Microglial depletion impairs glial scar formation and aggravates inflammation partly by inhibiting STAT3 phosphorylation in astrocytes after spinal cord injury

Astrocytes and microglia play an orchestrated role following spinal cord injury;however,the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood.Herein,microglia were pharmacologically depleted and the effects on the astrocytic response were examined.We further explored the potential mechanisms involving the signal transducers and activators of transcription 3(STAT3)pathway.For in vivo experiments,we constructed a contusion spinal cord injury model in C57BL/6 mice.To deplete microglia,all mice were treated with colony-stimulating factor 1 receptor inhibitor PLX3397,starting 2 weeks prior to surgery until they were sacrificed.Cell proliferation was examined by 5-ethynyl-2-deoxyuridine(EdU)and three pivotal inflammatory cytokines were detected by a specific Bio-Plex Pro^(TM) Reagent Kit.Locomotor function,neuroinflammation,astrocyte activation and phosphorylated STAT3(pSTAT3,a maker of activation of STAT3 signaling)levels were determined.For in vitro experiments,a microglia and astrocyte coculture system was established,and the small molecule STA21,which blocks STAT3 activation,was applied to investigate whether STAT3 signaling is involved in mediating astrocyte proliferation induced by microglia.PLX3397 administration disrupted glial scar formation,increased inflammatory spillover,induced diffuse tissue damage and impaired functional recovery after spinal cord injury.Microglial depletion markedly reduced EdU+proliferating cells,especially proliferating astrocytes at 7 days after spinal cord injury.RNA sequencing analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397.Double immunofluorescence staining confirmed that PLX3397 significantly decreased STAT3 expression in astrocytes.Importantly,in vitro coculture of astrocytes and microglia showed that microglia-induced astrocyte proliferation was abolished by STA21 administration.These findings suggest that microglial depletion impaired astrocyte proliferation and astrocytic scar formation,and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following spinal cord injury.

Axonal growth inhibitors and their receptors in spinal cord injury:from biology to clinical translation

Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelinassociated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19(that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the Rho A/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.

Up-regulation of intestinal nuclear factor kappa B and intercellular adhesion molecule-1 following traumatic brain injury in rats

AIM: Nuclear factor kappa B (NF-κB) regulates a large number of genes involved in the inflammatory response to critical illnesses, but it is not known if and how NF-κB is activated and intercellular adhesion molecule-1 (ICAM-1)expressed in the gut following traumatic brain injury (TBI).The aim of current study was to investigate the temporal pattern of intestinal NF-κB activation and ICAM-1expression following TBI.METHODS: Male Wistar rats were randomly divided into six groups (6 rats in each group) including controls with sham operation and TBI groups at hours 3, 12, 24, and 72, and on d 7. Parietal brain contusion was adopted using weight-dropping method. All rats were decapitated at corresponding time point and mid-jejunum samples were taken. NF-κB binding activity in jejunal tissue was measured using EMSA. Immunohistochemistry was used for detection of ICAM-1 expression in jejunal samples.RESULTS: There was a very low NF-κB binding activity and little ICAM-1 expression in the gut of control rats after sham surgery. NF-κB binding activity in jejunum significantly increased by 160% at 3 h following TBI (P＜0.05 vs control), peaked at 72 h (500% increase)and remained elevated on d 7 post-injury by 390% increase. Compared to controls, ICAM-1 was significantly up-regulated on the endothelia of microvessels in villous interstitium and lamina propria by 24 h following TBI and maximally expressed at 72 h post-injury (P＜0.001). The endothelial ICAM-1 immunoreactivity in jejunal mucosa still remained strong on d 7 post-injury. The peak of NF-κB activation and endothelial ICAM-1 expression coincided in time with the period during which secondary mucosal injury of the gut was also at their culmination following TBI.CONCLUSION: TBI could induce an immediate and persistent up-regulation of NF-κB activity and subsequent up-regulation of ICAM-1 expression in the intestine.Inflammatory response mediated by increased NF-κB activation and ICAM-1 expression may play an important role in the pathogenesis of acute gut mucosal injury following TBI.

FOXO3a as a sensor of unilateral nerve injury in sensory neurons ipsilateral, contralateral and remote to injury

Emerging evidence supports that the stress response to peripheral nerve injury extends beyond the injured neuron,with alterations in associated transcription factors detected both locally and remote to the lesion.Stress-induced nuclear translocation of the transcription factor forkhead class box O3a(FOXO3a)was initially linked to activation of apoptotic genes in many neuronal subtypes.However,a more complex role of FOXO3a has been suggested in the injury response of sensory neurons,with the injured neuron expressing less FOXO3a.To elucidate this response and test whether non-injured sensory neurons also alter FOXO3a expression,the temporal impact of chronic unilateral L4–6 spinal nerve transection on FOXO3a expression and nuclear localization in adult rat dorsal root ganglion neurons ipsilateral,contralateral or remote to injury relative to na?ve controls was examined.In na?ve neurons,high cytoplasmic and nuclear levels of FOXO3a colocalized with calcitonin gene related peptide,a marker of the nociceptive subpopulation.One hour post-injury,an acute increase in nuclear FOXO3a in small size injured neurons occurred followed by a significant decrease after 1,2 and 4 days,with levels increasing toward pre-injury levels by 1 week post-injury.A more robust biphasic response to the injury was observed in uninjured neurons contralateral to and those remote to injury.Nuclear levels of FOXO3a peaked at 1 day,decreased by 4 days,then increased by 1 week post-injury,a response mirrored in C4 dorsal root ganglion neurons remote to injury.This altered expression contralateral and remote to injury supports that spinal nerve damage has broader systemic impacts,a response we recently reported for another stress transcription factor,Luman/CREB3.The early decreased expression and nuclear localization of FOXO3a in the injured neuron implicate these changes in the cell body response to injury that may be protective.Finally,the broader systemic changes support the existence of stress/injury-induced humeral factor(s)influencing transcriptional and potentially behavioral changes in uninjured dorsal root ganglion neurons.Approval to conduct this study was obtained from the University of Saskatchewan Animal Research Ethics Board(protocol#19920164).

Sulforaphane protects liver injury induced by intestinal ischemia reperfusion through Nrf2-ARE pathway

AIM: To investigate the effect of sulforaphane (SFN) on regulation of NF-E2-related factor-2 (Nrf2)-antiox-idant response element (ARE) pathway in liver injury induced by intestinal ischemia/reperfusion (I/R). METHODS: Rats were divided randomly into four ex-perimental groups: control, SFN control, intestinal I/R and SFN pretreatment groups (n = 8 in each group). The intestinal I/R model was established by clamping the superior mesenteric artery for 1 h and 2 h reperfu-sion. In the SFN pretreatment group, surgery was performed as in the intestinal I/R group, with intraperitoneal administration of 3 mg/kg SFN 1 h before the op-eration. Intestine and liver histology was investigated. Serum levels of aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured. Liver tissue superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione (GSH) and glutathione peroxidase (GSH-Px) activity were assayed. The liver transcription factor Nrf2 and heme oxygenase-1 (HO-1) were determined by immunohistochemical analysis and Western blotting analysis.RESULTS: Intestinal I/R induced intestinal and liver injury, characterized by histological changes as well as a signif icant increase in serum AST and ALT levels (AST: 260.13 ± 40.17 U/L vs 186.00 ± 24.21 U/L, P < 0.01; ALT: 139.63 ± 11.35 U/L vs 48.38 ± 10.73 U/L, P < 0.01), all of which were reduced by pretreatment with SFN, respectively (AST: 260.13 ± 40.17 U/L vs 216.63 ± 22.65 U/L, P < 0.05; ALT: 139.63 ± 11.35 U/L vs 97.63 ± 15.56 U/L, P < 0.01). The activity of SOD in the liver tissue decreased after intestinal I/R (P < 0.01), which was enhanced by SFN pretreatment (P < 0.05). In ad-dition, compared with the control group, SFN markedly reduced liver tissue MPO activity (P < 0.05) and elevat-ed liver tissue GSH and GSH-Px activity (P < 0.05, P < 0.05), which was in parallel with the increased level of liver Nrf2 and HO-1 expression.CONCLUSION: SFN pretreatment attenuates liver injury induced by intestinal I/R in rats, attributable to the antioxidant effect through Nrf2-ARE pathway.

Identification of four differentially expressed genes associated with acute and chronic spinal cord injury based on bioinformatics data

Complex pathological changes occur during the development of spinal cord injury(SCI),and determining the underlying molecular events that occur during SCI is necessary for the development of promising molecular targets and therapeutic strategies.This study was designed to explore differentially expressed genes(DEGs)associated with the acute and chronic stages of SCI using bioinformatics analysis.Gene expression profiles(GSE45006,GSE93249,and GSE45550)were downloaded from the Gene Expression Omnibus database.SCI-associated DEGs from rat samples were identified,and Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed.In addition,a protein-protein interaction network was constructed.Approximately 66 DEGs were identified in GSE45550 between 3–14 days after SCI,whereas 2418 DEGs were identified in GSE450061–56 days after SCI.Moreover,1263,195,and 75 overlapping DEGs were identified between these two expression profiles,3,7/8,and 14 days after SCI,respectively.Additionally,16 overlapping DEGs were obtained in GSE450061–14 days after SCI,including Pank1,Hn1,Tmem150c,Rgd1309676,Lpl,Mdh1,Nnt,Loc100912219,Large1,Baiap2,Slc24a2,Fundc2,Mrps14,Slc16a7,Obfc1,and Alpk3.Importantly,3882 overlapping DEGs were identified in GSE932491–6 months after SCI,including 3316 protein-coding genes and 567 long non-coding RNA genes.A comparative analysis between GSE93249 and GSE45006 resulted in the enrichment of 1135 overlapping DEGs.The significant functions of these 1135 genes were correlated with the response to the immune effector process,the innate immune response,and cytokine production.Moreover,the biological processes and KEGG pathways of the overlapping DEGs were significantly enriched in immune system-related pathways,osteoclast differentiation,the nuclear factor-κB signaling pathway,and the chemokine signaling pathway.Finally,an analysis of the overlapping DEGs associated with both acute and chronic SCI,assessed using the expression profiles GSE93249 and GSE45006,identified four overlapping DEGs:Slc16a7,Alpk3,Lpl and Nnt.These findings may be useful for revealing the biological processes associated with SCI and the development of targeted intervention strategies.

Endoplasmic reticulum stress transducer old astrocyte specifically induced substance contributes to astrogliosis after spinal cord injury

Old astrocyte specifically induced substance(OASIS) is an endoplasmic reticulum(ER) stress transducer specifically expressed in astrocytes and osteoblasts. OASIS regulates the differentiation of neural precursor cells into astrocytes in the central nervous system. This study aimed to elucidate the involvement of ER stress responses stimulated via OASIS in astrogliosis following spinal cord injury. In a mouse model of spinal cord contusion injury, OASIS m RNA and protein expression were evaluated at days 7 and 14. A significant increase in OASIS m RNA on day 7 and an increase in protein on days 7 and 14 was observed in injured spinal cords. Immunostaining on day 7 revealed co-localization of OASIS and astrocytes in the periphery of the injury site. Furthermore, anti-OASIS small interfering RNA(si RNA) was injected at the injury sites on day 5 to elucidate the function of OASIS. Treatment with anti-OASIS si RNA caused a significant decrease in OASIS m RNA on day 7 and protein on days 7 and 14, and was associated with the inhibition of astrogliosis and hindlimb motor function recovery. Results of our study show that OASIS expression synchronizes with astrogliosis and is functionally associated with astrogliosis after spinal cord injury.

Major comorbid disease processes associated with increased incidence of acute kidney injury

Acute kidney injury(AKI) is commonly seen amongst critically ill and hospitalized patients. Individuals with certain co-morbid diseases have an increased risk of developing AKI. Thus, recognizing the co-morbidities that predispose patients to AKI is important in AKI prevention and treatment. Some of the most common co-morbid disease processes that increase the risk of AKI are diabetes, cancer, cardiac surgery and human immunodeficiency virus(HIV) acquired immune deficiency syndrome(AIDS). This review article identifies the increased risk of acquiring AKI with given co-morbid diseases. Furthermore, the pathophysiological mechanisms underlying AKI in relation to co-morbid diseases are discussed to understand how the risk of acquiring AKI is increased. This paper reviews the effects of various comorbid diseases including: Diabetes, cancer, cardiovascular disease and HIV AIDS, which all exhibit a significant increased risk of developing AKI. Amongst these comorbid diseases, inflammation, the use of nephrotoxic agents, and hypoperfusion to the kidneys have been shown to be major pathological processes that predisposes individuals to AKI. The pathogenesis of kidney injury is complex, however, effective treatment of the co-morbid disease processes may reduce its risk. Therefore, improved management of co-morbid diseases may prevent some of the underlying pathology that contributes to the increased risk of developing AKI.

Experimental study that trimetazidine inhibits Fas/FasL pathway to relieve the myocardial ischemia reperfusion injury in rats

Objective: To study the protective effect and molecular mechanism of trimetazidine on myocardial ischemia reperfusion injury in rats. Methods: Adult male SD rats were chosen as the experimental animals and randomly divided into control group, ischemia reperfusion group and trimetazidine group, and myocardial ischemia reperfusion models were established and then given intraperitoneal injection of trimetazidine hydrochloride for intervention. The expression levels of Fas/FasL pathway molecules as well as the contents of inflammatory and oxidative stress molecules in the myocardium, and the contents of myocardial enzymes in the blood circulation were measured 120 min after reperfusion. Results: Fas, FasL, Caspase-8 and Caspase-3 mRNA expression as well as NK-κB, TNF-α, ICAM-1, IL-17, IL-23, NOX2, NOX4, AOPP and MDA contents in myocardium, and LDH, CK and CK-MB contents in blood circulation of ischemia reperfusion group were significantly higher than those of control group, and Fas, FasL, Caspase-8 and Caspase-3 mRNA expression as well as NK-κB, TNF-α, ICAM-1, IL-17, IL-23, NOX2, NOX4, AOPP and MDA contents in myocardium, and LDH, CK and CK-MB contents in blood circulation of trimetazidine group were significantly lower than those of ischemia reperfusion group;LDH, CK and CK-MB contents in blood circulation as well as NK-κB, TNF-α, ICAM-1, IL-17, IL-23, NOX2, NOX4, AOPP and MDA contents in myocardium of trimetazidine group were positively correlated with Fas and FasL mRNA expression. Conclusion: Trimetazidine can inhibit Fas/FasL pathway to reduce the myocardial damage caused by inflammatory response and oxidative stress response during myocardial ischemia reperfusion in rats.

巨噬细胞移动抑制因子抑制剂对蛛网膜下腔出血大鼠脑损伤的保护作用

目的探讨巨噬细胞移动抑制因子(MIF)抑制剂对蛛网膜下腔出血(SAH)大鼠脑损伤的保护作用。方法将120只12月龄健康雄性大鼠按照随机数字表法分为假手术组、SAH组、小剂量、中剂量和大剂量抑制剂组,每组24只。采用枕大池二次注血法制作大鼠SAH动物模型,小剂量、中剂量和大剂量抑制剂组分别给予10 mg/kg、20 mg/kg和40 mg/kg剂量MIF抑制剂注射大鼠腹腔,测定五组大鼠炎症因子、神经细胞凋亡、脑含水量、血脑屏障通透性和神经功能情况。结果大鼠脑组织白介素-1β、肿瘤坏死因子-α及白介素-6蛋白表达、神经细胞凋亡指数、脑水肿指数、伊文氏蓝含量及神经行为学评分五组间比较,差异均有统计学意义(F=76.406、62.664、64.106、40.211、24.869、60.229、43.664,均P<0.01),与假手术组比较,SAH组白介素-1β、肿瘤坏死因子-α及白介素-6蛋白表达、神经细胞凋亡指数、脑水肿指数和伊文氏蓝含量增高,神经行为学评分下降,差异均有统计学意义(P<0.01),MIF抑制剂腹腔注射可逆转上述作用,且呈剂量-疗效依赖关系(P<0.05)。结论MIF抑制剂对SAH大鼠脑损伤具有明显的保护作用,且呈量效关系。

甲状腺全切术治疗对DTC患者炎性反应、神经营养因子的影响及其与喉返神经损伤的相关性

目的:探究甲状腺全切术治疗对分化型甲状腺癌(DTC)患者炎性反应、神经营养因子的影响及其与喉返神经(RLN)损伤的相关性。方法:选取285例DTC患者为研究对象。所有患者均行甲状腺全切术治疗。记录患者围术期指标、RLN损伤情况。比较患者术前、术后12、24 h及3、7、30 d的炎性反应指标[白细胞介素6(IL-6)、C反应蛋白(CRP)、肿瘤坏死因子α(TNF-α)]、神经营养因子[脑源性神经营养因子(BDNF)、神经生长因子(NGF)]水平,同时比较有无RLN损伤患者术后12 h的上述指标水平。采用Pearson法分析炎性反应指标、神经营养因子水平之间的相关性。结果:手术时间(88.35±7.64)min;术中出血量(23.24±6.98)mL;术后引流量(97.41±14.23)mL;术后住院时间(7.56±3.54)d。患者IL-6、CRP及TNF-α水平均随时间发展而呈先升高后降低的趋势(P<0.05)。患者BDNF、NGF水平均随时间发展而呈先降低后升高的趋势(P<0.05)。患者RLN损伤发生率为13.68%。RLN损伤组术后12 h的IL-6、CRP及TNF-α水平高于无RLN损伤组(P<0.05),BDNF、NGF水平低于无RLN损伤组(P<0.05)。RLN损伤组患者IL-6、CRP、TNF-α水平均随时间发展而呈先升高后降低的趋势(P<0.05),而BDNF、NGF水平均随时间发展而呈先降低后升高的趋势(P<0.05)。IL-6、CRP、TNF-α水平之间及BDNF、NGF之间均呈正相关关系(P<0.05),IL-6、CRP、TNF-α水平与BDNF、NGF水平均呈负相关关系(P<0.05)。结论:甲状腺全切术治疗DTC患者具有手术时间短、创伤小、术后恢复快的优点,对炎性反应、神经营养因子的影响较为轻微;但该手术会引起一定的RLN损伤,且炎性反应和神经营养因子均与RLN损伤有密切联系。

肾小管HIF-1α/miR-23a通路在脓毒血症急性肾损伤中的作用机制

目的探讨肾小管低氧诱导因子-1α(HIF-1α)/MicroRNA-23a(miR-23a)通路在脓毒血症急性肾损伤(SA-AKI)中的作用及相关作用机制。方法体外培养人近曲小管上皮细胞(HK-2细胞),采用脂多糖(LPS)处理HK-2细胞构建SA-AKI细胞模型。LPS处理的HK-2细胞分为LPS组、NC siRNA组、HIF-1αsiRNA组、anti-miR-NC组、anti-miR-23a组、HIF-1αsiRNA+miR-NC组、HIF-1αsiRNA+miR-23a组,以正常培养的HK-2细胞作为空白对照组(Control组)。采用qRT-PCR法检测细胞中HIF-1α、miR-23a基因表达;CCK-8法检测细胞活力;流式细胞术检测细胞凋亡;ELISA法检测细胞中炎性因子[白介素-1β(IL-1β)、白介素-6(IL-6)、肿瘤坏死因子α(TNF-α)]水平;Western blot法检测细胞中HIF-1α蛋白、NF-κB通路蛋白表达。结果与Control组比较,LPS组HK-2细胞中HIF-1α蛋白和mRNA表达水平、miR-23a mRNA表达水平均升高,细胞活力降低,细胞凋亡率、细胞中IL-6、IL-1β和TNF-α水平均升高(P<0.05)。与LPS组比较,HIF-1αsiRNA组HK-2细胞中HIF-1α蛋白和mRNA表达水平、miR-23a mRNA表达水平均降低,细胞活力升高,细胞凋亡率、细胞中IL-6、IL-1β和TNF-α水平均降低(P<0.05)。与LPS组比较,anti-miR-23a组HK-2细胞中miR-23a mRNA表达水平降低,细胞活力升高,细胞凋亡率、细胞中IL-6、IL-1β和TNF-α水平均降低(P<0.05)。与HIF-1αsiRNA组比较,HIF-1αsiRNA+miR-23a组HK-2细胞中miR-23a mRNA表达水平升高,细胞活力降低,细胞凋亡率、细胞中IL-6、IL-1β和TNF-α水平均升高(P<0.05)。与Control组比较,LPS组HK-2细胞p-NF-κB-p65/NF-κB-p65、p-IκBα/IκBα比值均升高(P<0.05)。与LPS组比较,HIF-1αsiRNA组和anti-miR-23a组HK-2细胞p-NF-κB-p65/NF-κB-p65、p-IκBα/IκBα比值均降低(P<0.05)。与HIF-1αsiRNA组比较,HIF-1αsiRNA+miR-23a组HK-2细胞p-NF-κB-p65/NF-κB-p65、p-IκBα/IκBα比值均升高(P<0.05)。结论肾小管HIF-1α通过调控miR-23a表达调节NF-κB信号通路,从而参与LPS诱导的肾小管上皮细胞损伤。

桑白皮多糖介导Nrf2/ARE通路减轻慢性低氧环境大鼠心肌损伤的作用

目的 探讨桑白皮多糖对慢性低氧环境所致的大鼠心肌损伤的影响,并观察其介导转录因子NF-E2相关因子(Nrf)2/抗氧化反应元件(ARE)通路的机制。方法 60只7~9周龄SD大鼠采用随机数字表分为对照组、模型组、阿托伐他汀组、桑白皮多糖低、中、高剂量组。除对照组外,其余各组均置于常压低氧舱内构建心肌损伤大鼠模型,对照组呼吸室内空气。自低氧第1天起,阿托伐他汀组给予20 mg/kg阿托伐他汀腹腔注射给药,桑白皮多糖低、中、高剂量组分别给予0.1、0.2、0.4μg/ml桑白皮多糖腹腔注射给药;对照组和模型组仅腹腔注射生理盐水。给药后24 h,苏木素-伊红(HE)染色观察各组心肌组织病变情况;原位末端标记法(TUNEL)检测各组心肌细胞凋亡率;酶联免疫吸附试验(ELISA)检测各组血清血浆肌酸激酶(CK)、乳酸脱氢酶(LDH)、心肌组织超氧化物歧化酶(SOD)活性和丙二醛(MDA)水平;检测心肌组织Nrf2、血红素加氧酶(HO)-1、B细胞淋巴瘤(Bcl)-2、Bcl-2相关X蛋白(Bax)mRNA及蛋白、p-Nrf2表达。结果 模型组心肌严重损伤,阿托伐他汀组和桑白皮多糖各剂量组均减轻;与对照组比较,模型组细胞凋亡率、CK、LDH活性和MDA水平、Bax mRNA和蛋白表达和p-Nrf2均明显上升,SOD活性、Nrf2、HO-1、Bcl-2 mRNA和蛋白表达明显下降(P<0.05);与模型组比较,阿托伐他汀组和桑白皮多糖各剂量组细胞凋亡率、CK、LDH活性和MDA水平、Bax mRNA和蛋白表达明显下降,SOD活性、Nrf2、HO-1、Bcl-2 mRNA和蛋白、p-Nrf2表达明显升高(P<0.05);桑白皮多糖对上述指标的作用与剂量有关。结论 桑白皮多糖可减轻慢性低氧环境大鼠心肌损伤,可能与激活Nrf2/ARE通路相关。

运动改善创伤性脑外伤的研究进展

创伤性脑外伤(traumatic brain injury,TBI)是由外力作用引起的脑功能改变或其他脑部病理学变化,是世界范围内的重大公共卫生问题。目前大量研究证明运动作为非药物治疗方式可有效改善TBI。损伤前后的运动均可通过相关机制有益于TBI患者的康复。本文综述了创伤前/后运动对TBI的影响及其潜在调控机制,以期为深入理解运动改善TBI的研究机制及运动疗法在TBI临床中的应用提供理论依据。

TRAF5通过调控IL-17A/NF-κB信号通路减轻氧-糖剥夺/再灌注引起的心肌细胞炎症和细胞凋亡

目的 探讨肿瘤坏死因子受体相关因子5(tumor necrosis factor receptor-associated factor 5,TRAF5)对氧-葡萄糖剥夺/再灌注(oxygen-glucose deprivation/reperfusion,OGD/R)引起的心肌细胞炎症和细胞凋亡的影响及相关分子机制。方法 体外培养大鼠心肌细胞系H9c2细胞,构建OGD/R诱导的H9c2细胞模型,以脂质体转染法将TRAF5过表达质粒(pcDNA-TRAF5)和空载质粒(pcDNA-NC)转染至OGD/R诱导的H9c2细胞。采用实时定量聚合酶链反应(quantitative real time polymerase chain reaction,qRT-PCR)法检测细胞中TRAF5基因表达,CCK-8法测定细胞活力,试剂盒法检测细胞中乳酸脱氢酶(lactate dehydrogenase,LDH)、心肌肌钙蛋白T(cardiac troponin T,cTnT)和炎性因子水平,流式细胞术检测细胞凋亡,Western blot法检测细胞中相关蛋白表达。结果 与对照组比较,OGD/R组H9c2细胞中TRAF5 mRNA和蛋白表达量降低,细胞活力降低,细胞中LDH、cTnT、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)、白细胞介素-1β(interleukin-1β,IL-1β)、单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)浓度升高,细胞凋亡率升高,细胞中B淋巴细胞瘤2(B-cell lymphoma-2,Bcl-2)蛋白表达量降低,Bcl-2相关蛋白(B-cell-lymphoma-2 related protein,Bax)、切割型半胱氨酸天冬氨酸蛋白水解酶-3(cleaved cysteine aspartate proteolytic enzyme-3,cleaved caspase-3)、白细胞介素-17A(interleukin-17A,IL-17A)蛋白表达量及磷酸化核转录因子-κB(phosphorylated nuclear factor-κB,p-NF-κB)/NF-κB比值升高,差异均有统计学意义(P<0.05)。与OGD/R组比较,OGD/R+pcDNA-TRAF5组H9c2细胞中TRAF5 mRNA和蛋白表达量升高,细胞活力升高,细胞中LDH、c TnT、TNF-α、IL-6、IL-1β、MCP-1浓度降低,细胞凋亡率降低,细胞中Bcl-2蛋白表达量升高,Bax、cleaved caspase-3、IL-17A蛋白表达量及p-NF-κB/NF-κB比值降低,差异均有统计学意义(P<0.05)。结论 TRAF5可减轻OGD/R引起的心肌细胞炎症和细胞凋亡,其作用机制可能与调控IL-17A/NF-κB信号通路有关。

Expression Changes of Early Response Genes in Lung Due to High Volume Ventilation

The expression changes of early response genes due to ventilation with high volume in adult rats in vivo were observed. Forty SD male rats were randomly divided into control and 30, 60, 90 and 120 min ventilation groups, respectively （n=8 in each group）. The animals were ventilated with tidal volume of 42 ml/kg and a PEEP level of 0 cmH2O at a rate of 40 breaths per minute in room air with a ventilator was given to the small animals. The expression of Egr-1, C-jun and IL-1β mRNA and proteins was detected by RT-PCR and immunohistochemical technique, respectively. The pathological changes in lung tissues were examined by HE staining. The results indicated that the expression of Egr-1, C-jun and IL-1β mRNA was detectable at 30th min after overventilation, but there was no significant difference in comparison with that in control group until overventilation for 60 min. However, at 90 and 120 min there was a significent increase as compared with 30 min or control group （P<0.05）. The expression of Egr-1, C-jun and IL-1β deteced by immunohistochemical assay also showed a similar tendency of the gradual increase. In the 120 min ventilation group, the expression intensity of Egr-1, C-jun and IL-1β proteins in lung cells was the strongest and the nuclear translocation was increased markedly in comparison with any other groups （P<0.05）. HE staining suggested that the degree of lung injury was aggravated gradually with the ventialtion going on and had a similar tendency to the expression of these early response genes and proteins. The current data suggested that overventilation activated and upregulated the expression of early response genes and the expression of these genes may be taken as the early signal to predict the onset and degree of lung injury. These results may demonstrated partially that the expression of early response genes induced by the mechanical stretch is associated with biochamic lung injury.

灯盏花素通过转录因子cAMP反应元件结合蛋白磷酸化促进细胞自噬保护心肌细胞缺血-再灌注损伤的机制研究

背景心肌缺血-再灌注(ischemia-reperfusion injury,I/R)损伤是心肌梗死血流再灌注后出现的常见临床问题,自噬在心肌I/R损伤过程中的作用机制尚不完全明确。目的研究灯盏花素在保护心肌缺血再灌注损伤过程中通过转录因子cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)促进自噬减轻细胞损伤的机制。方法在50μmol/L灯盏花素为最佳应答浓度的基础上进行实验。实验分为正常对照(Vehicle)组、I/R组、灯盏花素处理(Scu+I/R)组和CREB抑制并灯盏花素处理(KG501+Scu+I/R)组。通过糖氧剥夺培养方式建立小鼠心肌细胞I/R损伤模型,MTT法测定细胞活力;生化法测定超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)、乳酸脱氢酶(lactate dehydrogenase,LDH)和三磷酸腺苷(adenosine triphosphate,ATP)水平;RT-PCR测定线粒体内膜融合蛋白1(optic atrophy 1,Opa1)和线粒体动力相关蛋白1(dynamin-related protein 1,Drp1)表达;Western blot测定LC3、CREB和磷酸化cAMP反应元件结合蛋白(phosphorylated cAMP response element binding protein,p-CREB)表达。结果MTT测定发现:相比于其他浓度,50μmol/L灯盏花素可显著增加细胞活力,使I/R损伤心肌细胞SOD表达增加(P<0.01),MDA表达减少(P<0.01),LDH表达减少(P<0.01),ATP表达增加(P<0.01),LC3Ⅱ/Ⅰ比值增加(P<0.05),Drp1 mRNA表达减少(P<0.01),Opa1 mRNA表达增加(P<0.01),p-CREB/CREB比值增加(P<0.01)。CREB抑制剂(KG-501)可显著降低灯盏花素处理组的ATP、Opa1 mRNA表达(P均<0.01),使LC3Ⅱ/Ⅰ比值降低(P<0.05),细胞活力降低(P<0.01)。结论灯盏花素能够促进缺血心肌细胞自噬,缓解氧化损伤,提高心肌细胞活力。在保护心肌缺血-再灌注损伤过程中,灯盏花素可能通过CREB通路调节自噬作用。

基于Nrf2/HO-1通路探讨金雀异黄酮对糖尿病肾病大鼠肾损伤的影响及机制

目的探讨金雀异黄酮对糖尿病肾病大鼠肾组织损伤的影响及其可能的分子机制。方法取雄性SD大鼠55只,随机选择10只作为正常组,其余大鼠采用一次性腹腔注射链脲佐菌素60 mg/kg的方法复制糖尿病肾病模型。将40只成模大鼠随机分为模型组、金雀异黄酮组、鸦胆子苦醇组和金雀异黄酮+鸦胆子苦醇组,每组10只。金雀异黄酮组给予30 mg/kg金雀异黄酮灌胃,鸦胆子苦醇组给予鸦胆子苦醇2 mg/kg灌胃,金雀异黄酮+鸦胆子苦醇组给予30 mg/kg金雀异黄酮和鸦胆子苦醇2 mg/kg灌胃,均1次/d,连续灌胃6周。检测各组大鼠空腹血糖(FPG)水平及肾功能指标[血清肌酐(SCr)、尿素氮(BUN)和24 h尿微量白蛋白(24h U-mAlb)],计算肾脏指数,HE染色、PAS染色、Masson染色观察肾组织形态,透射电子显微镜观察肾细胞超微结构,分光光度法检测肾组织中总超氧化物歧化酶(T-SOD)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量,ELISA法检测肾组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-8(IL-8)含量,Western blot法检测肾组织中E2相关因子2(Nrf2)、p-Nrf2、血红素加氧酶1(HO-1)、醌氧化还原酶1(NQO1)、总核因子κB(NF-κB)、胞核NF-κB蛋白表达情况。结果与正常组比较,模型组大鼠FPG、SCr、BUN、24hU-mAlb水平和肾脏指数均明显升高(P均<0.05);肾小球肥大、硬化,炎性浸润,胶原沉积,肾小球细胞可见足突肿胀或消失、基底膜弥漫性增厚、线粒体嵴断裂或消失;肾组织中T-SOD、CAT活性均明显降低(P均<0.05),MDA、TNF-α、IL-1β、IL-8含量均明显升高(P均<0.05);肾组织中p-Nrf2、HO-1、NQO1蛋白相对表达量及p-Nrf2/Nrf2比值均明显降低(P均<0.05),胞核NF-κB蛋白相对表达量及胞核NF-κB/总NF-κB比值均明显升高(P均<0.05)。与模型组比较,金雀异黄酮组和金雀异黄酮+鸦胆子苦醇组大鼠FPG、SCr、BUN、24hU-mAlb水平和肾脏指数均明显降低(P均<0.05);肾组织病理改变和肾小球细胞超微结构病变均明显减轻;肾组织中T-SOD、CAT活性均明显升高(P均<0.05),MDA、TNF-α、IL-1β、IL-8含量均明显降低(P均<0.05);肾组织中p-Nrf2、HO-1、NQO1蛋白相对表达量及p-Nrf2/Nrf2比值均明显升高(P均<0.05),胞核NF-κB蛋白相对表达量及胞核NF-κB/总NF-κB比值均明显降低(P均<0.05)。鸦胆子苦醇组上述指标变化趋势与模型组相同且更加严重,鸦胆子苦醇可明显逆转金雀异黄酮对上述指标的调控作用。结论金雀异黄酮能够抑制糖尿病肾病大鼠肾组织氧化应激和炎症反应,减轻肾组织病变和肾细胞超微结构病变,其机制可能与促进Nrf2/HO-1通路活化、抑制NF-κB核转位有关。

蛛网膜下腔出血患者TLR4/NF-κB信号通路变化及意义

目的探究蛛网膜下腔出血患者Toll样受体4(Toll like receptor 4,TLR4)/核因子κB(nuclear factor kappa B,NF-κB)信号通路变化及意义。方法选取作者医院2018-05/2021-06月收治的60例蛛网膜下腔出血患者作为观察组,另选取同期体检健康者60例作为对照组。比较两组TLR4/NF-κB信号通路相关因子表达,分析蛛网膜下腔出血患者TLR4/NF-κB信号通路相关因子与神经功能损害美国国立卫生院卒中量表(national institutes of health stroke scale,NIHSS)评分的关系。比较不同预后患者发病第1、3、7、14天外周血TLR4/NF-κB信号通路相关因子,绘制受试者工作特征(receiver operating characteristic,ROC)曲线,评价TLR4/NF-κB信号通路相关因子对蛛网膜下腔出血患者预后的预测价值,分析TLR4/NF-κB信号通路相关因子与蛛网膜下腔出血患者预后不良风险的关系。结果观察组TLR4 mRNA、NF-κB mRNA表达均高于对照组,组间比较差异有统计学意义(P均<0.05)。蛛网膜下腔出血患者入院当天NIHSS评分为(11.24±3.12)分,根据Pearson相关性模型分析可知,蛛网膜下腔出血患者TLR4 mRNA、NF-κB mRNA表达均与NIHSS评分呈正相关关系(P<0.05)。不同组别间患者TLR4 mRNA、NF-κB mRNA相对表达量差异有统计学意义(F组间=8.037、19.348,P均<0.001),不同时间点患者TLR4 mRNA相对表达量差异有统计学意义(F时间=5.128、16.207,P均<0.001),且时间与组间存在交互效应(F交互=6.497、18.032,P=0.015、<0.001)。预后不良组患者发病第1天TLR4 mRNA、NF-κB mRNA相对表达量较预后良好组患者差异无统计学意义(P>0.05)。预后不良组患者发病第3天、7天、14天TLR4 mRNA、NF-κB mRNA相对表达量均高于预后良好组患者(P均<0.05)。预后不良组患者、预后良好组患者发病第3天TLR4 mRNA、NF-κB mRNA相对表达量升至最高(P均<0.05);发病第7天、14天TLR4 mRNA、NF-κB mRNA相对表达量均较发病第3天明显下降,且发病第14天降至最低(P均<0.05)。以预后不良作为阳性样本,预后良好作为阴性样本,绘制ROC曲线,结果显示,发病第3、7、14天TLR4 mRNA、NF-κB mRNA联合预测蛛网膜下腔出血患者预后的AUC分别为0.854、0.894、0.915,较各指标单独诊断价值明显提高。Logistic回归分析显示,TLR4 mRNA、NF-κB mRNA仍与蛛网膜下腔出血患者预后不良有关(P均<0.05)。结论蛛网膜下腔出血后TLR4/NF-κB信号通路被激活,TLR4 mRNA、NF-κB mRNA表达明显上调,且TLR4/NF-κB信号通路与蛛网膜下腔出血患者早期脑损伤有关,为临床治疗提供了新思路,有助于改善患者预后。

海藻糖抑制NF-κB介导的炎性反应缓解镉致大鼠肾脏损伤

为探究海藻糖对镉致大鼠肾脏炎性反应和组织损伤的保护作用及其机制,将32只体重110 g的雄性SD大鼠随机分为对照组、镉处理组、海藻糖组、镉与海藻糖共处理组,持续8周。试验结束后采集血清检测肾功能生化指标,ELISA检测肾脏组织中促炎因子含量,Western blot和免疫组织化学方法检测核因子κB(NF-κB)信号通路的变化,Real-time PCR检测NF-κB下游促炎因子转录水平。结果表明:与对照组相比,镉处理组血清肌酐(Scr)、尿素氮(BUN)和尿酸(UA)含量极显著升高,NF-κB信号通路激活介导肾脏炎性反应。与镉处理组相比,镉与海藻糖共处理组血清Scr、BUN、UA含量显著降低,肾脏组织白细胞介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α、巨噬细胞趋化蛋白(MCP)-1含量极显著降低,NF-κB核转位极显著减少并抑制下游促炎因子转录。综上,海藻糖可通过降低NF-κB介导的炎性反应而缓解镉致大鼠肾脏损伤。