High glucose‐induced excessive reactive oxygen species promote apoptosis through mitochondrial damage in rat cartilage endplate cells

Diabetes mellitus (DM) is an important factor in intervertebral disc degeneration (IDD). Apoptosis of cartilage endplate (CEP) cells is one of the initiators of IDD. However, the effects of high glucose on CEP cells are still unknown. Therefore, we conducted the present study to evaluate the effects of high glucose on CEP cells and to identify the mechanisms of those effects. Rat CEP cells were isolated and cultured in 10% foetal bovine serum (FBS, normal control) or high‐glucose medium (10% FBS + 0.1 M glucose or 10% FBS + 0.2 M glucose, experimental conditions) for 1 or 3 days. In addition, CEP cells were treated with 0.2 M glucose for 3 days in the presence or absence of alpha‐lipoic acid (ALA, 0.15 M). Flow cytometry was performed to identify and quantify the degree of apoptosis. The expression of reactive oxygen species (ROS) was assessed by flow cytometry, and mitochondrial damage (mitochondrial membrane potential) was assessed by fluorescence microscopy. Furthermore, the expression levels of cleaved caspase‐3, cleaved caspase‐9, Bcl‐2, Bax, and cytochrome c were evaluated by Western blotting. High glucose significantly increased apoptosis and ROS accumulation in CEP cells in a dose‐ and time‐dependent manner. Meanwhile, a disrupted mitochondrial membrane potential was detected in rat CEP cells cultured in the two high glucose concentrations. Incubating in high glucose enhanced the expression levels of cleaved caspase‐3, cleaved caspase‐9, Bax, and cytochrome c but decreased the level of the anti‐apoptotic protein Bcl‐2. ALA inhibited the expression of cleaved caspase‐3, cleaved caspase‐9, Bax, and cytochrome c but enhanced the expression of Bcl‐2. ALA also prevented disruption of the mitochondrial membrane potential in CEP cells. This study demonstrates that high glucose‐induced excessive reactive oxygen species promote mitochondrial damage, thus causing apoptosis in rat CEP cells in a dose‐ and time‐dependent manner. ALA could prevent mitochondrial damage and apoptosis caused by high glucose in CEP cells. The results suggest that appropriate blood glucose control may be the key to preventing IDD in diabetic patients. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2476–2483, 2018.

     

Alterations of ADAMTSs and TIMP‐3 in human nucleus pulposus cells subjected to compressive load: Implications in the pathogenesis of human intervertebral disc degeneration

Intervertebral disc degeneration (IDD) pertains to the loss of extracellular matrix (ECM), particularly the early loss of aggrecan, the turnover of which is regulated by ADAMTSs. Amongst the etiological factors of IDD, mechanical stress plays an important role in the physiological and pathological processes of nucleus pulposus (NP) cells. However, the role of ADAMTSs and their inhibitor in human NP cells under mechanical stress has not been elucidated to date. The purpose of this study was to investigate the role of ADAMTSs and TIMP‐3 in NP cells under mechanical stress. Human NP cells isolated from non‐degenerative and degenerative discs were subjected to dynamic compressive load. The expression of ADAMTSs, aggrecan, and TIMP‐3 was detected by quantitative real‐time PCR and/or Western blot. Consequently, the gene expression of ADAMTS‐1, 4, and 5 increased significantly in loaded NP cells compared with not‐loaded cells from either non‐degenerative or degenerative discs, whereas the gene expression of aggrecan decreased significantly. Moreover, Western blot indicated increased protein levels of ADAMTSs‐1, 4, and 5. However, the expression of TIMP‐3 altered insignificantly. Together, this study is the first addressing the underlying mechanisms of compressive load as a contributing factor to IDD in terms of ADAMTSs. Our results suggest that compressive load leads to the increase in ADAMTS‐1, 4, and 5 that contributes to the decrease of aggrecan and IDD via TIMP‐3 independent machinery. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:267–273, 2012     

Advanced glycation end products in degenerative nucleus pulposus with diabetes

Diabetes mellitus (DM) has been clinically proved as a risk factor of disc degeneration, and the accumulation of advanced glycation end products (AGEs) is known to be potentially involved in diabetes. The purpose of this study is to investigate the effect of AGEs in the degeneration process of diabetic nucleus pulposus (NP) in rats and humans. Diabetic NP cells from rat coccygeal discs were treated with different concentrations of AGEs (0, 50, and 100 µg/ml) for 3 days, and mRNA expressions of MMP‐2 and RAGE were measured by real‐time RT‐PCR. In addition, conditioned medium from NP cells was used to analyze protein expression of MMP‐2 activity and ERK by gelatin zymography and Western blot. These experiments were repeated using human intervertebral disc samples. The immunohistochemical expression of AGEs was significantly increased in diabetic discs. In response to AGEs, an increase of MMP‐2, RAGE, and ERK at both mRNA and protein expression levels was observed in diabetic NP cells. The findings suggest that AGEs and DM are associated with disc degeneration in both species. Hyperglycemia in diabetes enhances the accumulation of AGEs in the NP and triggers disc degeneration. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:238–244, 2014.     

沉默p53和p21基因延缓髓核细胞衰老退变实验研究

目的髓核细胞衰老凋亡是椎间盘退行性变的病理基础,探讨髓核细胞表型分子及延缓髓核细胞衰老退变的机制。方法原代培养8～10周龄雄性SD大鼠髓核细胞,免疫细胞化学染色鉴定髓核细胞表型分子低氧诱导因子1α(hypoxia inducible factor 1α,HIF-1α)、HIF-1β、基质金属蛋白酶2(matrix metalloproteinase 2,MMP-2)及Ⅱ型胶原表达。小干扰RNA(small interference RNA,siRNA)瞬时转染髓核细胞沉默p53、p21后行RT-PCR及Western blot检测沉默效率,siRNA转染前后细胞衰老相关β半乳糖苷酶(senescence associated-β-galactosidase,SA-β-gal)染色检测髓核细胞衰老变化,流式细胞仪检测髓核细胞周期变化,MTT法生长曲线分析髓核细胞增殖变化。结果免疫细胞化学染色显示髓核细胞表达HIF-1α、HIF-1β、MMP-2及Ⅱ型胶原。第35代髓核细胞转染p53、p21 siRNA后RT-PCR及Western blot检测示p53、p21表达明显受到抑制。第35代髓核细胞SA-β-gal染色阳性率明显高于第1代髓核细胞(P<0.001);正常第35代髓核细胞经p53 siRNA(p53转染组)和p21 siRNA(p21转染组)转染后,SA-β-gal阳性率明显低于正常第35代髓核细胞(正常组)和加入脂质体LipofectaminTM2000而无siRNA的第35代髓核细胞(阴性对照组),差异有统计学意义(P<0.001)。细胞周期分析显示,p53转染组及p21转染组G1期百分比均明显低于正常组和阴性对照组(P<0.05),S期百分比明显高于正常组和阴性对照组(P<0.05)。MTT生长曲线显示转染p53、p21 siRNA后可促进髓核细胞增殖。结论沉默p53、p21基因可通过调节细胞周期而抑制髓核细胞衰老退变,改善椎间盘退变过程;沉默p53、p21基因可能是潜在的治疗椎间盘退行性变的方法。     

The role of leptin on the organization and expression of cytoskeleton elements in nucleus pulposus cells

Obesity is an important risk factor for intervertebral disc degeneration and leptin is a biomarker of obesity. However, the expression of leptin receptors has not been determined in disc tissue. It is not known whether leptin has a direct effect on the nucleus pulposus (NP) cells. To determine whether the NP tissues and cells express leptin receptors (OBRa and OBRb) and whether leptin affects the organization and the expression of major cytoskeletal elements in NP cells. Messenger RNA (mRNA) and protein levels of OBRa and OBRb were measured by real‐time PCR and Western blot, respectively, in NP tissues and cells. Immunofluorescence and real‐time PCR and Western blot were performed to investigate the effect of leptin on cytoskeleton reorganization and expression. Results show that mRNA and proteins of OBRa and OBRb were expressed in all NP tissues and cells, and that OBRb expression was correlated with patients' body weight. Increased expression of β‐actin and reorganization of F‐actin were evident in leptin‐stimulated NP cells. Leptin also induced vimentin expression but had no effect on β‐tubulin in NP cells. These findings provide novel evidence supporting the possible involvement of leptin in the pathogenesis of intervertebral disc degeneration. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 847–857, 2013     

缺氧诱导因子在椎间盘退变过程中作用机制的研究进展

缺氧诱导因子（HIFs）是一类介导哺乳动物细胞内低氧反应的核转录复合体,目前的研究结果表明,其在椎间盘退变进程中起到重要作用。该文通过综述HIFs在椎间盘组织中的表达、调控以及与椎间盘细胞凋亡、自噬的关系,阐明其在椎间盘退变过程中的作用机制,从而为椎间盘退行性疾病的治疗开辟新思路和新途径。     

Altered expression of mitofusin 2 in penile tissues of diabetic rats

Diabetic erectile dysfunction (ED) is a common complication in diabetes mellitus, and the efficacy of first‐line therapies is not satisfactory. Recent studies revealed that corporal apoptosis was responsible for the nonresponsiveness of severe ED to phosphodiesterase type 5 inhibitors. Mitofusin 2 (Mfn2) is a versatile protein, regulating mitochondrial morphology and playing an important role in apoptosis. Several studies showed that expression of Mfn2 was decreased in STZ‐induced diabetic rats' kidney, myocardium and retina, which was associated with diabetic nephropathy, cardiomyopathy and retinopathy respectively. In this study, our aim was to explore the expression of Mfn2 and apoptosis in diabetic rats' penes. We found that erectile function (ICP/MAP) elicited by electrical stimulation of cavernous nerve was markedly impaired in diabetic rats compared with the normal rats. The mRNA and protein levels of Mfn2 were found to be significantly reduced in diabetic rats' penile tissues. Compared with normal rats, the content of smooth muscle and B‐cell lymphoma 2 (Bcl‐2)/Bcl‐2‐associated X protein (Bax) ratio were dramatically decreased, and penile apoptotic index and expression of activated‐caspase‐3 were dramatically increased in diabetic rats. This data indicated that repression of Mfn2 in diabetic rats' penes might be associated with excessive apoptosis in diabetes‐induced severe ED.     

P16INK4a和Fas在椎间盘组织细胞中的表达及意义

目的：分析衰老关键基因p16^INK4a及凋亡相关基因Fas在人类椎间盘退变过程中的表达变化。方法：分别取正常人和腰椎间盘退变患者的髓核及纤维环组织块制作石蜡切片，利用免疫组织化学及免疫荧光法检测p16^INK4a和Fas的表达情况；提取总蛋白及总RNA，利用Western blot及RT-PCR对p16^INK4a和Fas的表达以及视网膜母细胞瘤蛋白（pRb）的磷酸化状态进行分析。结果：p16m在正常人椎间盘髓核及纤维环组织中的表达阳性率分别为6．6％、4．7％，在内破裂椎间盘（IDD）及突出椎间盘（LIDP）组织中的表达分别为44．1％、38．9％和56．1％、46．7％，较正常人椎间盘明显升高（P〈0．05），尤以LIDP中的表达上调显著（P〈0．05）；Fas在正常椎间盘与IDD的髓核及纤维环组织中的表达阳性率均较低。分别为9．9％、8．1％和10．2％、10．9％，在LIDP组织中有相对较高表达阳性率。分别为25．2％和22．0％；Western blot及RT-PCR分析显示，p16^INK4a与Fas在各自蛋白及相应mRNA水平上的表达具有相同的变化趋势：p16^INK4a与Fas极少在同一个椎间盘细胞内表达；随着p16^INK4a表达的升高磷酸化pRb也逐渐减少。结论：p16^INK4a可能参与了椎间盘细胞的衰老过程，是导致椎间盘退变发生和发展的原因之一；Fas表达升高可能是突出椎间盘细胞中的一种继发改变。     

Role of caspases on cell death, inflammation, and cell cycle in glycerol-induced acute renal failure

Caspases are the main executioners of apoptosis as well as interleukin (IL)-1beta and IL-18 conversion to active forms. They are activated after acute kidney injuries. In this study, we evaluated the importance of the caspase family in the pathogenesis and recovery of glycerol-induced acute renal failure in rats (Gly-ARF). Rats were treated with pan-caspase or selective caspase 1 and 3 inhibitors at the moment we injected glycerol. Renal function, renal histology (HE), transferase-mediated deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling staining for apoptosis, leukocytes infiltration (immunohistochemistry), renal expression of IL-1beta and IL-18 (immunohistochemistry and Western blot), tubular regeneration (5-bromo-2'-deoxyuridine (BrdU) incorporation), and P27(Kip) expression (Western blot) were evaluated at appropriate times. All inhibitors reduced the renal function impairment. Pan-caspase and caspase-3 inhibitors reduced cellular death (necrosis and apoptosis) 24 h after Gly-ARF. All caspases inhibitors reduced macrophages infiltration. The expression of total IL-1beta was enhanced in Gly-ARF, but the active IL-1beta and IL-18 forms were abolished in pan-caspase treated rats. Caspase-1 inhibitor attenuated Gly-ARF but not tubular injury suggesting glomerular hemodynamic improvement. There was striking regenerative response 48 h after Gly-ARF characterized by enhanced BrdU incorporation and reduced expression of p27(Kip). This response was not blunted by caspases inhibition. Our findings demonstrate that caspases participate in important pathogenic mechanisms in Gly-ARF such as inflammation, apoptosis, vasoconstriction, and tubular necrosis. The early inhibition of caspases attenuates these mechanisms and reduces the renal function impairment in Gly-ARF.     

Cell adhesion to fibronectin induces mitomycin C resistance in bladder cancer cells

OBJECTIVE

To investigate whether cell adhesion to fibronectin induces drug resistance in human bladder cancer cells, and to study the survival signalling pathway in cell adhesion to fibronectin‐mediated chemotherapy resistance in vitro.

MATERIALS AND METHODS

T24 cells (human bladder cancer cell lines) were pre‐coated with fibronectin, and treated with mitomycin C (MMC) and the specific phosphoinositide‐3 kinase (PI3‐K) inhibitor LY294002. The apoptosis and cell cycles were analysed. The activity of the caspase‐8, ‐9 and apoptosis‐inducing factor (AIF) apoptosis pathways were assessed using colorimetric assay, immunofluorescence, Western blot and flow cytometry. The expression of glycogen synthase kinase‐3β (GSK‐3β) and cyclin D1, as the key regulator of G1/S phase transition, were determined by Western blot. The expression of PI3‐K, Akt, phospho‐Akt and β1‐integrin were also examined by Western blot.

RESULTS

Apoptosis induced by MMC was significantly resisted by fibronectin adhesion in T24 cells, and this effect was through inhibition of the caspase‐9 and AIF apoptosis pathways, but not the caspase‐8 pathway. Fibronectin antagonized MMC‐induced G0/G1‐phase arrest by inactivating GSK‐3β to stabilize cyclin D1 expression in T24 cells. Furthermore, fibronectin‐mediated protection of T24 cells was dependent on the activity of the PI3‐K/Akt signalling pathway, and the protection could be abolished by the PI3‐K inhibitor LY294002.

CONCLUSIONS

Fibronectin‐mediated PI3‐K/Akt activation protects T24 cells from MMC‐induced cell death through inhibition of both caspase‐9 and AIF‐mediated apoptosis and GSK‐3β/cyclin D1 involved G0/G1‐phase arrest.     

Quercetin and vitamin E attenuate diabetes-induced testicular anomaly in Wistar rats via the mitochondrial-mediated apoptotic pathway

The role of quercetin and vitamin E treatment against streptozotocin (STZ)-induced testicular abnormalities in diabetic rats and the possible mechanism of action they use for protection were investigated. Diabetes was induced by STZ (45 mg/kg i.p. once) and blood glucose was determined. Plasmatic insulin, testosterone, luteinising hormone and follicle-stimulating hormone (FSH) were determined by ELISA. Levels of cytochrome c, caspase 3 and caspase 9 were evaluated by immunohistochemistry, while lesions were viewed by histology. Insulin played a role in testicular protection against male infertility through modulation of luteinising hormone (LH). This consequently increased Leydig and Sertoli cells and maturation of germ cells with the attached epididymis having abundant spermatozoa. The study showed a positive correlation in the levels of LH, FSH and testosterone; it was further established that all treatments normalised diabetes-induced alterations. Treatment with quercetin and vitamin E resulted in 34% decrease of apoptogenic cytochrome c release. This protected the testes against excessive apoptosis by decreasing caspase 3 and caspase 9 activation by up to 30 and 28% respectively (p < .05). Histology also showed that treatment prevented testicular cell death. The findings show that quercetin/vitamin E possess free radical scavenging properties that protected against testicular damage in diabetes. This suggests the possibility of pharmaco-therapeutic intervention.     

Therapeutic effect of combination of alagebrium (ALT-711) and sildenafil on erectile function in diabetic rats

Recently, the relationship between advanced glycation end products (AGEs) and erectile dysfunction (ED) has been reported. The present study aimed to investigate whether a combination of an AGE cross-link breaker (alagebrium/ALT-711) and sildenafil could enhance the erectile capacity in streptozotocin (STZ) diabetic rats. Additionally, we assessed the effect of that treatment option on some molecules that have been suggested to have crucial roles in AGE-related ED pathways. Four groups of animals were utilized: (1) age-matched control rats, (2) STZ-induced diabetic rats (40 mg kg(-1) i.p.), (3) STZ rats+sildenafil (5 mg kg(-1) p.o.), (4) STZ rats treated with a combination of sildenafil (5 mg kg(-1) p.o)+alagebrium/ALT-711 (10 mg kg(-1) p.o.) for the final 1 month of the 2 months of diabetes period. At 2 months after i.p. injection of STZ, all animals underwent cavernosal nerve stimulation (CNS) to assess erectile function. Penile tissue AGEs, MDA (malondialdehyde), cyclic guanosine monophosphate (cGMP) (ELISA), endothelial nitric oxide (NO) synthase (eNOS), inducible NO synthase (iNOS) (western blot), nuclear factor (NF)-κB, mitogen-activated protein (MAP) kinase (immunohistochemistry) and apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) analyses were performed in all groups of rats. STZ diabetic rats had a significant decrease in erectile function as determined by the peak intracavernosal pressure (ICP) and total ICP (area under the erectile curve) after CNS when compared with control rats (P<0.05). The increase in both ICP and area under the erectile curve of STZ diabetic rats treated with a combination of sildenafil+alagebrium/ALT-711 as well as in STZ diabetic rats treated with sildenafil alone was significantly greater than STZ diabetic rats. Additionally, combination treatment decreased AGE, MDA, iNOS, NF-κB, MAP kinase and apoptosis levels, whereas it preserved cGMP contents in diabetic penile tissue. Decreased AGE, MDA, iNOS, NF-κB, MAP kinase and increased cGMP levels at the combination (sildenafil+alagebrium/ALT-711) therapy group increased both the peak ICP and total ICP to CNS in the STZ diabetic rats, which was similar to the response observed in control rats. These results may explain the role of AGEs in diabetes-related ED and the effect of an AGE cross-link breaker alagebrium/ALT-711+sildenafil therapy on some critical molecules related to AGE-related ED pathways.     

Antifibrogenic role of valproic acid in streptozotocin induced diabetic rat penis

We investigated the therapeutic effects of valproic acid (VPA) on erectile dysfunction and reducing penile fibrosis in streptozocin (STZ)‐induced diabetic rats. Eighteen male rats were divided into three experimental groups (Control, STZ‐DM, STZ‐DM plus VPA) and diabetes was induced by transperitoneal single dose STZ. Eight weeks after, VPA and placebo treatments were given according to groups for 15 days. All rats were anesthetised for the measurement of in vivo erectile response to cavernous nerve stimulation. Afterward penes were evaluated histologically in terms of immune labelling scores of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and transforming growth factor‐β1 (TGF‐β1). Slides were also evaluated in terms of collagen/smooth muscle ratio and penile apoptosis. After the treatment with VPA, erectile responses were found as improved when compared with STZ‐DM rats but not statistically meaningful. eNOS and VEGF immune expressions diminished in penile corpora of STZ‐DM rats and improved with VPA treatment. VPA led to decrease in TGF‐β1 expression and collagen content of diabetic rats’ penes. Penile apoptosis was not diminished with VPA. In conclusion, VPA treatment seems to be effective for reducing penile fibrosis in diabetic rats and more prolonged treatment period may enhance erectile functions.     

A ativação autofágica atenua a neurotoxicidade dos anestésicos locais ao diminuir a atividade da caspase‐3 em ratos

Background and objectivesThe mechanisms by which local anaesthetics cause neurotoxicity are very complicated. Apoptosis and autophagy are highly coordinated mechanisms that maintain cellular homeostasis against stress. Studies have shown that autophagy activation serves as a protective mechanism in vitro. However, whether it also plays the same role in vivo is unclear. The aim of this study was to explore the role of autophagy in local anaesthetic‐induced neurotoxicity and to elucidate the mechanism of neurotoxicity in an intrathecally injected rat model.MethodsEighteen healthy adult male Sprague‐Dawley rats were randomly divided into three groups. Before receiving an intrathecal injection of 1% bupivacaine, each rat received an intraperitoneal injection of vehicle or rapamycin (1 mg.kg^‐1) once a day for 3 days. The pathological changes were examined by Haematoxylin and Eosin (HE) staining. Apoptosis was analysed by TdT‐mediated dUTP Nick‐End Labelling (TUNEL) staining. Caspase‐3, Beclin1 and LC3 expression was examined by Immunohistochemical (IHC) staining. Beclin1 and LC3 expression and the LC3‐II/LC3‐I ratio were detected by western blot analysis.ResultsAfter bupivacaine was injected intrathecally, pathological damage occurred in spinal cord neurons, and the levels of apoptosis and caspase‐3 increased. Enhancement of autophagy with rapamycin markedly alleviated the pathological changes and decreased the levels of apoptosis and caspase‐3 while increasing the expression of LC3 and Beclin1 and the ratio of LC3‐II to LC3‐I.ConclusionsEnhancement of autophagy decreases caspase‐3‐dependent apoptosis and improves neuronal survival in vivo. Activation of autophagy may be a potential therapeutic strategy for local anaesthetic‐induced neurotoxicity.     

Inhibition of autophagy increases apoptosis during re‐warming after cold storage in renal tubular epithelial cells

Prolonged cold storage and re‐warming (CS/REW) of kidneys are risk factors for delayed graft function (DGF). Studies in renal tubular epithelial cells (RTECs) have determined apoptosis and autophagy in models of either cold storage (CS) or re‐warming alone. The effect of both cold storage and re‐warming on apoptosis and autophagy, in RTECS is not known and is relevant to DGF as the kidney is subjected to both CS and re‐warming. We hypothesized that CS/REW of RTECs would induce autophagy that protects against apoptosis. In CS/REW, there was increased autophagic flux of RTECs. Autophagy inhibition using an Atg5 siRNA resulted in increased cleaved caspase‐3 and increased apoptotic cells (on both morphology and annexin V staining) during CS/REW. The effect of autophagy inhibition on necrosis in RTECs is unknown. There were increased necrosis and caspase‐1, a mediator of necrosis, during CS/REW, and the Atg5 siRNA had no effect on necrosis and caspase‐1. In a kidney transplant model, there was an increase in LC3 II, a marker of autophagy, in kidneys transplanted after cold storage. In summary, autophagic flux is increased during CS/REW. Autophagy inhibition resulted in increased cleaved caspase‐3 and increased apoptosis during CS/REW without an effect on necrosis or caspase‐1. In conclusion, autophagy inhibition in RTECs after CS/REW induces apoptotic cell death and may be deleterious as a therapy to decrease DGF.     

Dysregulated miR‐98 Contributes to Extracellular Matrix Degradation by Targeting IL‐6/STAT3 Signaling Pathway in Human Intervertebral Disc Degeneration

Intervertebral disc degeneration (IDD) is associated with dysregulated expression of microRNAs (miRNAs). However, the precise molecular mechanisms underlying this disorder remain unclear. Therefore, we tested the hypothesis that miRNAs modulate IDD through effects on the IL‐6/STAT3 signaling pathway, a potential regulator of IDD. The miRNA expression profile was determined in nucleus pulposus (NP) tissues from patients with IDD and controls, employing miRNA microarray and quantitative real‐time PCR (RT‐qPCR). Biological functions of differential expression miRNAs were further investigated using immunofluorescent staining. Luciferase reporter assays and Western blotting were performed to determine miRNA targets. We identified 41 miRNAs that were differentially expressed in patients compared with controls. Following RT‐qPCR confirmation, miR‐98 was significantly downregulated in degenerative NP tissues. Moreover, its level was inversely correlated with grade of disc degeneration. Through gain‐of‐function and loss‐of‐function studies, miR‐98 was shown to significantly promote type II collagen expression in NP cells. Interleukin‐6 (IL‐6) was identified as a target of miR‐98. Knockdown of IL‐6 induced effects on NP cells similar to those induced by miR‐98. In contrast, IL‐6 treatment abrogated the effects induced by miR‐98 upregulation. Moreover, miR‐98 dramatically suppressed expression of STAT3 target gene, MMP2. IL‐6 treatment antagonized this effect, whereas knockdown of IL‐6 by IL‐6 short hairpin RNA (shIL‐6) induced inhibitory effects on the expression of p‐STAT3 and its main target genes, similar to miR‐98. The mRNA level of IL‐6 was inversely correlated with that of miR‐98 in degenerative NP tissues. These results suggest the downregulation of miR‐98 could promote IDD through the IL‐6/STAT3 signaling pathway. Our findings also highlight miR‐98 as a novel hopeful therapeutic target for IDD. © 2015 American Society for Bone and Mineral Research.     

Role of miR‐155 in the regulation of MMP‐16 expression in intervertebral disc degeneration

The molecular mechanisms of intervertebral disc degeneration (IDD) remain elusive. We found that miR‐155 is down‐regulated in degenerative nucleus pulposus (NP), and more severe degeneration is correlated with higher matrix metallopeptidase 16 (MMP‐16) expression. MMP‐16 also degraded matrix aggrecan. Here, we addressed the in vivo miR‐155‐mediated pathological impact on IDD using a classic puncture mouse model. Lentiviral upregulated‐miR‐155 or downregulated‐miR‐155 was transduced into the discs of C57 mice, which was validated by real‐time polymerase chain reaction (real‐time PCR) and in situ hybridization. Immunohistochemistry and western blotting revealed that up‐regulation of miR‐155 resulted in down‐regulation of MMP‐16 and an increase in aggrecan and collagen type II in mouse NP; whereas, down‐regulation of miR‐155 resulted in up‐regulation of MMP‐16 and a decrease in aggrecan in mouse NP. Radiographic and histological analysis showed that the up‐regulation of miR‐155 attenuated IDD, while down‐regulation of miR‐155 resulted in the deterioration of IDD. These findings indicate that decreased miR‐155 contributed to the up‐regulation of MMP‐16 in vivo, and MMP‐16 further degraded aggrecan and collagen type II, leading to the dehydration and degeneration of discs. Our findings revealed a therapeutic role for miR‐155 in IDD. © 2017 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1323–1334, 2017.

     

The possible role of interleukin-33 as a new player in the pathogenesis of contrast-induced nephropathy in diabetic rats

Introduction: Patients with diabetic kidney disease (DKD) are more prone to contrast-induced nephropathy (CN). Apoptosis and autophagy were found to be essential in the pathogenesis of DKD. Interleukin-33 (IL-33) is a cytokine, but its role in DKD and CN is unknown. As IL-33 is modulated by apoptosis, we aimed to determine the relationship between IL-33 apoptosis and autophagy in DKD with CN. Materials and methods: Thirty male Sprague–Dawley rats were enrolled and randomly allocated into three groups. The first group was comprised of healthy rats (HRs), whereas the other two groups were made up of diabetic rats (DRs) and diabetic rats with CN (DRs?+?CN). All groups except the HRs received 50?mg/kg/day of streptozotocin (STZ). The DRs?+?CN group was induced by administering 1.5?mg/kg of intravenous radiocontrast dye on the 35th day. Results: We observed increased IL-33 in the kidney tissue following induction of CN in the DRs. The DRs showed moderate immunopositivity, and the DRs?+?CN showed severe immunopositivity for caspase-3, cleaved caspase-3, caspase-8, caspase-9, LC3B, and Beclin-1 in tubular cells and glomeruli. The DRs also showed moderate immunopositivity in tubular cells, and the DRs?+?CN group showed severe immunopositivity for IL-33 in tubular cells. Increased caspase-3 was found in both glomeruli and tubuli; however, we could not demonstrate IL-33 in glomeruli. This could be secondary to inactivation of IL-33 via increased caspase-3 activity. Conclusion: The release of IL-33 from necrotic cells might induce autophagy, which can further balance the effects of increased apoptosis secondary to CN in DKD.     

Protective effect of CDDO-ethyl amide against high-glucose-induced oxidative injury via the Nrf2/HO-1 pathway

Background Context

Intervertebral disc degeneration (IDD) is the main cause of low back pain, and nucleus pulposus (NP) cell apoptosis is an important risk factor of IDD. However, the molecular mechanism of this disease remains unknown.

Glucosamine protects nucleus pulposus cells and induces autophagy via the mTOR‐dependent pathway

Although glucosamine has been suggested to be effective in the treatment of osteoarthritis, its effect on disc degeneration remains unclear. We sought to explore whether glucosamine can activate autophagy in rat nucleus pulposus (NP) cells and protect cells treated with IL‐1β or hydrogen peroxide (H₂O₂). Autophagy in cells was examined by detecting for LC3, Beclin‐1, m‐TOR, and p70S6K, as well as by analyzing autophagosomes. To inhibit autophagy, 3‐methyladenine (3‐MA) was used. In the cells treated with IL‐1β, the levels of Adamts‐4, Mmp‐13, aggrecan, and Col2a1 were analyzed by real‐time PCR and immunofluorescence. Apoptosis was analyzed by TUNEL. Cell senescence under H₂O₂ was revealed by SA‐β‐Gal staining. Glucosamine could activate autophagy in a dose‐dependent manner within 24 h and inhibit the phosphorylation of m‐TOR and p70S6K. Autophagy in IL‐1β or H₂O₂‐treated cells was increased by glucosamine. Glucosamine attenuated the decrease of aggrecan and prevented the apoptosis of the NP cells induced by IL‐1β, whereas 3‐MA partly reversed these effects. The percentage of SA‐β‐Gal‐positive cells induced by H₂O₂ treatment was decreased by glucosamine, accompanied by the decline of p70S6K phosphorylation. Glucosamine protects NP cells and up‐regulates autophagy by inhibiting the m‐TOR pathway, which might point a potential therapeutic agent for disc degeneration. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1532–1542, 2014.