Role of osteoprotegerin/receptor activator of nuclear factor kappa B/receptor activator of nuclear factor kappa B ligand axis in nonalcoholic fatty liver disease

Concomitantly with the increase in the prevalences of overweight/obesity, nonalcoholic fatty liver disease(NAFLD) has worldwide become the main cause of chronic liver disease in both adults and children. Patients with fatty liver display features of metabolic syndrome(Met S), like insulin resistance(IR), glucose intolerance, hypertension and dyslipidemia. Recently, epidemiological studies have linked obesity, Met S, and NAFLD to decreased bone mineral density and osteoporosis, highlighting an intricate interplay among bone, adipose tissue, and liver. Osteoprotegerin(OPG), an important symbol of the receptor activator of nuclear factor-B ligand/receptor activator of nuclear factor kappa B/OPG system activation, typically considered for its role in bone metabolism, may also play critical roles in the initiation and perpetuation of obesityrelated comorbidities. Clinical data have indicated that OPG concentrations are associated with hypertension, left ventricular hypertrophy, vascular calcification, endothelial dysfunction, and severity of liver damage in chronic hepatitis C. Nonetheless, the relationship between circulating OPG and IR as a key feature of Met S as well as between OPG and NAFLD remains uncertain. Thus, the aims of the present review are to provide the existent knowledge on these associations and to discuss briefly the underlying mechanisms linking OPG and NAFLD.

Receptor activator of nuclear factorκB ligand/osteoprotegerin axis and vascular calcifications in patients with chronic kidney disease

Vascular calcifications are commonly observed in patients with chronic kidney disease （CKD） and contri-bute to the excessive cardiovascular morbidity and mortality rates observed in these patients populations. Although the pathogenetic mechanisms are not yet fully elucidated, recent evidence suggests a link between bone metabolism and the development and progression of vascular calcifications. Moreover, accumulating data indicate that receptor activator of nuclear factor κB ligand/osteoprotegerin axis which plays essential roles in the regulation of bone metabolism is also involved in extra-osseous bone formation. Further studies are required to establish the prognostic significance of the above biomarkers as predictors of the presence and severity of vascular calcifications in CKD patients and of cardiovascular morbidity and mortality. Moreover, randomized clinical trials are needed to clarify whether inhibition of osteoclast activity will protect from vascular calcifcations.

Effect of Triptolide on Expression of Receptor Activator of Nuclear Factor-κB Ligand in Rat Adjuvant Induced Arthritis

The effect of triptolide （TP） on the expression of receptor activator of nuclear factor-κB ligand （RANKL） and osteoprotegerin （OPG） was explored in rat adjuvant induced arthritis （AA）. AA was induced in Wistar rats. Arthritis rats were treated with TP and methotrexate （MTX） at the onset （day 9） of arthritis. On the peak of arthritis （day 24）, the expression of RANKL and OPG protein in the joints and RANKL mRNA in peripheral blood mononuclear cells （PBMC） was detected. TNF-α and IL-1β levels in peripheral blood were determined. Bone erosion scores were also evaluated. The results showed that bone erosion scores in TP and MTX groups were lower than in AA group （.P〈0.01） ; The expression levels of RANKL in the synovium （P〈0.01） and bone （P〈0.05）, and OPG level in synovium （P〈0.05） were lower in TP group than in AA group （P〈0.05）. In TP group, the expression levels of RANKL mRNA and TNF-α, IL-1β in PBMC were lower than in AA group （all P〈0.01）. It was concluded that TP could inhibit rat adjuvant arthritis bone erosion by suppressing the expression of RANKL.

Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human periodontal ligament cells

Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering RNA(siRNA)eukaryotic expression vector targeted transforming growth factor βⅡ receptor(TGF-β RⅡ)was constructed and transfected into T cells.HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide(LPS)and baicalin.The obtained solution was divided into six groups according to the components(group Ⅰ:HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ:HPDL cells+LPS+T cells transfected with siRNA1;group Ⅲ:HPDL cells+LPS+T cells+baicalin;group Ⅳ:HPDL cells+LPS+T cells;group Ⅴ:HPDL cells+baicalin;group Ⅵ:HPDL cells)and was cultured for 48 hours.RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells.Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ(P<0.01)and higher than that in group Ⅲ(P<0.01);The ratio of RANKL/OPG in group Ⅲ was lower than that in group Ⅳ(P<0.01);the ratio of RANKL/OPG in group Ⅳ was higher than that in group Ⅵ(P<0.01);the ratio of RANKL/OPG in group Ⅴ was lower than that in group Ⅵ(P<0.05).Conclusion ① Baicalin could decrease the ratio of RANKL/OPG in HPDL cells.② The TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.③ Baicalin acts not only through TGF-β to regulate RANKL/OPG in HPDL cells,but also through other pathways.

Transcription factors specificity protein and nuclear receptor 4A1 in pancreatic cancer

Specificity protein(Sp)transcription factors(TFs)Sp1,Sp3 and Sp4,and the orphan nuclear receptor 4A1(NR4A1)are highly expressed in pancreatic tumors and Sp1 is a negative prognostic factor for pancreatic cancer patient survival.Results of knockdown and overexpression of Sp1,Sp3 and Sp4 in pancreatic and other cancer lines show that these TFs are individually pro-oncogenic factors and loss of one Sp TF is not compensated by other members.NR4A1 is also a prooncogenic factor and both NR4A1 and Sp TFs exhibit similar functions in pancreatic cancer cells and regulate cell growth,survival,migration and invasion.There is also evidence that Sp TFs and NR4A1 regulate some of the same genes including survivin,epidermal growth factor receptor,PAX3-FOXO1,α5-andα6-integrins,β1-,β3-andβ4-integrins;this is due to NR4A1 acting as a cofactor and mediating NR4A1/Sp1/4-regulated gene expression through GC-rich gene promoter sites.Several studies show that drugs targeting Sp downregulation or NR4A1 antagonists are highly effective inhibitors of Sp/NR4A1-regulated pathways and genes in pancreatic and other cancer cells,and the triterpenoid celastrol is a novel dual-acting agent that targets both Sp TFs and NR4A1.

Imbalance of osteoprotegerin/receptor activator of nuclear factor-κB ligand and oxidative stress in patients with obstructive sleep apnea-hypopnea syndrome

Background:Obstructive sleep apnea-hypopnea syndrome (OSAHS) is associated with a higher prevalence of osteoporosis.However,the underlying mechanisms linking OSAHS with bone loss are still unclear.The aim of this study was to investigate the changes of receptor activator of nuclear factor-κB ligand (RANKL,an osteoclastogenesis-promoting factor) and osteoprotegerin (OPG,the decoy receptor for RANKL),oxidative stress and bone metabolism markers in OSAHS,in order to understand the potential mechanisms underlying bone loss in OSAHS patients.Methods:Forty-eight male patients with OSAHS,confirmed by polysomnography (PSG) study,were enrolled.Twenty male subjects who were confirmed as not having OSAHS served as the controls.The subjects’bone mineral density (BMD) was assessed in lumbar spine and femoral neck using dual-energy X-ray absorptiometry (DXA).Blood samples were collected from all subjects for measurement of RANKL,OPG,the bone formation marker bone-specific alkaline phosphatase (BAP),the bone resorption marker tartrate-resistant acid phosphatase 5b (TRAP-5b),and total antioxidant capacity (TAOC).Results:The BMD and the T-score of the femoral neck and the lumbar spine were significantly lower in OSAHS patients as compared to the control group (P< 0.05).The serum level of BAP was significantly decreased in the OSAHS group (15.62 ± 5.20 μg/L) as compared to the control group (18.83 ± 5.50 μg/L,t= -2.235,P< 0.05),while the levels of TRAP-5b did not differ between the two groups (t= -1.447,P> 0.05).The serum level of OPG and the OPG/RANKL ratio were lower in the OSAHS group compared to the control group (bothP< 0.05).TAOC level was also decreased significantly in the OSAHS group (P< 0.05).Correlation analysis showed that the TAOC level was positively correlated with BAP in the OSAHS group (r= 0.248,P= 0.04),but there were no correlations between TAOC and the BMD or the T-scores.The correlations between the level of OPG (or the OPG/RANKL ratio) and BMD or TAOC did not reach significance.Conclusion:In OSAHS patients,lower levels of TAOC were associated with decreased bone formation,suggesting a role of oxidative stress in bone loss,while the role of OPG/RANKL imbalance in bone metabolism in OSAHS needs further evaluation .

Cannabinoid receptor-2 selective antagonist negatively regulates receptor activator of nuclear factor kappa B ligand mediated osteoclastogenesis

Background The cannabinoid receptor-2 （CB2） is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist （AM630） on receptor activator of nuclear factor kappa B （RANK） ligand （RANKL）induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.Methods RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase （TRAP） staining using a commercial kit. Total ribonucleic acid （RNA）was isolated and real-time reverse transcriptase-polymerase chain reaction （RT-PCR） was done to examine the expression of RANK, cathepsin K （CPK） and nuclear factor kappa B （NF-κB）. The extracellular signal-regulated kinase （ERK）,phosphorylation of ERK （P-ERK） and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazoliumbromide （MTT） assay.Results AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of 〉100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.Conclusion AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.

Role of Osteoprotegerin and Receptor Activator of Nuclear Factor-kB Ligand in Bone Loss Related to Advanced Chronic Obstructive Pulmonary Disease

Background： Osteoporosis is a common complication of chronic obstructive pulmonary disease （COPD）. Recent clinical and biological researches have increasingly delineated the biomolecular pathways of bone metabolism regulation in COPD. We extended this work by examining the specific association and potential contribution of the osteoprotegerin （OPG）/receptor activator of nuclear factor-riB ligand （RANKL） axis to the pathogenesis of osteoporosis in advanced COPD. The aim of this study was to assess the relationships of serum OPG, RANKL, and tumor necrosis factor-alpha （TNF-a） with bone turnover in men with very severe COPD. Methods： Pulmonary function, T-score at the lumbar spine （LS） and femoral neck （FN）, serum OPG, RANKL, soluble receptor of tumor necrosis factor-alpha-1 and 11 （sTNFR-I, sTNFR-II）, osteocalcin （OC）, and β-CrossLaps （βCL） levels were measured in 45 men with very severe stage COPD and 36 male non-COPD volunteers. COPD patients and healthy controls were compared using all independent t-test and Mann-Whitney U-test. The Pearson coefficient was used to assess the relationships between variables. Results： OPG and OC were lower in male COPD patients than in control subjects whereas RANKL, serum ~CL, TN F-o~, and its receptors were higher. OPG directly correlated with forced expiratory volume in I s （FEVI） % predicted （r = 0.46, P 〈 0.005）, OC （r= 0.34, P 〈 0.05）, LS （I.= 0.56, P 〈 0.001 ）, and FN T-score （r= 0.47, P 〈 0.01 ）. In contrast, serum RANKL inversely associated with LS and FN T-score （r = -0.62, P 〈 0.001 and r = -0.48, P 〈 0.001 ） but directly correlated with [3CL （r = 0.48, P 〈 0.001 ）. In addition, OPG was inversely correlated with RANKL （r = -0.39, P 〈 0.01 ）, TNF-a （r = -0.56, P 〈 0.001 ）, and sTNFR-I （r = -0.40, P 〈 0.01 ）. Conelusion： Our results suggest that serum OPG and RANKL levels are inversely associated with bone loss in men with advanced stage COPD.

Receptor activator of nuclear factor kappa B ligand and osteoprotegerin expression in chronic apical periodontitis：possible association with inflammatory cells

Background Receptor activator of nuclear factor kappa B （NF-κB） ligand （RANKL） and osteoprotegerin （OPG） have been recently shown to play important roles in bone resorption. The aim of this study was to investigate the possible association between the expression of bone resorption regulators （RANKL and OPG） and inflammatory cell infiltration in chronic apical periodontitis.Methods The samples of chronic periapical lesions （n=40） and healthy periapical tissues （n=10） were examined for immunohistochemical analysis of RANKL and OPG. Lesion samples were further analyzed for the inflammatory infiltration condition. The inflammatory cell infiltration was scored in relation to immunohistochemical reactivity for CD3, CD20 and CD68.Results The number of RANKL-positive cells and the ratio of RANKL/OPG in chronic apical periodontitis were significantly higher than those in healthy periapical tissues （P＜0.001）. The number of RANKL-positive cells was higher in lesions with severe inflammatory infiltration than in those with light inflammatory infiltration （P＜0.05）. Significantly increased RANKL expression was found with T lymphocytes （CD3＋）, macrophages （CD68＋） and B lymphocytes （CD20＋）infiltration （P＜0.05）. No association was found between the ratio of RANKL/OPG and inflammatory cell infiltration.Conclusions RANKL expression was increased with T, B lymphocytes and macrophages infiltration, respectively in chronic periapical lesions. RANKL appears to be closely related to periapical inflammatory infiltrates. The relative ratio of RANKL/OPG may be a key determinant of RANKL-mediated bone resorption.

Effect of Wenhua Juanbi Recipe(温化蠲痹方) on Expression of Receptor Activator of Nuclear Factor Kappa B Ligand,Osteoprotegerin,and Tumor Necrosis Factor Receptor Superfamily Member 14 in Rats with Collagen-Induced Arthritis

Objective： To study the effect of Wenhua Juanbi Recipe（温化蠲痹方, WJR） on expression of receptor activator of nuclear factor kappa B ligand（RANKL）, osteoprotegerin（OPG）, and tumor necrosis factor receptor superfamily member 14（TNFRSF14, also known as LIGHT） in rats with collagen-induced arthritis（CIA）. Methods： CIA rats were generated by subcutaneous injection of bovine collagen type-Ⅱ at the tail base. Sixty CIA rats were randomly assigned（10 animals/group） to： model, methotrexate（MTX）-treated（0.78 mg/kg body weight）, and WJR-treated（22.9 g/kg） groups. Healthy normal rats（n=10） were used as the normal control. Treatments or saline were administered once daily by oral gavage. Rats were sacrificed at day 28 post-treatment and knee synovium and peripheral blood serum were collected. Toe swelling degree and expression of RANKL, OPG, and LIGHT were determined by Western blot and immunohistochemistry. Results： Compared with the normal group, toe swelling degree was significantly increased in the model group（P〈0.01）. After treatment, toe swelling degree decreased significantly in the WJR and MTX groups compared with the model group（P〈0.01）. Compared with the normal group, expression of RANKL and LIGHT were significantly increased and OPG significantly decreased in peripheral blood and synovium of the model group（P〈0.01）. Conversely, RANKL and LIGHT expression were significantly reduced and OPG increased in the WJR and MTX groups compared with the model group（P〈0.01）. No statistically significant difference existed between WJR and MTX groups. Conclusion： WJR likely acts by reducing RANKL expression and increasing OPG expression, thus inhibiting RANKL/RANK interaction and reducing LIGHT expression, thereby inhibiting osteoclast formation/activation to block bone erosion.

Biochanin A attenuates spinal cord injury in rats during early stages by inhibiting oxidative stress and inflammasome activation

Previous studies have shown that Biochanin A,a flavonoid compound with estrogenic effects,can serve as a neuroprotective agent in the context of cerebral ischemia/reperfusion injury;howeve r,its effect on spinal cord injury is still unclea r. In this study,a rat model of spinal cord injury was established using the heavy o bject impact method,and the rats were then treated with Biochanin A(40 mg/kg) via intrape ritoneal injection for 14 consecutive days.The res ults showed that Biochanin A effectively alleviated spinal cord neuronal injury and spinal co rd tissue injury,reduced inflammation and oxidative stress in spinal cord neuro ns,and reduced apoptosis and pyroptosis.In addition,Biochanin A inhibited the expression of inflammasome-related proteins(ASC,NLRP3,and GSDMD)and the Toll-like receptor 4/nuclear factor-κB pathway,activated the Nrf2/heme oxygenase 1 signaling pathway,and increased the expression of the autophagy markers LC3 Ⅱ,Beclin-1,and P62.Moreove r,the therapeutic effects of Biochanin A on early post-s pinal cord injury were similar to those of methylprednisolone.These findings suggest that Biochanin A protected neurons in the injured spinal cord through the Toll-like receptor 4/nuclear factor κB and Nrf2/heme oxygenase 1 signaling pathways.These findings suggest that Biochanin A can alleviate post-spinal cord injury at an early stage.

Yemazhui(Herba Eupatorii Lindleyani)ameliorates lipopolysaccharide-induced acute lung injury via modulation of the toll-like receptor 4/nuclear factor kappa-B/nod-like receptor family pyrin domain-containing 3 protein signaling pathway and intestinal flor

OBJECTIVE:To investigate the impact of Yemazhui(Herba Eupatorii Lindleyani,HEL)against lipopolysaccharide(LPS)-induced acute lung injury(ALI)and explore its underlying mechanism in vivo.METHODS:The chemical constituents of HEL were analyzed by ultra-high performance liquid chromatographyquadrupole time-of-flight mass spectrometry method.Then,HEL was found to suppress LPS-induced ALI in vivo.Six-week-old male Sprague-Dawley rats were randomly divided into 6 groups:control,LPS,Dexamethasone(Dex),HEL low dose 6 g/kg(HEL-L),HEL medium dose 18 g/kg(HEL-M)and HEL high dose 54 g/kg(HEL-H)groups.The model rats were intratracheally injected with 3 mg/kg LPS to establish an ALI model.Leukocyte counts,lung wet/dry weight ratio,as well as myeloperoxidase(MPO)activity were determined followed by the detection with hematoxylin and eosin staining,enzyme linked immunosorbent assay,quantitative real time polymerase chain reaction,western blotting,immunohistochemistry,and immunofluorescence.Besides,to explore the effect of HEL on ALI-mediated intestinal flora,we performed 16s rRNA sequencing analysis of intestinal contents.RESULTS:HEL attenuated LPS-induced inflammation in lung tissue and intestinal flora disturbance.Mechanism study indicated that HEL suppressed the lung coefficient and wet/dry weight ratio of LPS-induced ALI in rats,inhibited leukocytes exudation and MPO activity,and improved the pathological injury of lung tissue.In addition,HEL reduced the expression of tumor necrosis factoralpha,interleukin-1beta(IL-1β)and interleukin-6(IL-6)in bronchoalveolar lavage fluid and serum,and inhibited nuclear displacement of nuclear factor kappa-B p65(NF-κBp65).And 18 g/kg HEL also reduced the expression levels of toll-like receptor 4(TLR4),myeloid differentiation factor 88,NF-κBp65,phosphorylated inhibitor kappa B alpha(phospho-IκBα),nod-like receptor family pyrin domain-containing 3 protein(NLRP3),IL-1β,and interleukin-18(IL-18)in lung tissue,and regulated intestinal flora disturbance.CONCLUSIONS:In summary,our findings revealed that HEL has a protective effect on LPS-induced ALI in rats,and its mechanism may be related to inhibiting TLR4/NF-κB/NLRP3 signaling pathway and improving intestinal flora disturbance.

Down-regulation of peroxisome proliferator-activated receptor γ coactivator-1α expression in fatty acid-induced pancreatic betacell apoptosis involves nuclear factor-κB pathway

Background Pancreatic beta-cell apoptosis induced by lipotoxicity, to a large extent, contributes to the progression of type 2 diabetes. To investigate the mechanism of free fatty acid induced apoptosis, we aimed to study the effects of palmitic acid （PA） on the apoptosis and peroxisome proliferator-activated receptor y coactivator-1α （PGC-1α） expression in βTC3 cells as well as the possible role of nuclear factor-KB （NF-KB） in this process. Methods Hoechst 33258 was used to detect βTC3 cell apoptosis, which was induced by PA stimulation for 12 hours. PGC-1α expression was analyzed by reverse transcription polymerase chain reaction, IκB kinase β （IKKβ）, IκBα NF-KB-inducing kinase （NIK） and ReI-B expressions were analyzed by Western blotting. MGβ2 was employed to block the endogenous IκBαdegradation before PA administration, and then its effect on PA-inducing cell apoptosis and PGC-1α mRNA expression was analyzed. Results Significant increased cell apoptosis was found at the concentration of 0.5 mmol/L and 1.0 mmol/L PA administration. PA （0.5 mmol/L） could extensively reduced the expression of PGC-1α mRNA. After exposing βTC3 cells to 0.5 mmol/L PA for different time periods （0, 4, 6, 8, 10 and 12 hours）, IKKβ protein expression increased while IκBα NIK and ReI-B protein expression declined in a time-dependent manner. Pretreatment with MGβ2 to inhibit the degradation of IκBα partially prevented the down-regulation of PGC-1α mRNA expression after 12-hour PA treatment in accordance with the decrease of PA induced apoptosis. Conclusions NF-KB canonical pathway was activated in PA-mediated βTC3 cell apoptosis, whereas non-canonical pathway was inhibited. Reduced PGC-1α expression by PA in βTC3 cells could involve the activation of canonical NF-KB pathway, so as to deteriorate the PA induced apoptosis.

黄芪补肾活血汤对芳香化酶抑制剂诱导骨质疏松模型小鼠破骨细胞活性的影响

背景:芳香化酶抑制剂尽管显著提高了激素受体阳性乳腺癌患者的临床获益,但其相关的不良事件——骨质疏松严重影响了患者的生活质量,黄芪补肾活血汤能有效预防芳香化酶抑制剂所致骨质疏松的发生,但是其作用机制尚不清楚。目的:探究黄芪补肾活血汤对芳香化酶抑制剂所致骨质疏松模型小鼠破骨细胞活性的影响及机制。方法:选取60只8周龄C57BL/6J雌性小鼠随机分为假手术组、模型组、黄芪补肾活血汤高、中、低剂量组、阳性对照组各10只,除假手术组外,其余组小鼠均切除双侧卵巢联合皮下注射来曲唑构建绝经后芳香化酶抑制剂所致骨质疏松模型,黄芪补肾活血汤高、中、低剂量组分别给予19.24,9.62,4.81 g/(kg·d)黄芪补肾活血汤进行灌胃(1次/d),阳性对照组给予阿仑膦酸钠5 mg/kg灌胃(1次/周)。给药3个月后,Micro-CT检测胫骨骨密度和骨微结构,对股骨进行苏木精-伊红染色、抗酒石酸酸性磷酸酶染色及免疫组化检测核因子κB受体活化因子配体、骨保护素蛋白表达,ELISA检测血清中Ⅰ型胶原交联羧基端肽、抗酒石酸酸性磷酸酶5b水平。结果与结论:①与假手术组相比,模型组小鼠骨密度显著下降、骨小梁形态疏松断裂、血清中Ⅰ型胶原交联羧基端肽、抗酒石酸酸性磷酸酶5b水平显著上升,表明芳香化酶抑制剂所致骨质疏松模型构建成功;②与模型组相比,黄芪补肾活血汤高、中、低剂量组和阳性对照组小鼠骨密度、骨微结构显著改善,骨小梁形态增粗致密,血清中Ⅰ型胶原交联羧基端肽、抗酒石酸酸性磷酸酶5b水平显著下降,破骨细胞数量减少,核因子κB受体活化因子配体蛋白表达下降,骨保护素蛋白表达升高。结果表明,黄芪补肾活血汤可能调控核因子κB受体活化因子配体/核因子κB受体活化因子/骨保护素信号通路抑制破骨细胞活性,改善骨小梁形态和骨微结构,提高骨密度,进而预防芳香化酶抑制剂所致骨质疏松模型的发生发展。

白藜芦醇激活细胞外信号调节激酶5信号蛋白促进小鼠MC3T3-E1细胞增殖

背景:细胞外信号调节激酶5信号蛋白对生物体的存活不可或缺,白藜芦醇能通过多种途径促进成骨细胞增殖,但其是否能通过细胞外信号调节激酶5信号蛋白调控成骨细胞功能还需进一步验证。目的:探究细胞外信号调节激酶5对MC3T3-E1细胞增殖以及相关分泌蛋白的调控作用,进一步验证白藜芦醇通过激活细胞外信号调节激酶5完成上述过程。方法:小鼠MC3T3-E1前成骨细胞分别用完全培养基、XMD8-92(细胞外信号调节激酶5抑制剂)、表皮生长因子(细胞外信号调节激酶5激活剂)和白藜芦醇单独干预及XMD8-92+表皮生长因子、白藜芦醇+XMD8-92干预后,通过Western blot检测各组细胞内细胞外信号调节激酶5、磷酸化细胞外信号调节激酶5蛋白,增殖相关蛋白Cyclin D1、CDK4、PCNA,以及成骨细胞分泌蛋白骨保护素、核因子κB受体活化因子配体的表达情况,使用细胞免疫荧光染色检测各组细胞外信号调节激酶5、骨保护素和核因子κB受体活化因子配体荧光强度,使用EdU染色检测各组细胞增殖情况。白藜芦醇干预MC3T3-E1细胞的适宜浓度及时间由细胞形态学观察和CCK-8实验确定。结果与结论:①细胞外信号调节激酶5信号蛋白的激活能有效促进MC3T3-E1细胞增殖、上调骨保护素/核因子κB受体活化因子配体比值;②白藜芦醇干预MC3T3-E1细胞的适宜浓度及时间为5μmol/L,24 h;③白藜芦醇可以激活细胞外信号调节激酶5信号蛋白,进而促进成骨细胞增殖,并上调骨保护素/核因子κB受体活化因子配体比值;④研究结果表明,白藜芦醇可以通过激活细胞外信号调节激酶5信号蛋白促进MC3T3-E1细胞增殖,并通过激活细胞外信号调节激酶5信号蛋白上调骨保护素/核因子κB受体活化因子配体比值。

miR-338-3p靶向核因子κB受体活化因子配体影响牙槽骨成骨细胞增殖及凋亡

背景:miR-338-3p能够抑制破骨细胞分化,下调核因子κB受体活化因子配体水平能够促进骨形成。然而miR-338-3p是否通过调控核因子κB受体活化因子配体表达影响牙槽骨成骨细胞增殖、凋亡尚不清楚。目的:探究miR-338-3p靶向核因子κB受体活化因子配体对牙槽骨成骨细胞增殖、凋亡的影响及机制。方法:分离人牙槽骨成骨细胞,对其进行细胞转染及Wnt-C59(Wnt/β-catenin通路抑制剂)处理,分为转染对照组、miR-338-3p组、miR-338-3p+对照组、miR-338-3p+核因子κB受体活化因子配体组和miR-338-3p+Wnt-C59组。双荧光素酶实验验证miR-338-3p对核因子κB受体活化因子配体的调控作用;CCK-8、EdU染色检测细胞增殖水平;流式细胞术检测细胞周期和凋亡水平;RT-qPCR检测细胞miR-338-3p、核因子κB受体活化因子配体、Wnt-3a、β-catenin、糖原合酶激酶3βmRNA表达水平;Western Blot检测细胞核因子κB受体活化因子配体、增殖细胞核抗原、Ki67、细胞周期蛋白D1、Bcl-2、Bax、天冬氨酸半胱氨酸蛋白酶3、Wnt-3a、β-catenin、糖原合酶激酶3β蛋白表达水平。结果与结论:①miR-338-3p靶向调控核因子κB受体活化因子配体;②过表达miR-338-3p后,细胞存活率、EdU阳性细胞率、S期细胞比例升高,细胞凋亡率降低,miR-338-3p、增殖细胞核抗原、Ki67、细胞周期蛋白D1、Bcl-2、Wnt-3a、β-catenin mRNA和蛋白水平升高,Bax、天冬氨酸半胱氨酸蛋白酶3、核因子κB受体活化因子配体、糖原合酶激酶3βmRNA和蛋白水平降低(均P<0.05);③过表达核因子κB受体活化因子配体或Wnt-C59处理能够减弱过表达miR-338-3p对细胞增殖、凋亡的影响(均P<0.05);④结果表明,miR-338-3p靶向核因子κB受体活化因子配体表达可促进牙槽骨成骨细胞增殖,并抑制细胞凋亡,其可能通过激活Wnt/β-catenin信号通路发挥作用。

Puerarin partly counteracts the inflammatory response after cerebral ischemia/reperfusion via activating the cholinergic anti-inflammatory pathway

Puerarin, a major isoflavonoid derived from the Chinese medical herb radix puerariae （Gegen）, has been reported to inhibit neuronal apoptosis and play an anti-inflammatory role in focal cerebral ischemia model rats. Recent findings regarding stroke pathophysiology have recognized that anti-inflammation is an important target for the treatment of ischemic stroke. The cholinergic anti-inflammatory pathway is a highly robust neural-immune mechanism for inflammation control. This study was to investigate whether activating the cholinergic anti-inflammatory pathway can be involved in the mechanism of inhibiting the inflammatory response during puerarin-induced cerebral ischemia/reperfusion in rats. Results showed that puerarin pretreatment （intravenous injection） re- duced the ischemic infarct volume, improved neurological deficit after cerebral ischemia/reperfusion and decreased the levels of interleukin-1β, interleukin-6 and tumor necrosis factor-a in brain tissue. Pretreatment with puerarin （intravenous injection） attenuated the inflammatory response in rats, which was accompanied by janus-activated kinase 2 （JAK2） and signal transducers and activators of transcription 3 （STAT3） activation and nuclear factor kappa B （NF-KB） inhibition. These observa- tions were inhibited by the alpha7 nicotinic acetylcholine receptor （a7nAchR） antagonist a-bungarotoxin （a-BGT）. In addition, puerarin pretreatment increased the expression of a7nAchR mRNA in ischemic cerebral tissue. These data demonstrate that puerarin pretreatment strongly protects the brain against cerebral ischemia/reperfusion injury and inhibits the inflammatory re- sponse. Our results also indicated that the anti-inflammatory effect of puerarin may partly be medi- ated through the activation of the cholinergic anti-inflammatory pathway.

Neutrophil extracellular traps contribute to myofibroblast differentiation and scar hyperplasia through the Toll-like receptor 9/nuclear factor Kappa-B/interleukin-6 pathway

Background:Inflammation is an important factor in pathological scarring.The role of neutrophils,one of the most important inflammatory cells,in scar hyperplasia remains unclear.The purpose of this article is to study the correlation between neutrophil extracellular traps(NETs)and scar hyperplasia and identify a new target for inhibiting scar hyperplasia.Methods:Neutrophils were isolated from human peripheral blood by magnetic-bead sorting.NETs in plasma and scars were detected by enzyme-linked immunosorbent assays(ELISAs),immunofluorescence and flow cytometry.Immunohistochemistry was used to assess neutrophil(CD66B)infiltration in hypertrophic scars.To observe the entry of NETs into fibroblasts we used immunofluorescence and flow cytometry.Results:We found that peripheral blood neutrophils in patients with hypertrophic scars were more likely to form NETs(p<0.05).Hypertrophic scars showed greater infiltration with neutrophils and NETs(p<0.05).NETs activate fibroblasts in vitro to promote their differentiation and migration.Inhibition of NETs with cytochalasin in wounds reduced the hyperplasia of scars in mice.We induced neutrophils to generate NETs with different stimuli in vitro and detected the proteins carried by NETs.We did not find an increase in the expression of common scarring factors[interleukin(IL)-17 and transforming growth factor-β(TGF-β),p>0.05].However,inhibiting the production of NETs or degrading DNA reduced the differentiation of fibroblasts intomyofibroblasts.In vitro,NETs were found to be mediated by Toll-like receptor 9(TLR-9)in fibroblasts and further phosphorylated nuclear factor Kappa-B(NF-κB).We found that IL-6,which is downstream of NF-κB,was increased in fibroblasts.Additionally,IL-6 uses autocrine and paracrine signaling to promote differentiation and secretion.Conclusions:Our experiments found that NETs activate fibroblasts through the TLR-9/NF-κB/IL-6 pathway,thereby providing a new target for regulating hypertrophic scars.

Mindin is upregulated during colitis and may activate NF-κB in a TLR-9 mediated manner

AIM:To investigate the regulation of mindin expression and the signaling pathway involved during inflammation.METHODS:C57BL/6 mice were treated with 3% dextran sulfate sodium (DSS) in drinking water for 6 d to induce acute colitis,and then the colon was harvested for histological analysis or for RNA isolation.mRNA expression of mindin and nuclear factor (NF)-κB p65 was analyzed by quantitative real time polymerase chain reaction (RT-PCR) and mindin expression construct was conf irmed by Western blotting.Mouse macrophage and intestinal epithelial lineage cells were stimulated with different cytokines and toll-like receptor (TLR) ligands,before pNF-κB-luciferase activity was assessed using the Dual-Luciferase reporter assay system.RESULTS:mRNA expression of mindin was upregulated 4.7 ± 1.1 fold compared with the baseline during DSS-induced intestinal inflammation in the mice.Stimulation with CpG-ODN (a known TLR-9 ligand) induced 4.2 ± 0.3 fold upregulation of mindin expression in RAW 264.7 cells.Full-length of mindin was cloned from cDNA of mouse mesenteric lymph node,then the pCMV-Mindin-Flag expression vector was established and the protein expression level was confi rmed.Transfection of the mindin construct and stimulation with CpG-ODN signifi cantly increased the NF-κB-luciferase activity by 2.5 ± 0.3 and 4.5 ± 0.5 fold in RAW264.7 and CMT93 cells,respectively (P < 0.01).CONCLUSION:Mindin expression is upregulated during intestinal inflammation and may induce NF-κB promoter activation in a TLR-9 mediated manner.

Pioglitazone ameliorates retinal ischemia/reperfusion injury via suppressing NLRP3 inflammasome activities

AIM：To explore the role of Pioglitazone（Pio） on a mouse model of retinal ischemia/reperfusion（I/R） injury and to elucidate the potential mechanism.METHODS：Retinal ischemia was induced in mice by increasing the intraocular pressure,and Pio was administered 4 h though periocular injection before I/R.The number of cells in the ganglion cell layer（GCL） was counted 7 d after retinal I/R injury.Glial fibrillary acidic protein（GFAP）,nuclear factor-kappa B（NF-κB）,p38,phosphorylated-p38,PPAR-γ,interleukin-1β（IL-1β）,Toll-like receptor 4（TLR4）,NLRP3,cleaved caspase-1,caspase-1 were determined by real-time polymerase chain reaction and Western blotting.RESULTS：Pio promoted the survival of retinal cells in GCL following retinal I/R injury（P〈0.05）.Besides,retinal I/R injury stimulated the expression of GFAP and TLR4,which were partially reversed by Pio treatment（P0.05）.Retinal I/R injury-upregulated expression of NLRP3,cleaved caspase-1,IL-1β was attenuated after Pio treatment（P〈0.05）.Moreover,I/R injury induced activation of NF-κB and p38 were inhibited by Pio treatment（P〈0.05）.CONCLUSION：Pio promotes retinal ganglion cells survival by suppressing I/R-induced activation of TLR4/NLRP3 inflammasomes via inhibiting NF-κB and p38 phosphorylation.