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慢性肾衰竭幼鼠胫骨生长板软骨细胞线粒体自噬增强对软骨细胞凋亡的影响
王小健, 徐威威, 李荣山, 李爱中, 路晓, 马月红, 田伟, 张宇明, 常峰, 苏云星
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慢性肾衰竭幼鼠胫骨生长板软骨细胞线粒体自噬增强对软骨细胞凋亡的影响
Effect of increased mitophagy of chondrocytes in tibial growth plate of young rats with chronic renal failure on apoptosis
目的 探讨慢性肾衰竭（chronic renal failure，CRF）幼鼠胫骨生长板软骨细胞线粒体自噬水平及对细胞凋亡的影响。方法 雄性4周龄Sprague-Dawley（SD）幼鼠40只，按照随机数字表法分为正常对照组（蒸馏水灌胃）和CRF组（腺嘌呤150 mg·kg-1·d-1灌胃），连续灌胃6周后处死幼鼠，X线测量胫骨长度，组织学切片测量比较胫骨生长板宽度，应用原位末端转移酶标记技术（TUNEL）检测生长板软骨细胞凋亡率；体外培养正常对照组和CRF组幼鼠胫骨生长板软骨细胞至第3代，应用TUNEL技术检测软骨细胞凋亡率，应用荧光双染技术观测软骨细胞线粒体与自噬溶酶体共定位情况，应用Western印迹法检测线粒体标志蛋白线粒体外膜转位酶（translocase of the outer mitochondrial membrane-20，Tom-20）和自噬标志轻链蛋白-3（light chain-3，LC-3）表达情况，应用透射电镜观察软骨细胞线粒体自噬情况。结果 与正常对照组相比，CRF组幼鼠胫骨长度较短[（27.32±5.81）mm 比（35.43±3.61）mm，t=5.226，P<0.001]，生长板相对宽度较窄（0.56±0.19比1.00±0.21，t=6.744，P<0.001），软骨细胞凋亡率较高（17.2%±4.8%比5.1%±3.4%，t=6.505，P<0.001）。CRF组体外培养生长板软骨细胞凋亡率高于正常对照组（11.8%±6.2%比3.1%±1.2%，t=4.357，P<0.001），荧光双染技术结果显示CRF组软骨细胞线粒体与自噬溶酶体出现明显的共定位现象，CRF组软骨细胞LC-3蛋白（t=8.944，P<0.001）及Tom-20蛋白（t=6.708，P<0.001）表达均少于正常对照组，电镜结果显示CRF组软骨细胞内出现较多的自噬囊泡吞噬线粒体并降解的现象。结论 CRF幼鼠胫骨生长板软骨细胞线粒体自噬水平出现增高现象，导致线粒体减少，引起软骨细胞凋亡、数量减少，最终导致胫骨发育不良。
Objective To explore the level of tibial growth plate chondrocyte mitophagy in young rats with chronic renal failure (CRF) and its effect on chondrocyte apoptosis. Methods Male 4-week-old Sprague-Dawley rats were randomly divided into two groups according to random number table method: normal control group (n=20, intragastric administration with distilled water) and CRF group (n=20, given adenine suspension 150 mg·kg-1·d-1). All the young rats were sacrificed after continuous gavage for 6 weeks. The length of tibia was measured on X ray film, the width of tibia growth plate was measured and compared on histological section, and the apoptosis rate of chondrocytes in growth plate was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The growth plate chondrocytes of two groups were isolated and cultured to the third generation in vitro, and the apoptosis rate of chondrocytes was detected by TUNEL assay. The co-localization of mitochondria and autophagy lysosomes in chondrocytes was observed by double fluorescence staining. Western blotting was used to detect the level of mitochondrial marker protein translocate of the outer mitochondrial membrane-20 (Tom-20) and autophagy marker light chain-3 protein (LC-3). The mitophagy of growth plate chondrocytes was observed by transmission electron microscope. Results Compared with the normal control group, the tibia length of CRF group was shorter [(27.32±5.81) mm vs (35.43±3.61) mm, t=5.226, P<0.001], and the relative width of growth plate in histological section was narrower (0.56±0.19 vs 1.00±0.21, t=6.744, P<0.001). The apoptosis rate of chondrocytes in growth plate in CRF group was higher than that in the normal control group (17.2%±4.8% vs 5.1%±3.4%, t=6.505, P<0.001). The apoptosis rate of chondrocytes cultured in vitro in CRF group was higher than that in the normal control group (11.8%±6.2% vs 3.1%±1.2%, t=4.357, P<0.001). The result of double influorescence staining showed that there was co-localization between mitochondria and autophagy lysosomes in CRF group. Western blotting results showed that the levels of LC-3 protein (t=8.944, P<0.001) and Tom-20 protein (t=6.708, P<0.001) in CRF group were lower than those in the normal control group. Conclusion The level of tibial growth plate chondrocyte mitophagy in young rats with CRF increases, which will lead to a decrease in the number of mitochondria, an increase in the apoptosis and a decrease in the number of chondrocytes, and eventually lead to dysplasia of tibia.
肾功能不全，慢性 / 线粒体 / 自噬 / 生长板 {{custom_keyword}} /
Renal insufficiency, chronic / Mitochondria / Autophagy / Growth plate {{custom_keyword}} /
杨克魁 , {{custom_editor}}
表1 两组幼鼠实验第6周末各指标比较（ |
| 组别 | 血尿素氮(mmol/L) | 血肌酐(μmol/L) | 血钙(mmol/L) | 血磷(mmol/L) | iPTH(ng/L) | 尿蛋白量(mg/24 h) |
|---|---|---|---|---|---|---|
| 正常对照组(n=20) | 7.6±1.8 | 30.2±11.6 | 2.68±0.65 | 2.98±1.12 | 134.2±16.9 | 47.2±9.8 |
| 慢性肾衰竭组(n=18) | 17.3±6.2a | 71.2±20.1a | 2.71±0.76 | 3.02±0.94 | 128.3±17.1 | 149.6±32.4a |
| t值 | 6.392 | 7.585 | 0.131 | 0.118 | 1.068 | 12.870 |
| P值 | <0.001 | <0.001 | 0.896 | 0.906 | 0.293 | <0.001 |
| 注：iPTH：全段甲状旁腺素；与正常对照组比较，aP<0.01 |
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Background: Zinc is an essential micronutrient for human beings and its deficiency affects their normal growth and development. Objective: The main aim was to evaluate the effect of two doses of zinc supplementation (ZS) on the nutritional status in chronic kidney disease (CKD) children. Methods: A randomized-trial multicentric study was conducted in 48 CKD (23 females) patients under 18-years-old, for a year. At random, participants took 30 or 15 mg/day of ZS, respectively. Anthropometric measurements and biochemical analysis were performed. Hypozincemia was determined by serum zinc concentration (SZC) using atomic absorption spectrophotometry. The positive or negative change in patients’ body mass index (BMI) Z-score, serum albumin, zinc and C-reactive protein (CRP) levels were used to evaluate the effect of ZS. Results: Mean SZC was normal before and after ZS. Despite ZS, there were no significant changes in serum albumin, zinc and CRP levels. A positive and significant association was observed between SZC and serum albumin before (p = 0.000) and after (p = 0.007) ZS. In both groups of ZS, there was a small but positive and significant change in body mass and normalization in BMI Z-score, hypoalbuminemia, hypozincemia and high CRP, especially with 30 mg/day of ZS. Conclusions: Zinc supplementation may be beneficial for nutritional status in children and adolescents with CKD.
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Synovitis, a common clinical symptom for osteoarthritis (OA) patients, is highly related to OA pathological progression and pain manifestation. The activated synovial macrophages have been demonstrated to play an important role in synovitis, but the mechanisms about macrophage activation are still not clear. In this study, we found that the exosome-like vesicles from osteoarthritic chondrocytes could be a new biological factor to stimulate inflammasome activation and increase mature IL-1β production in macrophages. The degraded cartilage explants produced more exosome-like vesicles than the nondegraded ones, while the exosome-like vesicles from chondrocytes could enter into joint synovium tissue and macrophages. Moreover, the exosome-like vesicles from osteoarthritic chondrocytes enhanced the production of mature IL-1β in macrophages. These vesicles could inhibit ATG4B expression via miR-449a-5p, leading to inhibition of autophagy in LPS-primed macrophages. The decreased autophagy promoted the production of mitoROS, which further enhanced the inflammasome activation and subsequent IL-1β processing. Ultimately, the increase of mature IL-1β may aggravate synovial inflammation and promote the progression of OA disease. Our study provides a new perspective to understand the activation of synovial macrophages and synovitis in OA patients, which may be beneficial for therapeutic intervention in synovitis-related OA patients.
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Pyruvate kinase M2 isoform (PKM2) catalyzes the last step of glycolysis and plays an important role in tumor cell proliferation. Recent studies have reported that PKM2 also regulates apoptosis. However, the mechanisms underlying such a role of PKM2 remain elusive. Here we show that PKM2 translocates to mitochondria under oxidative stress. In the mitochondria, PKM2 interacts with and phosphorylates Bcl2 at threonine (T) 69. This phosphorylation prevents the binding of Cul3-based E3 ligase to Bcl2 and subsequent degradation of Bcl2. A chaperone protein, HSP90α1, is required for this function of PKM2. HSP90α1's ATPase activity launches a conformational change of PKM2 and facilitates interaction between PKM2 and Bcl2. Replacement of wild-type Bcl2 with phosphorylation-deficient Bcl2 T69A mutant sensitizes glioma cells to oxidative stress-induced apoptosis and impairs brain tumor formation in an orthotopic xenograft model. Notably, a peptide that is composed of the amino acid residues from 389 to 405 of PKM2, through which PKM2 binds to Bcl2, disrupts PKM2-Bcl2 interaction, promotes Bcl2 degradation and impairs brain tumor growth. In addition, levels of Bcl2 T69 phosphorylation, conformation-altered PKM2 and Bcl2 protein correlate with one another in specimens of human glioblastoma patients. Moreover, levels of Bcl2 T69 phosphorylation and conformation-altered PKM2 correlate with both grades and prognosis of glioma malignancy. Our findings uncover a novel mechanism through which mitochondrial PKM2 phosphorylates Bcl2 and inhibits apoptosis directly, highlight the essential role of PKM2 in ROS adaptation of cancer cells, and implicate HSP90-PKM2-Bcl2 axis as a potential target for therapeutic intervention in glioblastoma.
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Mitochondrial dysfunction leads to osteoarthritis (OA) and disc degeneration. Hypoxia inducible factor-1α (HIF-1α) mediated mitophagy has a protective role in several diseases. However, the underlying mechanism of HIF-1α mediated mitophagy in OA remains largely unknown. This current study was performed to determine the effect of HIF-1α mediated mitophagy on OA. Therefore, X-ray and tissue staining including HE staining, safranin O-fast green (S-O) and Alcian Blue were used to assess imageology and histomorphology differences of mouse knee joint. Transcriptional analysis was used to find the possible targets in osteoarthritis. Western blot analysis, RT-qPCR and immunofluorescence staining were used to detect the changes in gene and protein levels in the vitro experiment. The expression of HIF-1α was increased in human and mouse OA cartilage. HIF-1α knockdown by siRNA further impair the hypoxia-induced mitochondrial dysfunction; In contrast, HIF-1α mediated protective role was reinforced by prolylhydroxylase (PHD) inhibitor dimethyloxalylglycine (DMOG). In addition, HIF-1α stabilization could alleviate apoptosis and senescence via mitophagy in chondrocytes under hypoxia condition, which could also ameliorate surgery-induced cartilage degradation in mice OA model. In conclusion, HIF-1α mediated mitophagy could alleviate OA, which may serve as a promising strategy for OA treatment.
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Osteoarthritis is the most common degenerative joint disease and causes major pain and disability in adults. It has been reported that mitochondrial dysfunction in chondrocytes is associated with osteoarthritis. Sirtuins are a family of nicotinamide adenine dinucleotide-dependent histone deacetylases that have the ability to deacetylate protein targets and play an important role in the regulation of cell physiological and pathological processes. Among sirtuin family members, sirtuin 3, which is mainly located in mitochondria, can exert its deacetylation activity to regulate mitochondrial function, regeneration, and dynamics; these processes are presently recognized to maintain redox homeostasis to prevent oxidative stress in cell metabolism. In this review, we provide present opinions on the effect of mitochondrial dysfunction in osteoarthritis. Furthermore, the potential protective mechanism of SIRT3-mediated mitochondrial homeostasis in the progression of osteoarthritis is discussed.
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Hydroxytyrosol (HT), a major phenolic antioxidant found in olive oil, can afford protection from oxidative stress in several types of non-tumoral cells, including chondrocytes. Autophagy was recently identified as a protective process during osteoarthritis (OA) development and critical for survival of chondrocytes. Therefore we have investigated the possibility to modulate chondrocyte autophagy by HT treatment.DNA damage and cell death were estimated in human C-28/I2 and primary OA chondrocytes exposed to hydrogen peroxide. Autophagic flux and mitophagy were monitored by measuring levels and location of autophagy markers through western blot, immunostaining and confocal laser microscopy. Late autophagic vacuoles were stained with monodansylcadaverine. The involvement of sirtuin 1 (SIRT-1) was evaluated by immunohistochemistry, western blot and gene silencing with specific siRNA.HT increases markers of autophagy and protects chondrocytes from DNA damage and cell death induced by oxidative stress. The protective effect requires the deacetylase SIRT-1, which accumulated in the nucleus following HT treatment. In fact silencing of this enzyme prevented HT from promoting the autophagic process and cell survival. Furthermore HT supports autophagy even in a SIRT-1-independent manner, by increasing p62 transcription, required for autophagic degradation of polyubiquitin-containing bodies.These results support the potential of HT as a chondroprotective nutraceutical compound against OA, not merely for its antioxidant ability, but as an autophagy and SIRT-1 inducer as well.HT may exert a cytoprotective action by promoting autophagy in cell types that may be damaged in degenerative diseases by oxidative and other stress stimuli.Copyright © 2016 Elsevier B.V. All rights reserved.
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Osteoarthritis (OA) is a degenerative rheumatic disease that is associated with extracellular matrix degradation and chondrocyte apoptosis in the articular cartilage. The role of mitochondria in degenerative diseases is widely recognized. We undertook this study to evaluate mitochondrial function in normal and OA chondrocytes and to examine age‐related changes in mitochondria.
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To define apoptotic chondrocyte death and the expression of Bcl-2, Bax, and Fas in human osteoarthritis (OA) cartilage.Cartilage samples were obtained from patients with knee OA at the time of joint replacement surgery and from normal autopsy cases. In OA, sections were obtained both from the lesional area, usually within 1 cm of bony exposure, and from the non-lesional area, which had macroscopically normal appearance or only mild surface irregularities. Apoptosis was verified by microscopic examination of hematoxylin and eosin stained specimens, TUNEL staining, electron microscopy, and DNA ladder analysis by electrophoresis. Immunohistochemistry was done to study the expression of Bcl-2, Bax, and Fas. Apoptotic cells and Bcl-2, Bax, and Fas positive cells were counted within defined microscopic fields. Expression of Bcl-2 and Bax was verified by Western blot.The percentage of apoptotic cells in the lesional area was significantly higher than in the non-lesional area in cartilage from the same patient with OA, while apoptotic cells were rarely seen in normal cartilage. This result was confirmed by TUNEL stain. Many chondrocytes with chromatin condensation were verified in electron microscopy, and DNA from OA lesional cartilage revealed a DNA ladder on electrophoresis. Bcl-2 and Fas expressions were significantly higher in the OA lesional area than in the non-lesional area. On the other hand, Bcl-2 expression in normal cartilage was significantly higher than in OA cartilage. There was no significant difference in Bax expression among normal, OA lesional, and OA non-lesional cartilage.These results show that apoptotic chondrocyte death occurs more frequently in OA compared to normal cartilage and in OA lesional compared to OA non-lesional cartilage. The different expression patterns of Bcl-2 and Fas in OA lesional and non-lesional cartilage suggest that they might be involved in the pathogenesis of OA.
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所有作者声明无利益冲突
王小健：酝酿和设计实验、实施研究、采集数据、分析/解释数据、起草文章；徐威威、路晓、马月红、田伟、张宇明：实施研究；李荣山：获取研究经费、指导；李爱中：酝酿和设计实验、实施研究；常峰、苏云星：对文章的知识性内容作批评性审阅、获取研究经费、指导
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