Heat Shock Protein and Apoptosis in Supraspinatus Tendinopathy

Heat shock proteins (HSPs) are often upregulated following oxidative and other forms of stress. Based on reports of excessive apoptosis in torn supraspinatus tendon and mechanically loaded tendon cells, we hypothesized heat shock proteins may be present in rodent and human models of tendinopathy due to their central role in caspase dependent apoptotic cell signaling. We used a running rat supraspinatus tendinopathy overuse model with custom microarrays to investigate the process at a genetic level. Additionally torn supraspinatus tendon and matched intact subscapularis tendon samples were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from 10 patients undergoing arthroscopic stabilization surgery and evaluated using semiquantative RT-PCR and immunohistochemistry. We identified substantial upregulation of heat shock proteins and apoptotic genes in the rodent model. We further confirmed increased levels of heat shock protein and apoptotic regulatory genes in human supraspinatus and subscapularis tendon. This finding suggests heat shock proteins play a role in the cascade of stress-activated programmed cell death and degeneration in tendinopathy and may provide a novel target in preventing tendinopathies. Each author certifies that he or she has no commercial associations (eg, consultancies, stock ownership, equity interest, patent/licensing arrangements, etc) that might pose a conflict of interest in connection with the submitted article. Each author certifies that his or her institution has approved the animal and human protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research, and that informed consent for participation in the study was obtained.     

Hypoxia suppresses serum deprivation‐induced degradation of the nucleus pulposus cell extracellular matrix through the JNK and NF‐κB pathways

Intervertebral disc (IVD) degeneration is associated with the imbalance between anabolism and catabolism of the nucleus pulposus (NP) extracellular matrix (ECM). Serum deprivation (SD) has been reported to exacerbate IVD degeneration; however, the effect of SD on ECM metabolism is not fully understood. Hypoxia plays important roles in maintaining the physiological functions of IVD cells; however, whether hypoxia has any effect on NP ECM production under conditions of SD is still unclear. In the current study, we established an in vitro SD model by exposing NP cells to serum‐free medium. SD decreased the expression of aggrecan and collagen II, as well as the production of sulfated glycosaminoglycan (sGAG) in a time‐dependent manner. However, hypoxia abolished SD‐mediated down‐regulation of aggrecan and collagen II expression via JNK1/2 activation. Moreover, hypoxia abolished SD‐induced MMP‐3 and MMP‐13 expression by inhibiting NF‐κB activation, p65 translocation, and MMP‐3 and MMP‐13 promoter activity. These results indicated that, hypoxia maintained ECM production under conditions of SD. This effect was elicited in part through JNK1/2‐mediated up‐regulation of matrix gene expression and down‐regulation of MMP expression, through the inhibition of NF‐κB. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2059–2066, 2017.

     

Expression and enzymatic activity of MMP-2 during healing process of the acute supraspinatus tendon tear in rabbits.

We investigated the spontaneous healing process of a surgically created supraspinatus tendon tear in rabbits with specific reference to the expression of matrix metalloproteinase-2 (MMP-2) and its time-course change in enzymatic activity along with the expression of tissue inhibitors of metalloproteinases (TIMPs). A transverse, full thickness tear of the supraspinatus tendon was created and examined. Immunohistochemical analysis revealed that MMP-2 positive cells were mainly localized at both cutting ends of the tendon, and reparative tissue encroached into the gap from the bursal side. The expression of TIMP-1 was induced in the cells at not only the tendon edges but also the reparative tissue during the healing process. TIMP-2 was constitutively expressed in both the tendon and the reparative tissue. Gelatin zymography using tissue culture media demonstrated latent and active forms of MMP-2 and characteristic time-linked changes of the enzymatic activity. Western blotting confirmed the bands as the latent form of MMP-2. These results suggest that MMP-2 is expressed and activated during the healing process of acute supraspinatus tendon tear and can play an important role in the remodeling process.     

Fibrogenic stresses activate different mitogen-activated protein kinase pathways in renal epithelial, endothelial or fibroblast cell populations

Fibrogenic stresses promote progression of renal tubulointerstitial fibrosis, disparately affecting survival, proliferation and trans-differentiation of intrinsic renal cell populations through ill-defined biomolecular pathways. We investigated the effect of fibrogenic stresses on the activation of cell-specific mitogen-activated protein kinase (MAPK) in renal fibroblast, epithelial and endothelial cell populations. The relative outcomes (cell death, proliferation, trans-differentiation) associated with activation or inhibition of extracellular-regulated protein kinase (ERK) or stress activated/c-Jun N terminal kinase (JNK) were analysed in each renal cell population after challenge with oxidative stress (1 mmol/L H2O2), transforming growth factor-beta1 (TGF-beta1, 10 ng/mL) or tumour necrosis factor-alpha (TNF-alpha, 50 ng/mL) over 0-20 h. Apoptosis increased significantly in all cell types after oxidative stress (P < 0.05). In fibroblasts, oxidative stress caused the activation of ERK (pERK) but not JNK (pJNK). Inhibition of ERK by PD98059 supported its role in a fibroblast death pathway. In epithelial and endothelial cells, oxidative stress-induced apoptosis was preceded by early induction of pERK, but its inhibition did not support a pro-apoptotic role. Early ERK activity may be conducive to their survival or promote the trans-differentiation of epithelial cells. In epithelial and endothelial cells, oxidative stress induced pJNK acutely. Pretreatment with SP600125 (JNK inhibitor) verified its pro-apoptotic activity only in epithelial cells. Transforming growth factor-beta1 did not significantly alter mitosis or apoptosis in any of the cell types, nor did it alter MAPK activity. Tumor necrosis factor-alpha caused increased apoptosis with no associated change in MAPK activity. Our results demonstrate renal cell-specific differences in the activation of ERK and JNK following fibrotic insult, which may be useful for targeting excessive fibroblast proliferation in chronic fibrosis.     

Effects of crystalline glucocorticoid triamcinolone acetonide on cultered human supraspinatus tendon cells

Background Rotator cuff tears are a common cause of shoulder pain and impairment. Subacromial glucocorticoid injections are widely used for treatment of epiphenomenons of chronic impingement syndrome with the possible side effects of tendon rupture and impaired tendon healing.Methods Using qRT-PCR, western blot, immunoflourescence, and measurement of ³H-thymidine uptake we investigated the effects of the crystalline glucocorticoid triamcinolone acetonide (TAA) when added to the culture medium of isolated human rotator cuff tendon cells.Results After 2 weeks of incubation, the cells had lost their fibroblastic appearance and parallel orientation, which is characteristic of cellular degeneration in vivo. Moreover, expression and secretion of collagen I was strongly reduced, and there was a decrease in proliferation rate. Cell migration was blocked and the rate of expression of the matrix metalloproteinases MMP2, MMP8, MMP9, and MMP13 was reduced, but expression of TIMP1 (a tissue inhibitor of MMPs) was upregulated, indicating a reduction in the cellular capacity for tendon repair. In addition, changes in cellular differentiation were observed: the number of adipocytes increased and levels of the protein Sox9—a marker of differentiating and mature chondrocytes—were elevated in triamcinolone acetonide treated cells.Interpretation These results may indicate that the use of TAA is one reason for weaker mechanical tendon properties and for the high rate of re-rupture after supraspinatus tendon repair.     

Effects of crystalline glucocorticoid triamcinolone acetonide on cultered human supraspinatus tendon cells

Background Rotator cuff tears are a common cause of shoulder pain and impairment. Subacromial glucocorticoid injections are widely used for treatment of epiphenomenons of chronic impingement syndrome with the possible side effects of tendon rupture and impaired tendon healing.

Methods Using qRT-PCR, western blot, immunoflourescence, and measurement of ³H-thymidine uptake we investigated the effects of the crystalline glucocorticoid triamcinolone acetonide (TAA) when added to the culture medium of isolated human rotator cuff tendon cells.

Results After 2 weeks of incubation, the cells had lost their fibroblastic appearance and parallel orientation, which is characteristic of cellular degeneration in vivo. Moreover, expression and secretion of collagen I was strongly reduced, and there was a decrease in proliferation rate. Cell migration was blocked and the rate of expression of the matrix metalloproteinases MMP2, MMP8, MMP9, and MMP13 was reduced, but expression of TIMP1 (a tissue inhibitor of MMPs) was upregulated, indicating a reduction in the cellular capacity for tendon repair. In addition, changes in cellular differentiation were observed: the number of adipocytes increased and levels of the protein Sox9—a marker of differentiating and mature chondrocytes—were elevated in triamcinolone acetonide treated cells.

Interpretation These results may indicate that the use of TAA is one reason for weaker mechanical tendon properties and for the high rate of re-rupture after supraspinatus tendon repair.     

Growth factor and protease expression during different phases of healing after rabbit deep flexor tendon repair

The purpose of the study was to contribute to the mapping of molecular events during flexor tendon healing, in particular the growth factors insulin‐like growth factor‐1 (IGF‐1), vascular endothelial growth factor (VEGF) and nerve growth factor (NGF), matrix metalloproteinases (MMP‐3 and MMP‐13) and their inhibitors (tissue inhibitors of metalloproteinases, TIMP‐1 and TIMP‐3, and the protease cathepsin K. In a rabbit model of flexor tendon injury, the mRNA expression for the growth factors, MMPs and TIMPs were measured in tendon and tendon sheath tissue at several time points (3, 6, 21, and 42 days) representing different phases of the healing process. We found that MMP‐13 remained increased during the study period, whereas MMP‐3 returned to normal levels within the first week after injury. TIMP‐3 was down‐regulated in the tendon sheaths. Cathepsin K was up‐regulated in tendons and sheaths after injury. NGF was present in both tendons and sheaths, but unaltered. IGF‐1 exhibited a late increase in the tendons, while VEGF was down‐regulated at the later time points. In conclusion, we have demonstrated the presence of NGF in flexor tendons. MMP‐13 expression appears to play a more protracted role in flexor tendon healing than MMP‐3. The relatively low levels of endogenous IGF‐1 and VEGF mRNA following injury support their potential beneficial role as exogenous modulators to optimize tendon healing and strength without increasing adhesion formation. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:886–892     

An Essential Role of NRF2 in Diabetic Wound Healing

The high mortality and disability of diabetic nonhealing skin ulcers create an urgent need for the development of more efficacious strategies targeting diabetic wound healing. In the current study, using human clinical specimens, we show that perilesional skin tissues from patients with diabetes are under more severe oxidative stress and display higher activation of the nuclear factor-E2–related factor 2 (NRF2)–mediated antioxidant response than perilesional skin tissues from normoglycemic patients. In a streptozotocin-induced diabetes mouse model, Nrf2^−/− mice have delayed wound closure rates compared with Nrf2^+/+ mice, which is, at least partially, due to greater oxidative DNA damage, low transforming growth factor-β1 (TGF-β1) and high matrix metalloproteinase 9 (MMP9) expression, and increased apoptosis. More importantly, pharmacological activation of the NRF2 pathway significantly improves diabetic wound healing. In vitro experiments in human immortalized keratinocyte cells confirm that NRF2 contributes to wound healing by alleviating oxidative stress, increasing proliferation and migration, decreasing apoptosis, and increasing the expression of TGF-β1 and lowering MMP9 under high-glucose conditions. This study indicates an essential role for NRF2 in diabetic wound healing and the therapeutic benefits of activating NRF2 in this disease, laying the foundation for future clinical trials using NRF2 activators in treating diabetic skin ulcers.     

Activation of stress-activated protein kinases (SAPK) in tendon cells following cyclic strain: the effects of strain frequency, strain magnitude, and cytosolic calcium.

Cyclic strain has been shown to benefit tendon health. However, repetitive loading has also been implicated in the etiology of tendon overuse injuries. Recent studies demonstrated that in several cell lines cyclic strain was associated with an activation of stress-activated protein kinases (SAPKs). These SAPKs, in turn, were shown to be important upstream regulators of a variety of cell processes including apoptosis. To examine the effect of repetitive loading on SAPK activation in tendon cells in vitro, canine patellar tendon cells were cyclically strained, and the cellular stress response evaluated by measuring c-Jun N-terminal kinase (JNK) activation. The effects of strain frequency and strain magnitude as well as the role of calcium signaling in this mechanotransduction mechanism were also examined. Cyclic strain resulted in an immediate activation of JNK, which peaked at 30 min and returned to resting levels by 2 h. This activation was regulated by a magnitude-dependent but not frequency-dependent response and appeared to be mediated through a calcium-dependent mechanotransduction pathway. While transient JNK activation is associated with normal cell processes. persistent JNK activation has been linked to the initiation of the apoptotic cascade. A similar mechanism could be responsible for initiating the pathological events (localized cell death) seen in tendon overuse injury.     

Anti‐inflammatory effect of platelet‐rich plasma on nucleus pulposus cells with response of TNF‐α and IL‐1

The purpose of this study was to investigate the anti‐inflammatory effect of platelet‐rich plasma (PRP) with collagen matrix on human nucleus pulposus (NP) cell in response to pro‐inflammatory cytokines such as tumor necrosis factor‐alpha (TNF‐α) and interleukin‐1 (IL‐1). NP cells from human disks were cultured in a monolayer and maintained in the collagen matrix prior to the addition of recombinant human IL‐1 and TNF‐α. After applying IL‐1 and TNF‐α, PRP prepared by using a commercially available platelet concentration system was added. The response was investigated using real‐time PCR for mRNA expression of type II collagen, aggrecan, matrix metalloproteinase‐3 (MMP‐3), and cyclooxygenase‐2 (COX‐2). The combination of IL‐1β and TNF‐α led to decrease of matrix synthesis gene expression such as collagen type II and aggrecan and increase of the degradation gene expression of COX‐2 and MMP‐3, compared to the control. Consecutive PRP exposure significantly recovered the down‐regulated gene expression of collagen type II and aggrecan and significantly reduced the increased MMP‐3 and COX‐2 gene expression, compared to that of control groups with pro‐inflammatory cytokines. The administration of PRP with collagen matrix markedly suppressed cytokine‐induced pro‐inflammatory degrading enzymes and mediators in the NP cell. It also rescued gene expression concerning matrix synthesis, thereby stabilizing NP cell differentiation. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:551–556, 2014.     

Enhancement of Achilles tendon repair mediated by matrix metalloproteinase inhibition via systemic administration of doxycycline

Collagenases or matrix metalloproteinases (MMPs) have been shown to play an important role in the matrix degradation cascade associated with Achilles tendon rupture and disease. The goal of this study was to examine the effects of daily administration of doxycycline (Doxy) through oral gavage on MMP activity and on the repair quality of Achilles tendons in vivo. Our findings indicate that Achilles tendon transection resulted in increasing MMP‐8 activity from 2 to 6 weeks post‐injury, with peak increases in activity occurring at 4 weeks post‐injury. Doxy adiministration at clinically relevant serum concentrations was found to significantly inhibit MMP activity after continuous treatment for 4 weeks, but not for continuous administration for shorter durations (96 h or 2 weeks). Extended doxy administration was also associated with improved collagen fibril organization, and enhanced biomechanical properties (stiffness, ultimate tensile strength, maximum load to failure, and elastic toughness). Our findings indicate that a temporal delay exists between Achilles tendon transection and associated increases in MMP‐8 activity in situ. Our findings suggest that inhibition of MMP‐8 at its peak activity levels ameliorates fibrosis development and improves biomechanical properties of the Achilles tendon. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:500–506, 2014.     

Fibronectin, MMP-1 and histologic changes in rotator cuff disease.

This study was designed to investigate human surgical specimens from patients with impingement (n = 16), ruptured supraspinatus tendons (n = 7), frozen shoulder (n = 2) and controls (n = 9) with respect to histological changes and the presence of fibronectin and Matrix metalloprotease-1 (MMP-1). The biopsy of the middle part of the supraspinatus tendons was analyzed microscopically after staining with hematoxyline eosin, Van Giesons hematoxyline and Phospho Tungstic Acid Hematoxyline for visualization of fibrin. Immunofluorescent stainings for fibronectin and MMP-1 were performed. Histology and immunofluorescence were assessed blindly. Necrotic tendinous tissue and fibrin were found only in some specimens from ruptures. The staining for fibronectin was significantly increased among patients with a rupture. MMP-1 was, however, only infrequently found in specimens from patients with impingement and ruptures. Fibrosis and thinning of fascicles seemed to be a more non-specific finding, appearing in control, impingement and rupture specimens. In conclusion, necrotic tendinous tissue, fibrin and fibronectin appear to be signs of tendon degeneration, whereas fibrosis and thinning of fascicles were found also in controls.     

Inhibition of c-Jun N-terminal kinase ameliorates apoptosis induced by hydrogen peroxide in the kidney tubule epithelial cells (NRK-52E)

BACKGROUND: Hydrogen peroxide (H2O2)-induced apoptosis has been shown to be involved in ischemic and toxic tubular damage. Recent studies have revealed that oxidative stress can activate c-Jun N-terminal kinase (JNK), and the oxidative stress-JNK pathway is an important apoptotic pathway of cells exposed to various stresses. The present study was designed to investigate JNK activation and the effects of the JNK pathway inhibition during H2O2-induced apoptosis in kidney epithelial tubule cells (NRK-52E). MATERIALS AND METHODS: NRK-52E cells were treated with 0-500 microM H2O2 and/or 100 microM quercetin (an inhibitor of the JNK pathway). Apoptosis was assessed by flow cytometry analysis and DNA ladder. JNK activity was assessed by the GST-c-Jun (1-169) binding/protein kinase assay. RESULTS: H2O2 induced apoptosis in NRK-52E cells in a concentration-dependent manner, which was demonstrated by the reduced DNA PI staining, externalization of phosphatidylserine and DNA ladder. Apoptosis induced by H2O2 was accompanied by JNK activation and up-regulated JNK activity. Quercetin treatment suppressed the JNK activity and ameliorated apoptosis induced by H2O2. CONCLUSIONS: H2O2 induced apoptosis in NRK-52E cells, which was associated with activation and up-regulation of JNK. Quercetin treatment could decrease JNK activity and ameliorate H2O2-induced apoptosis.     

MGF enhances tenocyte invasion through MMP‐2 activity via the FAK‐ERK1/2 pathway

Tendon regeneration and healing requires tenocytes to move to the repair site followed by proliferation and synthesis of the extracellular matrix. A novel synthetic growth factor, mechano‐growth factor (MGF), has been discovered to have positive roles in tissue repair through the improvement of cell proliferation and migration and the protection of cells against injury‐induced apoptosis. However, it remains unclear whether MGF has the potential to accelerate tendon repair. In this study, using a transwell system, we found that MGF‐C25E (a synthetic mechano‐growth factor E peptide) significantly promotes tenocyte invasion, which was accompanied by the increased phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2) as well as the increased activity of matrix metalloproteinases‐2 (MMP‐2). The MMP‐2 inhibitor OA‐Hy blocked MGF‐C25E‐promoted tenocyte invasion. Inhibitors of FAK or ERK1/2 blocked MGF‐C25E‐promoted tenocyte invasion and MMP‐2 activity as well. These results indicate that MGF‐C25E promotes tenocyte invasion by increasing MMP‐2 activity via the FAK‐ERK1/2 signaling pathway. Taken together, our findings provide the first evidence that MGF‐C25E enhances tenocyte invasion and indicate that it may serve as a potential repair material for promoting the healing and regeneration of injured tendons.     

Postoperative Tendon Loading With Treadmill Running Delays Tendon‐to‐Bone Healing: Immunohistochemical Evaluation in a Murine Rotator Cuff Repair Model

Mechanical stress has an important effect on tendon‐to‐bone healing. The purpose of the present study was to compare tendon‐to‐bone healing in animals exposed to either tendon unloading (botulinum toxin injection) or excessive loading (treadmill running) in a murine rotator cuff repair model. Forty‐eight C57BL/6 mice underwent unilateral supraspinatus tendon detachment and repair. Mice in the unloaded group were injected with botulinum toxin to the supraspinatus muscle. The contralateral shoulder of the unloaded group was used as a control. Mice were euthanized at 1, 2, and 4 weeks after surgery and evaluated with hematoxylin–eosin and immunohistochemical (IHC) staining for Ihh, Gli1, Wnt3a, and β‐catenin. The positive staining area on IHC and the Modified Tendon Maturing Score were measured. The score of the unloaded group was significantly higher (better healing) than that of the treadmill group at 4 weeks. Ihh and the glioma‐associated oncogene homolog 1 (Gli1) positive area in the unloaded group were significantly higher than those of the control group at 1 week. The peak time‐points of the Ihh and Gli1 positive area was 1 week for the unloaded group and 2 weeks for the treadmill group. The Wnt3a positive area in the unloaded group was significantly higher than that of the control group at 2 weeks. The β‐catenin positive area in the unloaded group was significantly higher than that of the treadmill group and the control group at 1 week. Our data indicated that the unloaded group has superior tendon maturation compared to the treadmill running group. Excessive tendon loading may delay the tendon healing process by affecting the activity of Ihh and Wnt/β‐Catenin pathways. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1628–1637, 2019.     

Apoptosis induced by chemotherapeutic agents involves c-Jun N-terminal kinase activation in sarcoma cell lines.

Molecular mechanisms underlying chemotherapeutic agent-induced apoptosis in sarcoma cells are not well known. Induction of apoptosis is regulated by several components including mitogen-activated protein kinases (MAPKs) comprising ERK, p38MAPKs, and c-Jun N-terminal kinase (JNK). In the present study, we examined whether activation of JNK is induced by the chemotherapeutic agents cis-diaminedichloroplatinum (cisplatin, CDDP) or doxorubicin (DXR), and whether the ectopic expression of constitutively active (MKK7-JNK1) or dominant-negative form of JNK (dnJNK) influenced apoptosis in response to the CDDP or DXR in sarcoma cell lines MG-63 and SaOS-2. The CDDP or DXR induced JNK activation in the both cell lines, as assessed by Western blotting using phosphospecific antibodies. A transient expression of the activated form of JNK sensitized the MG-63 and SaOS-2 cells to the drug-induced apoptosis, while dnJNK1 reduced the proportion of apoptotic cell death. Apoptosis was determined by flow cytometry using annexin-V Cy5. Collectively, our results indicate that JNK activation is involved in apoptotic cell death in sarcoma cell lines following stimulation with CDDP or DXR.     

Mechanical environment of the supraspinatus tendon: a two-dimensional finite element model analysis

We performed 2-dimensional finite element model analysis to estimate the mechanical environment of the supraspinatus tendon. The geometric shape of the finite element model was determined by magnetic resonance imaging of a normal human shoulder obtained at 0 degrees, 30 degrees, and 60 degrees of abduction, whereas the histologic location of noncalcified and calcified fibrocartilage was determined from a cadaveric specimen. The supraspinatus tendon was pulled proximally with the force of 10 N at 0 degrees, 53 N at 30 degrees, and 115 N at 60 degrees of abduction. The area of high principal stress maximum was observed on the articular side of the supraspinatus tendon, which shifted toward the insertion as the arm was abducted. High stress concentration on the articular side of the supraspinatus tendon near its insertion during arm elevation may explain the frequent occurrence of rotator cuff tears at this site.     

The small GTPase Rho mediates articular chondrocyte phenotype and morphology in response to interleukin‐1α and insulin‐like growth factor‐I

Small GTPases regulate the cytoskeleton and numerous other cellular functions. In this study, the role of Rho GTPase was examined in articular chondrocytes. Chondrocytes grown in monolayer were treated with interleukin‐1α (IL‐1α), insulin‐like growth factor‐I (IGF‐I), C3 Transferase, Y27632, or transfected with Rho wild type or two constitutively active mutants of Rho (Q63L and G14V). Quantitative PCR was used to determine changes in matrix metalloproteinase‐13 (MMP‐13), collagen types IIB (COL2A1) and type I (COL1A1), aggrecan (AGG), and SOX‐9 gene expression. Affinity assays were performed to measure endogenous GTP‐bound Rho, and confocal microscopy was used to assess changes in organization of the actin cytoskeleton. IL‐1α and RhoG14V increased cytoplasmic actin stress fiber formation, which was blocked by C3 Transferase, and Y27632. IL‐1α treatment also increased Rho activity. Conversely, IGF‐I lead to formation of a cortical rim of actin and decreased Rho activity. Inhibition of Rho signaling with C3 Transferase significantly decreased Rho activity and returned IL‐1α‐induced Rho activity to a level not different from control. C3 Transferase treatment also increased mRNA expression of AGG, COL2A1, and SOX‐9, and decreased expression of MMP‐13. Expression of RhoQ63L or RhoG14V resulted in increased MMP‐13 expression; however, inhibition of Rho with Y27632 was unable to inhibit IL‐1α‐induced MMP‐13 expression. Together, these results indicate a role for increased Rho activity in mediation of chondrocyte catabolic signaling pathways. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:58–64, 2009     

Ganoderma lucidum polysaccharide ameliorated diabetes mellitus-induced erectile dysfunction in rats by regulating fibrosis and the NOS/ERK/JNK pathway

BackgroundDiabetes mellitus-induced erectile dysfunction (DMED) is a frequent complication of diabetes mellitus (DM), with limited therapy at present. This study aimed to explore the role and mechanism of Ganoderma lucidum polysaccharide (GLP) on DMED.MethodsDMED was induced in the experimental rats [male 12-week-old Sprague-Dawley (SD) rats] by treatment with streptozotocin (60 mg/kg) and apomorphine (APO). Next, rats in the GLP low dose (GLP-L)/GLP high dose (GLP-H) groups were treated with GLP (100 or 400 mg/kg/d, respectively) for 8 weeks. Subsequently, erectile function was assessed by APO and electrostimulation of the cavernous nerve (CN). Serum or penile testosterone (T), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and cyclic guanosine monophosphate (cGMP) contents were evaluated by enzyme-linked immunosorbent assay (ELISA). The levels of oxidative stress indicators in the corpus cavernosum (CC) were measured by corresponding kits, and histological changes in the CC were observed by hematoxylin-eosin (HE) and Masson staining. Additionally, the apoptosis index, caspase-3, caspase-9, and eNOS expression, and mitochondrial membrane potential (MMP) were also detected. Furthermore, quantitative polymerase chain reaction (qPCR) and western blot assays were conducted to determine the NOS, TGF-β1 mRNA expression, ERK1/2, eNOS, JNK phosphorylation, and arginase II protein expression.ResultsThe erectile function test revealed that erectile dysfunction (ED) was alleviated in the DMED rats following treatment with GLP. Moreover, GLP upregulated the T and cGMP content, improved the oxidative stress and histological injuries of CC, and also inhibited the apoptosis and MMP loss of penile tissues in DMED rats. Furthermore, GLP treatment enhanced the mRNA expression of NOS and TGF-β1 and suppressed the phosphorylation of ERK1/2, eNOS, and JNK, as well as the protein expression of arginase II in DMED rats.ConclusionsGLP ameliorated DMED by repairing the CC pathological damage and upregulating NOS expression and ERK/JNK phosphorylation, indicating that GLP may be a candidate drug for DMED therapy.