Cordycepin prevented IL-β-induced expression of inflammatory mediators in human osteoarthritis chondrocytes

Purpose

Cordycepin, a nucleoside derivative isolated from Cordyceps, has been reported to exert anti-inflammatory, antitumor, antidiabetic and renoprotective effects. Osteoarthritis (OA) is a degenerative joint disease with an inflammatory component that drives the degradation of cartilage extracellular matrix. This study aimed to assess the effects of cordycepin on human OA chondrocytes.

Methods

In this study, human OA chondrocytes were pretreated with cordycepin at 10, 50 or 100 μM and subsequently stimulated with interleukin-1β (IL-1β) (5 ng/ml) for 24 h. Production of prostaglandin E₂ (PGE2) and nitric oxide (NO) were evaluated by the Griess reaction and an enzyme-linked immunosorbent assay (ELISA). Gene expression of matrix metalloproteinase (MMP)-13, IL-6, inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) was measured by real-time polymerase chain reaction (PCR). MMP-13 and IL-6 proteins in culture medium were determined using cytokine-specific ELISA. Western immunoblotting was used to analyse the iNOS and COX-2 protein production in culture medium. Nuclear factor kappa-B (NF-κB) activity regulation was explored using Western immunoblotting.

Results

Pretreatment with cordycepin significantly inhibited the production of PGE2 and NO induced by IL-1β. Cordycepin also significantly decreased the IL-1β-stimulated gene expression and production of MMP-13, IL-6, iNOS and COX-2 in OA chondrocytes. Pretreatment with cordycepin attenuated IL-1β-induced activation of NF-κB by suppressing degradation of its inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκB-α) in the cytoplasm.

Conclusions

We show for the first time the anti-inflammatory activity of cordycepin in human OA chondrocytes. Thus, with this unique profile of actions, cordycepin may prove to be a potentially attractive and new therapeutic/preventive agent for OA.     

MiR-702-3p inhibits the inflammatory injury in septic H9c2 cells by regulating NOD1

BackgroundCardiac insufficiency is a common complication of sepsis and septic shock and is the most common cause of death in critically ill patients. Recent studies have found that microRNAs (miRNAs) play a potential role in sepsis as markers, but little is known about their functional effects on sepsis-induced cardiomyopathy (SIC).ObjectiveThis study is designed to explore the possible role and underlying mechanisms of miR-702-3p in septic cardiomyopathy.MethodsAs expected, H9c2 cells were induced with lipopolysaccharide (LPS) to construct the model of septic cardiomyopathy. The expression of miR-702-3p was detected by qRT-PCR assay and those of IL-1β, IL-6 and TNF-α by ELISA assay. The viability, proliferation and apoptosis of LPS-treated H9c2 cells were determined by CCK-8, EdU, flow cytometry and western blot assays. Moreover, Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) was predicted and confirmed as a direct target of miR-702-3p by TargetScan, miRwalk and miRDB prediction and dual-luciferase reporter gene assays.ResultsWhile LPS can weaken the viability of H9c2 cells, miR-702-3p enhances that of LPS-treated H9c2 cells by inhibit the expressions of TNF-α, IL-6, IL-1β. We found NOD1 is a target gene of miR-702-3p, and over-expression of NOD1 restores the inhibitory effects of miR-702-3p on the LPS-treated H9c2 cells.ConclusionMiR-702-3p played an important role in the pathogenesis of sepsis cardiomyopathy via targeting NOD1, suggesting that miR-702-3p may be a potential new target for the treatment of SIC.     

长链非编码RNA MEG3靶向下调miR-21对IL-1β诱导的软骨细胞凋亡及炎症反应的影响

目的探究长链非编码RNA母系表达基因3(MEG3)靶向miR-21的作用对白细胞介素1β(IL-1β)诱导的软骨细胞凋亡及炎症反应的影响及其作用机制。方法将细胞分为cTRL组、IL-1β组、LV-MEG3组、miR-21 mimic组和LV-MEG3+mimic组,用IL-1β处理软骨细胞后,加入对应的慢病毒或miRNA mimic处理细胞。RT-PCR检测MEG3、miR-21、基质金属蛋白酶13(MMP-13)、II型胶原蛋白(Collagen II)、聚蛋白聚糖(Aggrecan)基因表达水平,Hoechst检测细胞凋亡,Western blot检测活化半胱天冬酶3(cl-Caspase-3)、cl-Caspase-9、MMP-13、Collagen II、Aggrecan蛋白表达水平和p65、信号传导及转录激活因子3(STAT3)磷酸化比率,试剂盒检测丙二醛(MDA)、乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、肿瘤坏死因子α(TNF-α)、IL-6、IL-10水平,免疫荧光检测p65的核定位情况。结果与cTRL组比较,IL-1β组miR-21表达,细胞凋亡率,MMP-13基因表达,cl-Caspase-3、cl-Caspase-9、MMP-13蛋白表达,MDA、LDH、TNF-α、IL-6水平,p65和STAT3磷酸化比率,p65核内信号水平升高,MEG3表达,Collagen II、Aggrecan基因和蛋白表达,SOD、GSH、IL-10水平降低;与IL-1β组比较,LV-MEG3组miR-21表达,细胞凋亡率,MMP-13基因表达,cl-Caspase-3、cl-Caspase-9、MMP-13蛋白表达,MDA、LDH、TNF-α、IL-6水平,p65和STAT3磷酸化比率,p65核内信号水平降低,MEG3表达水平,Collagen II、Aggrecan基因和蛋白表达水平,SOD、GSH、IL-10水平升高;miR-21 mimic组各项检测指标的变化与LV-MEG3组相反;与miR-21 mimic组比较,LV-MEG3+mimic组miR-21表达,细胞凋亡率,MMP-13基因表达,MMP-13、cl-Caspase-3、cl-Caspase-9蛋白表达,MDA、LDH、TNF-α、IL-6水平,p65和STAT3磷酸化比率,p65核内信号水平降低,MEG3表达水平,Collagen II、Aggrecan基因和蛋白表达,SOD、GSH、IL-10水平升高。结论MEG3可下调miR-21表达,从而抑制IL-1β诱导的软骨细胞凋亡,缓解炎症反应,其作用机制可能与抑制NF-κB信号通路激活有关。     

CircHIPK3 alleviates inflammatory response and neuronal apoptosis via regulating miR-382-5p/DUSP1 axis in spinal cord injury

BackgroundSpinal cord injury (SCI) is one of the serious neurological diseases with high morbidity which may be treated with hematopoietic stem cell (HSC) transplants. Circular RNAs (circRNAs) play vital roles in SCI. The study aimed to reveal the function and mechanism of circRNA homeodomain interacting protein kinase 3 (HIPK3) in SCI.MethodsSCI model in vitro was established by treating neuronal cells AGE1.HN with oxygen-glucose deprivation (OGD) and CoCl2. The levels of circHIPK3, miR-382-5p and dual specificity phosphatase 1 (DUSP1) were examined using quantitative real-time PCR (qRT-PCR) or western blot assay. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors (IL-6 and TNF-α). Cell proliferation and apoptosis were evaluated by 5′-ethynyl-2′-deoxyuridine (EdU) assay and flow cytometry. Caspase-3 Colorimetric Assay Kit was used to detect aaspase-3 activity. The interactions among circHIPK3, miR-382-5p and DUSP1 were confirmed by dual-luciferase reporter and RNA immunoprecipitation assays.ResultsCircHIPK3 and DUSP1 were down-regulated, while miR-382-5p was up-regulated in OGD-induced AGE1.HN cells. Overexpression of circHIPK3 suppressed inflammatory response and cell apoptosis and promoted proliferation in OGD-induced AGE1.HN cells by sponging miR-382-5p. CircHIPK3 regulated DUSP1 expression by targeting miR-382-5p. MiR-382-5p inhibition hindered inflammatory response of IL-6 and TNF-α and neuronal apoptosis and promoted apoptosis via targeting DUSP1.ConclusionCircHIPK3 overexpression alleviated OGD-induced AGE1.HN cell inflammatory response and neuronal apoptosis via regulating miR-382-5p/DUSP1 axis, indicating that circHIPK3 might be a promising therapeutic target for SCI.     

七叶皂苷A在小鼠软骨细胞及骨关节炎中的作用及相关分子机制

目的:探讨七叶皂苷A(EsA)在小鼠骨关节炎(OA)中的作用及相关分子机制.方法:分离培养C57BL/6小鼠原代软骨细胞,将细胞用10 mg/L白细胞介素(IL)-1β及不同浓度(10、50、100μmol/L)EsA进行处理后,CCK-8检测细胞活性,ELISA法检测细胞培养液上清中IL-6和肿瘤坏死因子-α(TNF...     

Glucosamine inhibits IL-1beta-induced NFkappaB activation in human osteoarthritic chondrocytes

OBJECTIVE: Glucosamine sulfate (GS) is a commonly used drug for the treatment of osteoarthritis. The mechanism of the action of this drug does, however, remain to be elucidated. In human osteoarthritic chondrocytes (HOC) stimulated with a proinflammatory cytokine, we studied whether GS could modify the NFkappaB activity and the expression of COX-2, a NFkappaB-dependent gene. METHODS: Using HOC in culture stimulated with interleukin-1 beta (IL-1beta), the effects of GS on NFkappaB activation, nuclear translocation of NFkappaB/Rel family members, COX-1 and COX-2 expressions and syntheses and prostaglandin E2 (PGE2) concentration were studied. RESULTS: GS significantly inhibited NFkappaB activity in a dose-dependent manner, as well as the nuclear translocation of p50 and p65 proteins. Furthermore, GS-preincubated IL-1beta-stimulated HOC showed an increase in IkappaBalpha in the cell cytoplasm in comparison with HOC incubated with IL-1beta alone. GS also inhibited the gene expression and the protein synthesis of COX-2 induced by IL-1beta, while no effect on COX-1 synthesis was seen. GS also inhibited the release of PGE2 to conditioned media of HOC stimulated with IL-1beta. CONCLUSIONS: GS inhibits the synthesis of proinflammatory mediators in HOC stimulated with IL-1beta through a NFkappaB-dependent mechanism. Our study further supports the role of GS as a symptom- and structure-modifying drug in the treatment of OA.     

Inhibition of microRNA-19a-3p alleviates the neuropathic pain (NP) in rats after chronic constriction injury (CCI) via targeting KLF7

Background/purposeNeuropathic pain(NP) is derived from the dysfunctions of nerve system. The current research is to explore the impact and mechanism of miR-19a-3p in neuropathic pain in rats.MethodsThe NP was induced through the chronic constriction injury (CCI) surgery in rats. The pro-inflammatory factors (IL-1β, IL-6, TNF-α) in spinal cord tissues from rats were measured using Elisa kits. Moreover, the different levels of thermal hyperalgesia and mechanical allodynia in rats were examined through paw withdrawal latency (PWL) and paw withdrawal threshold (PWT). To investigate into the role of miR-19a-3p and KLF7 in NP of rats, the knockdown of miR-19a-3p alone or along with KLF7 downregulation in rats were achieved through lentivirus injection. The miR-19a-3p and KLF7 expression in spinal cord of rats on Day 3,7,14 after CCI were detected using RT-qPCR. The protein expression of KLF7 were measured by Western blot. Bioinformatics and luciferase assays were used for the prediction and verification of bindings between KLF7 and miR-19a-3p.ResultsCCI surgery caused neuropathic pain in rats with the levels of inflammatory cytokines increased and PWL and PWT decreased. Moreover, miR-19a-3p expression was increased while the protein and mRNA levels were decreased in spinal cord tissues in rats after CCI surgery. In rat microglial cells, miR-19a-3p downregulation could promote the KLF7 in both mRNA and protein expression. In spinal cord tissues of rats, the inhibition of miR-19a-3p enhanced the KLF7 expression. Furthermore, miR-19a-3p downregulation suppressed the IL-1β, IL-6 and TNF-α concentrations, and could decrease the NP but inhibition of KLF7 could partially reverse this in CCI rats.ConclusionmiR-19a-3p inhibition may alleviate NP via KLF7 in CCI rats.     

miR-1287-5p upregulation inhibits the EMT and pro-inflammatory cytokines in LPS-induced human nasal epithelial cells (HNECs)

BackgroundChronic rhinosinusitis is an intractable symptom that influences daily lives of patients. miR-1287-5p was discovered to play a suppressive role in cervical cancer and HBV-related infection.PurposeThis study investigated the potential role of miR-1287-5p in the in-vitro model of chronic rhinosinusitis.MethodsGSE169376 dataset was analyzed and differential miRNAs in nasal mucosa tissues in the chronic rhinosinusitis group were screened out. LPS was used to treat HNECs for 12h, 24h and 48h. Cells underwent LPS treatment after SNAI1 downregulation, miR-1287-5p upregulation or pretreatment of the HMGB1 inhibitor, Glycyrrhizin. RT-PCR was used to measure the RNA expression of miR-1287-5p, SNAI1 and HMGB1. ELISA was used for the detection of IL-6, IL-8, TNF-α changes. Targetscan and starBase were used to predict the targets (SNAI1 and HMGB1) of miR-1287-5p. Dual-luciferase reporter assays were applied to validate this. Western blot was used to analyze the protein changes of Snai1, Vimentin, E-cadherin and HMGB1.ResultsmiR-1287-5p was downregulated in the chronic rhinosinusitis group and decreased after LPS treatment in HNECs. The upregulation of miR-1287-5p inhibited IL-6, IL-8, TNF-α and EMT. miR-1287-5p targeted and inhibited SNAI1 and HMGB1. SNAI1 downregulation led to inhibition in EMT while loss of HMGB1 contributed to the decrease in pro-inflammatory cytokines. Knockdown of SNAI1 decreased HMGB1, resulting in the reduction of pro-inflammatory cytokines while HMGB1 inhibitor reduced SNAI1 and thus suppressed the EMT process.ConclusionmiR-1287-5p downregulation was associated with chronic rhinosinusitis and its upregulation inhibited the EMT and inflammation in LPS-induced HNECs through Snai1/HMGB1 pathway.     

硫酸镁与氯化钠溶液灌洗对创伤性关节炎软骨损伤修复的影响

目的通过股骨髁间钻孔构建兔创伤性关节炎(PTOA)模型,比较相同渗透压下(400 mOsm/L)硫酸镁与氯化钠溶液持续灌洗对PTOA软骨损伤修复的影响。方法对18只新西兰白兔采用股骨髁间钻孔构建PTOA模型,随机分为PTOA组、氯化钠组和硫酸镁组,每组6只。ELISA检测关节腔积液白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、Ⅱ型胶原蛋白(CollagenⅡ)的表达,qPCR检测滑膜组织IL-1β、TNF-α、金属蛋白酶-3(MMP-3)基因表达。行软骨切片染色组织学观察及骨关节炎(OA)评分。结果关节腔积液IL-1β、TNF-α、CollagenⅡ分泌硫酸镁组少于PTOA组和氯化钠组,滑膜组织IL-1β、TNF-α和MMP-3 mRNA水平硫酸镁组低于PTOA组和氯化钠组;软骨组织切片OA评分硫酸镁组低于PTOA组和氯化钠组,差异均有统计学意义(P<0.05)。结论与相同渗透压下氯化钠溶液相比,硫酸镁溶液有利于减轻关节内炎症,减少CollagenⅡ及蛋白聚糖丢失,对软骨退变起到保护作用,有利于延缓PTOA病情进展。     

Pulsed Electromagnetic Field Modulates Tendon Cells Response in IL-1β-Conditioned Environment

Strategies aiming at controlling and modulating inflammatory cues may offer therapeutic solutions for improving tendon regeneration. This study aims to investigate the modulatory effect of pulsed electromagnetic field (PEMF) on the inflammatory profile of human tendon-derived cells (hTDCs) after supplementation with interleukin-1β (IL-1β). IL-1β was used to artificially induce inflammatory cues associated with injured tendon environments. The PEMF effect was investigated varying the frequency (5 or 17 Hz), intensity (1.5, 4, or 5 mT), and duty-cycle (10% or 50%) parameters to which IL-1β-treated hTDCs were exposed to. A PEMF actuation with 4 mT, 5 Hz and a 50% duty cycle decreased the production of IL-6 and tumor necrosis factor-α (TNF-α), as well as the expression of TNFα, IL-6, IL-8, COX-2, MMP-1, MMP-2, and MMP-3, while IL-4, IL-10, and TIMP-1 expression increased. These results suggest that PEMF stimulation can modulate hTDCs response in an inflammatory environment holding therapeutic potential for tendon regenerative strategies. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:160–172, 2020     

IL-1β mediates NGF and COX-2 expression through transforming growth factor-activating kinase 1 in subacromial bursa cells derived from rotator cuff tear patients

BackgroundIncreased interleukin (IL)-1β expression in the subacromial bursa (SAB) is associated with severe pain in rotator cuff tears (RCTs). Additionally, transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is essential for cytokine-mediated cascades. TAK1 also regulates the expression of pain-associated molecules such as cycloxygenase-2 (COX-2) and nerve growth factor (NGF) in synovial fibroblasts; however, this regulation in the SAB is not fully understood.MethodsSAB samples were harvested from 18 subjects with RCTs. The expression and localization of NGF and COX-2 was determined using polymerase chain reaction (PCR) analysis and immunohistochemistry. Regulation of COX-2 and NGF by IL-1β in subacromial bursa cells (SABCs) was investigated by culturing and stimulating SABCs with vehicle control (culture medium), 50 ng/ml recombinant human IL-1β (rhIL1-β), 50 ng/ml rhIL-1β and 10 μM celecoxib (COX-2 inhibitor), or 10 μM prostaglandin E2 (PGE2) for 24 h. The effects of TAK1 inhibition on rhIL-1β stimulation were determined by culturing and treating SABCs with control, 50 ng/ml rhIL-1β, or 50 ng/ml rhIL-1β and 10 μM (5Z)-7-oxozeaenol (TAK1 inhibitor) for 24 h. NGF and COX-2 mRNA expression was monitored using quantitative PCR.ResultsCOX-2 and NGF mRNA expression was observed in all SAB specimens. Immunohistochemical analysis showed that COX-2-positive cells were in the lining and sublining layers. NGF-positive cells were observed in the sublining layer. rhIL-1β treatment significantly increased NGF and COX-2 mRNA levels compared with control cells. The COX-2 inhibitor did not suppress rhIL-1β-induced NGF expression, and PGE2 stimulation did not alter NGF mRNA expression. In contrast, the TAK1 inhibitor significantly reduced rhIL-1β-stimulated COX-2 and NGF mRNA expression.ConclusionIL-1β regulates the expression of NGF and COX-2, pain-related molecules in the SAB, through TAK1. Therefore, TAK1 may be one potential therapeutic target for reducing pain in patients with RCTs.     

肿瘤坏死因子α对人腹膜间皮细胞基质金属蛋白酶及其组织抑制物表达的影响

目的 观察肿瘤坏死因子α（TNF-α）对人腹膜间皮细胞(HPMC) 基质金属蛋白酶（MMP）2、MMP-9及其组织抑制物（TIMP）2、TIMP-1 mRNA和Ⅰ型胶原表达的影响，同时观察TNF-α、TGF-β、IL-1单独或协同作用对HPMC的MMP-9活性的影响。 方法 采用半定量RT-PCR法测定细胞MMP-2、MMP-9、TIMP-2及TIMP-1 mRNA 的表达。采用Biotrak MMP-9 活性检测系统来精确定量测定MMP-9活性及MMP-9原的含量。ELISA法检测Ⅰ型胶原蛋白的表达。 结果 TNF-α(1~10 μg/L)分别刺激HPMC 4、16、24及48 h后， HPMC的MMP-9 mRNA表达显著上调，为基础的2.3～4.9倍（P ＜ 0.05），呈时间依赖性。TNF-α（1 μg/L）作用48 h后显著下调TIMP-1、TIMP-2 mRNA 表达，为基础的77.2％、61.3％（P ＜ 0.05），而MMP-2 mRNA表达没有显著变化。TNF-α+TGF-β１ （1~10 μg/L）、 TNF-α+TGF-β1+IL-1(1~10 μg/L)和TNF-α(5~10 μg/L)刺激HPMC 24 h后，促其分泌MMP-9 的作用最为明显。同时，TNF-α明显上调Ⅰ型胶原蛋白表达（P ＜ 0.05）。 结论 TNF-α 明显上调HPMC的MMP-9 mRNA的表达和MMP-9 的活性；联合其他细胞因子后促MMP-9分泌的作用更明显。TNF-α单独或协同其他因子在腹膜纤维化的过程中可能发挥了重要作用。     

Ginsenoside Rb1 prevents interleukin-1 beta induced inflammation and apoptosis in human articular chondrocytes

Purpose

Osteoarthritis (OA) is an age-related joint disease that is characterised by the degeneration of articular chondrocytes. Ginsenosides, the most important pharmacological ingredients of ginseng, have been proven to provide effective therapy for neurodegenerative diseases and can inhibit cell apoptosis. We investigated whether ginsenoside Rb1 can modulate inflammation and apoptosis in human chondrocytes.

Methods

Chondrocytes were isolated from OA patients undergoing total knee replacement surgery. Apoptosis was assessed by TUNEL (terminal deoxyribonucleotide transferasemediated dUTP nick end-labelling)-positive staining. Levels of PGE2 and NO²- were detected by ELISA. Gene expression levels were measured for type II collagen (Col2A1), aggrecan, MMP-13, COX-2, iNOS, caspase-3, and PARP.

Results

The results showed that TUNEL-positive staining chondrocytes were decreased by Rb1 compared with IL-1β. Both 10 or 100 μg/ml Rb1 inhibited the effect of IL-1β on chondrocytes by decreasing levels of PGE2, NO²-, MMP-13, COX-2, iNOS, caspase-3 and PARP and increasing aggrecan and Col2A1 gene expression levels, to block IL-1β-induced cell inflammation and apoptosis.

Conclusions

The results suggest that Rb1 possesses potential anti-inflammatory and anti-apoptotic properties in human chondrocytes, possibly by binding to oestrogen receptors to exert its pharmacological effects.     

微小RNA-27b-3p与基质金属蛋白酶13在人软骨细胞的对应关系

目的探讨微小RNA-27b-3p(miR-27b-3p)与基质金属蛋白酶-13(MMP-13)在人软骨细胞表达及其靶向对应关系。 方法运用蛋白质印迹法(WB)与实时定量PCR技术(qRT-PCR)明确miR-27b-3p与MMP13在正常和骨关节炎(OA)人软骨细胞的表达。利用不同浓度的白介素(IL)1β干预原代人软骨细胞24 h,或利用不同时间点的IL-1β(10 ng/ml)干预原代人软骨细胞。利用原位杂交、转染及双荧光素酶报告技术确定miR-27b-3p与MMP13的靶向对应关系;结合运用核转录因子-κB(NF-κB)和丝裂原活化蛋白激酶(MAPK)信号通路抑制剂评估其作用机制。两组资料比较采用独立样本t检验,多组资料比较采用单因素方差分析,LSD法多重比较检验。 结果WB、qRT-PCR和原位杂交检测结果显示,与正常软骨相比,OA软骨中miR-27b-3p表达降低(t＝5.07,P<0.01),MMP13表达升高(t＝－6.31,P<0.01)。IL-1β干扰后的结果显示miR-27b-3p表达降低(F＝129.54,P<0.05),MMP-13表达升高(F＝394.50,P<0.05)。通过TargetScan数据库和荧光素酶报告基因检测结果分析,野生型-MMP13组荧光素酶活性降低(F＝55.27,P<0.001),突变型-MMP-13荧光素酶活性变化没有统计学意义(P＝0.654)。利用特异性MAPK信号抑制剂和NF-kB抑制剂干预IL-1β诱导软骨细胞模型结果提示,与对照组相比,抑制剂组的MMP13表达水平降低(F＝28.43,P<0.001),miR-27b-3p表达水平增高(F＝35.04,P<0.001)。 结论miR-27b-3p在OA软骨细胞呈现低表达,并负向调控MMP13的表达,其作用机制可能是通过NF-κB和MAPK信号通路,这结果提示这miR-27b-3p可能作为OA诊断与治疗的潜在靶点。     

Mechanism of miR-365 in regulating BDNF-TrkB signal axis of HFD/STZ induced diabetic nephropathy fibrosis and renal function

Purpose

Diabetic nephropathy (DN) is one of the most serious complications of diabetes that leads to decline of renal function. Although numerous studies have revealed that microRNAs (miRNAs) play essential roles in the progression of DN, whether miR-365 is involved remains elusive.

Methods

The successful construction of DN model was confirmed by ELSIA, hematoxylin–eosin (HE) and Masson staining assay. The expression of miR-365 was detected through RT-qPCR. The levels of BDNF, p-TrkB, α-smooth muscle actin (SMA), collagen IV (Col.IV), transforming growth factor-β1 (TGF-β1), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6) were evaluated by western blot, IF or ELISA assays. Luciferase reporter assay was used to detect the interaction between miR-365 and BDNF.

Results

The DN mice model was induced by streptozotocin (STZ). Then miR-365 expression was found to upregulate in tissues of DN rat. Furthermore, elevated expression of miR-365 was found in high glucose (HG)-treated HK-2 cells. Silencing of miR-365 suppressed the accumulation of ECM components and secretion of inflammatory cytokines in HK-2 cells. In addition, it was demonstrated that miR-365 could target BDNF. The protein levels of BDNF and p-TrkB were negatively regulated by miR-365 in HK-2 cells. Moreover, inhibition of miR-365 suppressed the levels of SMA, Col.IV, TGF-β1, TNF-α, and IL-6, indicating the renal fibrosis was inhibited by miR-365 knockdown.

Conclusion

MiR-365 could regulate BDNF-TrkB signal axis in STZ induced DN fibrosis and renal function. The results of the current study might provide a promising biomarker for the treatment of DN in the future.

     

miR-31-5p靶向Notch1调节骨关节炎软骨细胞增殖和凋亡的作用

目的明确miR-31-5p在骨关节炎软骨细胞中的作用。方法采用Hulth法进行大鼠骨关节炎造模,并分离原代软骨细胞,白细胞介素(interleukin,IL)-1β诱导体外模型,检测miR-31-5p的表达变化;进一步通过在线数据库预测miR-31-5p靶点并验证,细胞增殖检测试剂盒(cell counting kit-8,CCK-8)和流式细胞术检测miR-31-5p对原代软骨细胞增殖和凋亡的影响。结果骨关节炎大鼠中miR-31-5p表达下调;miR-31-5p mimics促进软骨细胞增殖,而miR-31-5p inhibitor诱导软骨细胞凋亡增加;此外,miR-31-5p inhibitor促进IL-6和基质金属蛋白酶13(matrix metalloprotein,MMP-13)表达增加;荧光素酶报告基因实验证实Notch1是miR-31-5p的直接靶点,Notch1过表达质粒会部分抵消miR-31-5p mimics对软骨细胞的增殖保护作用。结论miR-31-5p下调表达介导骨关节炎软骨细胞增殖受限和凋亡发生,可能参与骨关节炎的发病。     

Metformin alleviated EMT and fibrosis after renal ischemia–reperfusion injury in rats

Purpose The purpose of this study is to assess the potential effects of metformin on the development of EMT and tubulointerstitial fibrosis 12 weeks after acute renal ischemia–reperfusion. Methods Male Sprague–Dawley rats were randomly assigned to four groups: Sham, IRI, transient administration of metformin (TAM), and continuous administration of metformin (CAM). Metformin was administered i.p. at a dose of 125?μg kg?^??1 d^???1 3 d prior to suffering from IRI (TAM), or from 3 d before suffering from IRI to 12 weeks after reperfusion (CAM). Renal function, histology, and expressions of IL-6, TNF-α, α-SMA, TGF-β1, Vimentin, and E-cadherin were analyzed. Results Tubulointerstitial fibrosis worsened further in IRI, accompanied by the increased expressions of interleukin-6, TNF-α, α-SMA, TGF-β1, Vimentin, and loss of E-cadherin. Although there were no significant differences between IRI and TAM (p?>?0.05). Compared with the IRI, expressions of IL-6, TNF-α, α-SMA, TGF-β1, and Vimentin were reduced and the expression of E-cadherin was restored in CAM (p?0.05). CAM also significantly promoted activation of AMPK (p?0.05), which showed no difference among Sham, IRI, and TAM (p?>?0.05). Conclusions CAM significantly attenuated tubulointerstitial fibrosis and EMT in rats, potentially via activation of AMPK and down-regulation of TGF-β1.     

Characterization of opticin digestion by proteases involved in osteoarthritis development

ObjectiveOpticin is a class III member of the small leucine-rich repeat proteoglycan (SLRP) family, produced in articular joint tissues. In normal and osteoarthritic (OA) cartilage, opticin is degraded. This study aimed to assess whether human cartilage opticin is degraded by the main proteases involved in OA pathophysiology, and to determine the protease cleavage sites of this SLRP.MethodsWe analyzed the proteolytic activity of matrix metalloproteinases (MMPs)-1, -2, -3, -7, -8 and -9, and ADAMTS-4 and -5 on proteoglycan extracts from normal and moderately fibrillated OA human cartilage, and on recombinant human opticin. Opticin degradation was analyzed by Western blotting and cleavage sites were determined by sequence analysis.ResultsAll eight proteases digested opticin from proteoglycan extracts from both normal and OA samples, as well as recombinant human opticin, MMP-2 and MMP-7 are the proteases that degrade recombinant human opticin most efficiently. The opticin cleavage site determined for these MMPs was between the glycosylation and leucine-rich repeat domains. MMP-7 had two additional digestion sites near the N-terminal end of opticin.ConclusionOpticin is a substrate for several MMPs and aggrecanases involved during OA cartilage degradation, and seems to be a preferential substrate for MMP-7. The role of opticin in cartilage degeneration could be related to decreased levels of intact opticin, followed by its proteolytic degradation, which in turn may stimulate some of the modifications observed in the OA cartilage, such as neovascularisation and changes in the extracellular matrix.