages ( < 0.01) and CD4- positive T cells ( < 0.05) in the kidney tissues.@*CONCLUSIONS@#In cisplatin induced acute kidney injury mice model, Pannexin-1 expression is up-regulated in the kidneys tissue, and blocking pannexin-1 alleviates the acute kidney injury reducing renal inflammatory cell infiltration.

Effects of Exercise Preconditioning on Blood-brain Barrier Permeability in Rats after Cerebral Ischemia-reperfusion Injury / 中国康复理论与实践

Objective:To observe the effects of exercise preconditioning on blood-brain barrier (BBB) permeability, and connexin 43 (Cx43) and pannexin 1 (Panx1) protein after cerebral ischemia-reperfusion injury in rats. Methods:Fifty-four male Sprague-Dawley rats were randomly divided into sham group (n = 18), model group (n = 18) and exercise preconditioning group (n = 18). The exercise preconditioning group was trained with treadmill for three weeks before modeling. The middle cerebral arteries were occluded in the model group and the exercise preconditioning group using the modified Koizumi suture. After reperfusion of 24 hours, the rats were assessed with modified Neurological Severity Score (mNSS). The permeability of BBB was observed with Evans blue (EB). The expression of Cx43 and Panx1 was detected with Western blotting and immunohistochemistry in the ischemic tissues. Results:Compared with the model group, the mNSS score decreased in the exercise preconditioning group (P < 0.05), while the Evans blue content and the expression of Cx43 and Panx1 decreased (P < 0.05), as well as the the positive areas of Cx43 and Panx1 (P < 0.05). Conclusion:Exercise preconditioning can improve the permeability of BBB in cerebral ischemia-reperfusion rats, which may associate with down-regulation of Cx43 and Panx1, to protect brain from injury.

An experiment on the pathogenesis of early biotrauma in ventilator-induced lung injury / 中华危重病急救医学

Objective To investigate the pathogenesis of early biotrauma in ventilator-induced lung injury (VILI).Methods Twenty-four 8-week-old male specific-pathogen-free Sprague-Dawley (SD) rats weighing 250-300 g were randomly divided into sham group (S group), conventional mechanical ventilation group (L group) and high tidal volume (VT) mechanical ventilation group (H group) with 8 rats in each group. All rats received tracheostomy after anesthesia. Rats in S group received no mechanical ventilation but breathe room air spontaneously. All other parameters of the ventilator were the same in both mechanical ventilation groups, and the fraction of oxygen was set to 0.21, the rats in L group received 7 mL/kg VT, and those in H group received 28 mL/kg VT. Four hours after ventilation all rats were sacrificed and the lung tissues were harvested for wet/dry (W/D) ratio. Pathological injury score was evaluated by hematoxylin and eosin (HE) staining. Transferase-mediated deoxyuridine triphosphate-biotin nick end labeling stain (TUNEL) was performed to count the apoptosis cell in lung epithelial. Western Blot was performed to evaluate hemi-channel protein Pannexin-1 expression in lung homogenate. Bronchoalveolar lavage fluid (BALF) was collected, and the concentration of lactate dehydrogenase (LDH), isoprostane, adenosine triphosphate (ATP) and white cell count in BALF were measured. Yo-pro-1/propidium iodide (PI) double stain was performed to evaluate early apoptosis cell in BALF.Results There was no significant difference in lung injury between S group and L group. Compared with S group and L group, rats in H group showed significant lung injury, represented as alveolar rupture, inflammatory cell infiltration, interstitial edema and airway epithelial exfoliation, and the lung W/D ratio was increased significantly (5.1±0.2 vs. 4.4±0.2, 4.3±0.4, bothP< 0.01), pathological score was significantly increased [4.00 (4.00, 8.00) vs. 1.00 (0, 4.00), 2.00 (0, 4.75), bothP< 0.01], the white cell in BALF was significantly increased (×106/L: 2.97±0.46 vs.1.03±0.26, 0.79±0.19, bothP< 0.01), the level of LDH was significantly increased (U/L: 148.6±38.2 vs. 34.4±13.5, 78.6±13.9, bothP< 0.01), and the expression of Pannexin-1 in lung homogenate was significantly increased (Pannexin-1/GAPDH: 0.89±0.21 vs. 0.48±0.25, 0.61±0.17, bothP< 0.01), the ATP concentration in BALF was also significantly increased (nmol/L: 456.84±148.72 vs. 19.23±13.34, 113.26±57.90, bothP< 0.01). There was no significant difference in the apoptosis cell in lung tissue or the apoptosis cell rate, isoprostane level in BALF among the three groups [apoptosis cell in lung (cells/HP): 4.00 (3.00, 5.00) vs. 5.00 (4.00, 6.00), 4.00 (3.25, 6.00); apoptosis cell rate in BALF: (0.57±0.20)％ vs. (0.42±0.16)％, (0.58±0.19)％; isoprostane in BALF (μg/L): 3.85±0.46 vs. 3.83±0.60, 3.59±0.69, allP > 0.05].Conclusion The early pathogenesis of biotrauma in VILI is related to the release of inflammation mediator via membrane channel after activating by pressure stress, but not apoptosis and lipid peroxidation.

Regulatory effect of hemichannels protein Pannexin-1 on P2X7 receptor activity in the lungs of mice with lung injury / 中华危重病急救医学

Objective To investigate the regulative effect of hemichannels protein Pannexin-1 on P2X7 receptor activation and caspase-1 mediated inflammatory response in the lungs of mice with lung injury. Methods Sixty male C57BL/6 mice were randomly divided into five groups with 12 mice in each group: sham operation group (Sham group), mechanical ventilation (MV) group, MV + low dose lipopolysaccharide (LPS) group (MLL group), MV + medium and high dose LPS group (MML group) and MV + high dose LPS group (MHL group). A "two-hit" lung injury model was reproduced by MV with high tidal volume combined with LPS injection in airway. All the mice underwent tracheotomy and intubation. After operation, the mice in Sham group were maintained spontaneous breathing, and those in other four groups were put on small animal ventilators to give MV with a large tidal volume of 28 mL/kg. After stable respiration in mice, those in the Sham group and MV group were injected 8 mL/kg of normal saline (NS) into the airway, and those in MLL, MML and MHL groups were given 2, 5 and 8 mg/kg of LPS respectively (diluted with NS into 8 mL/kg). After 4 hours on MV, the mice were sacrificed, and bronchoalveolar lavage fluid (BALF) was extracted to determine intracellular and extracellular ATP concentration. Lung tissue was harvested and water containing ratio of lungs was measured. The degree of lung pathological damage was observed after hematoxylin-eosin (HE) staining, and lung injury score was calculated. The expression of Pannexin-1 protein in lung tissue was calculated with immunohistochemistry. Western Blot and fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) were used to detect the protein and mRNA expressions of Pannexin-1, P2X7 receptor, caspase-1 and interleukin-1β (IL-1β). Results There was no obvious pathological change in lung tissue in Sham group, intracellular ATP concentration was higher than extracellular ATP concentration, water content in lung tissue was lower, Pannexin-1 expression was low in lung tissue by immunohistochemical staining, and Pannexin-1, P2X7 receptor, caspase-1 and IL-1β were only expressed in micro-protein and mRNA in lung tissue. Compared with the Sham group, the alveolar lesions and hemorrhages in the MV group were not obvious, and lung injury score was slightly increased. There was no significant fluctuation between intracellular ATP concentration and extracellular ATP concentration. The water content in lung tissue was increased significantly, while the expressions of Pannexin-1, P2X7 receptor, caspase-1 and IL-1β in lung tissue were increased slightly. After LPS intervention, progressively increased lung exudation, ruptured alveoli, dilated capillaries, and inflammatory cells were found, and lung injury score was increased without significant difference among the three LPS doses groups. With the increase in LPS dosage, the concentration of extracellular ATP in BALF was increased, the concentration of intracellular ATP was decreased, the water containing ratio of lung tissue was increased gradually, and the protein and mRNA expressions of Pannexin-1, P2X7 receptor, caspase-1 and IL-1β in lung tissue were increased gradually in a dose-dependent manner. The parameters in MHL group showed significant differences as compared with those in MV group [lung injury score: 8.25±0.45 vs. 3.50±0.52; intracellular ATP concentration (μmol/L): 198.76±150.77 vs. 896.69±281.11, extracellular ATP concentration (μmol/L): 336.57±90.28 vs. 141.52±42.22; lung water containing rate: (6.37±0.11)% vs. (5.05±0.14)%; Pannexin-1 protein (gray value): 3.20±0.70 vs. 1.54±0.76, Pannexin-1 mRNA (2-ΔΔCT): 7.86±0.86 vs. 2.47±0.92; P2X7 receptor protein (gray value): 3.18±0.88 vs. 1.80±0.72, P2X7 receptor mRNA (2-ΔΔCT): 7.17±0.96 vs. 2.31±0.45; caspase-1 protein (gray value): 3.00±0.45 vs. 0.93±0.51, caspase-1 mRNA (2-ΔΔCT): 4.39±0.91 vs. 2.74±0.41; IL-1β protein (gray value): 2.54±1.08 vs. 1.16±0.53, IL-1β mRNA (2-ΔΔCT): 132.34±41.48 vs. 19.67±8.67; all P < 0.05]. Conclusion Pannexin-1 may be involved in LPS and MV induced lung injury, which may be regulated by intracellular release of ATP to the extracellular site and binding to P2X7 receptor on the cell surface, thereby regulating active caspase-1 production and release, involving in the production of IL-1β and other inflammatory factors eventually which leads to the occurrence and development of lung injury.

Progress in relationship between pannexin and tumors / 中国病理生理杂志

Pannexin is a new member of gap junction families which was discovered in 2000 and was widely distributed in humans. Pannexin forms hemichannels and participates in transmission of small molecules and many other pathophysiological processes. Recent studies have found that the abnormal expression of pannexin is related to occurrence and development of tumors. This article reviews the relationship between pannexin and tumors,and aims to provide new i-deas for treatment of tumors.

Change of pannexin1 expression in dorsal horn of spinal cord in rats with neuropathic pain / 重庆医学

Objective To observe the expression of pannexin1(PX1) in the dorsal horn of spinal cord in model ratwith neu-ropathipain afteselective ligation of sciatinerve branche.Method50 male SD ratwere randomly divided into 3 group,inclu-ding the control group(Wgroup ,n= 10) ,sham operation group(sham group ,n= 10) and sciatinerve branch selective injury group(SNI group ,n=30) .30 ratwere killed on postoperative 3 ,5 ,7 ,14 d and the lumbasegmenof the spinal cord wataken fodetecting the expression of PX1 by using Western blo.Othe20 ratwere killed on 7 d afteSNI and the expression of glial fibril-lary acidiprotein(GFAP) in the spinal cord wadetected with immunohistology .Among them ,10 ratin the SNI group were trea-ted with intrathecal intubation before operation and administrated with saline 20 μL ocarbenoxolone(CBX) 20 μL by intrathecal injection on postoperative 7 d fodetermining the expression of GFAP by the immunohistology .ResultThe expression of PX1 in the SNI group waincreased and enhanced with time ,which wasignificantly highethan thain the Wgroup and the sham group (P＜0 .05);the GFAP expression on 7 d in the SNI group waobviously increased compared with the Wgroup and the sham group(P＜0 .05);afteintrathecal injection of CBX ,the expression of GFAP wasignificantly decreased compared with thain the normal saline group(P＜0 .05) .No statistically significandifferencein the expression of PX1 and GFAP were found in the Wgroup and the sham group .Conclusion PX1 may be involved in the activation of astrocyte,prompting thaPX1 playan importanrole in the neuropathipain caused by the peripheral nervel injury .

Spinal Gap Junction Channels in Neuropathic Pain

Damage to peripheral nerves or the spinal cord is often accompanied by neuropathic pain, which is a complex, chronic pain state. Increasing evidence indicates that alterations in the expression and activity of gap junction channels in the spinal cord are involved in the development of neuropathic pain. Thus, this review briefly summarizes evidence that regulation of the expression, coupling, and activity of spinal gap junction channels modulates pain signals in neuropathic pain states induced by peripheral nerve or spinal cord injury. We particularly focus on connexin 43 and pannexin 1 because their regulation vastly attenuates symptoms of neuropathic pain. We hope that the study of gap junction channels eventually leads to the development of a suitable treatment tool for patients with neuropathic pain.