Experimental immune inflammation in the synovial membrane: I. The immunological mechanism

Synovitis was produced in immunized guinea-pigs and rats by injection of antigen into the knee joint. The reaction was mainly mononuclear at 48 hours and, in the guinea-pig, progressed to chronic granuloma formation. Antigens principally used were bovine γ-globulin and tuberculin PPD. Immune deviation, giving a diminished delayed hypersensitivity response, also gave diminished synovial inflammation when compared with undeviated control animals. Immune synovitis to tuberculin PPD was successfully transferred with peritoneal cells taken from immunized animals and given intravenously to normal recipients whose knee joints were injected with antigen. Intravenous transfer of immune serum gave rise to a synovial reaction to bovine γ-globulin, injected into recipients' joints. Transfer of both cells and serum gave rise to particularly severe reactions. Transfer of either cells or serum or cells with serum failed to give reactions extending beyond 3–4 days. This suggests that active immunization is a requisite for the chronic inflammatory reaction of synovitis. Although the 48-hour synovial membrane reaction of rats was similar to that seen in the guinea-pig, chronic inflammatory reactions were not found in that species.     

Effects of nicotine on a rat model of early stage osteoarthritis

The objective of the study was to investigate the effects of nicotine on articular cartilage degeneration and inflammation in a rat model of early stage osteoarthritis (OA), using T2 mapping. In this study, a rat model of early stage OA was established by immobilizing the left knee joints of adult male rats for two weeks. Subsequently, rats were fed with nicotine for two and four weeks. Changes in the articular cartilage from the medial femoral condylar region of the knee were evaluated by gross observation and histological grading with the contents of cartilage matrix detected. T2 values of the articular cartilage were estimated through high-field magnetic resonance imaging (MRI) (7.0T). Levels of serum tumor necrosis factor-α (TNF-α) were assessed by ELISA. The expression of TNF-α and the cholinergic receptor, α7nAChR, in the synovial tissue was measured by RT-PCR. Nicotine treatment ameliorated cartilage destruction, promoted matrix production, reduced the serum level of TNF-α and the expression of TNF-α in the synovial tissue, and increased the expression of α7nAChR in the synovial tissue in the rat model of early stage OA. In conclusion, nicotine prevented cartilage damage and had an anti-inflammatory effect in a rat model of early stage OA. Thus nicotine may have potential as a therapeutic strategy for early stage OA.     

Inhibitory effects of intrathecal p38β antisense oligonucleotide on bone cancer pain in rats

Objective: To evaluate the effects of intrathecal administration p38β antisense oligonucleotide on the development of bone cancer pain rats. Methods: Forty female SD rats weighing 180~220 g were randomly divided into 4 groups (n = 10 each): Group A (control group): intra-tibial injection of 3 μl Hank’s solution; group B (model group): intra-tibial injection of 3 μl MADB-106 mammary gland carcinoma cells of rats (4.8 × 10³/μl); group C (p38β-SODN 20 μg); group D (p38β-ASODN 20 μg). The model procedures in group C and D were same to those in the group B. From the 14^th day after operation, p38β-SODN 20 μg and p38β-ASODN 20 μg were respectively intrathecally administrated in group C and D once daily for 6 days whereas normal saline was for group A and B. Mechanical withdrawal threshold and radiant heat threshold of rat hind paws were measured before operation and every other day until 22 d of post-operation. The lumbar 4-6 spinal cord was removed on the 22^nd day. The expression of spinal p38β protein was determined by Western blot. Results: No significant differences in mechanical withdrawal threshold and radiant heat threshold were found at all time points in control group. During the first 6 days after operation there were obvious differences in radiant heat stimulus between control group between the other groups (P < 0.05); During 14-22 days after operation, mechanical pain threshold and radiant heat threshold between p38β-SODN group and Model group were significantly changed compared with that in control group (P < 0.05). However, the differences were not remarkable between control group and p38β-ASODN group (P > 0.05). The expression of p38β protein in lumbar spinal cord was significantly higher between p38β-SODN group and Model group than that in control group (P < 0.05). There was no significant difference in p38β protein expression between p38β-ASODN group and control group (P > 0.05). Conclusions: Hyperalgesia induced by bone cancer can be inhibited by intrathecal administration of p38β antisense oligonucleotide, which is achieved by reducing expression of p38β protein.     

Comparison of the efficacy of bone marrow mononuclear cells and bone mesenchymal stem cells in the treatment of osteoarthritis in a sheep model

Objective: To evaluate the therapeutic efficacy of uncultured bone marrow mononuclear cells (BMMCs) and bone mesenchymal stem cells in an osteoarthritis (OA) model of sheep. Methods: Induction of sheep OA was performed surgically through anterior cruciate ligament transection and medial meniscectomy. After 12 weeks, concentrated BMMCs obtained from autologous bone marrow harvested from anterior iliac crest or a single dose of 10 million autologous bone mesenchymal stem cells (BMSCs) suspended in phosphate-buffered saline (PBS) was delivered to the injured knee via direct intra-articular injection. Animals of the PBS group received vehicle alone. The contra-lateral joints were selected randomly as the control group. Knees of the four groups were compared macroscopically and histologically, and glycosaminoglycan (GAG) contents normalized to cartilage wet weight were measured at lesions of cartilage from medial condyle of the femur head. Gene expression levels of type II collagen (Col2A1), Aggrecan and matrix metalloproteinase-13 (MMP-13) in cartilage were measured based on RT-PCR and prostaglandin E2 (PGE2), Tumor Necrosis Factor-α (TNF-α) and Transforming Growth Factor beta (TGF-β) concentrations in synovial fluid were determined with ELISA assays at 8 weeks after injection. Results: At 8 weeks post cell transplantation, partial cartilage repair was observed in the cell therapy, but not the PBS group (P<0.05). The BMSCs group showed higher regeneration of cartilage and lower proteoglycan loss than the BMMCs group (P<0.05). Concentrated BMMCs injection led to a weaker treatment effect, but also inhibited PGE2, TNF-α and TGF-β levels in synovial fluid and promoted higher levels of Aggrecan and Col2A1 and downregulation of MMP-13 in sheep chondrocytes in a similar manner to BMSCs, compared with the PBS group. Conclusions: Bone marrow cells showed therapeutic efficacy in a sheep model of OA. Despite similar therapeutic potential, the easier and faster process of collection and isolation of BMMCs supports their utility as an effective alternative for OA treatment in the clinic.     

Effect of losartan with folic acid on plasma homocysteine and vascular ultrastructural changes in spontaneously hypertensive rats

Elevated homocysteine (Hcy) is a high risk factor of hypertension due to its function in endothelial dysfunction. Its level in the blood is strongly influenced by folic acid. In order to investigate the effects of losartan with folic acid on plasma level of Hcy and vascular ultrastructural changes, thirty spontaneously hypertensive rats (SHR) involved and randomly divided into three groups (n=10): SHR-C group (control), SHR-L group (losartan 25 mg·kg^-1·d^-1), SHR-L+Y group (losartan 25 mg·kg^-1·d^-1 + folic acid 0.4 mg·kg^-1·d^-1). Another 10 Wistar Rats involved as WKY-C group for normal control. The level of plasma Hcy was measured dynamically by LS-MS, the vascular ultrastructural changes were analyzed by light and electron microscopy. Moreover, the thickness and area of aorta was measured. The results showed the Hcy levels in four groups were WKY-C 7.49 ± 1.95 μmol/L; SHR-C 8.45 ± 1.90 μmol/L; SHR-L 8.28 ± 2.11 μmol/L; SHR-L+Y 7.53 ± 2.02 μmol/L at 80 days. There was no significant change for plasma Hcy (P>0.05). The morphological change showed the subendothelial space didn’t increased significantly, the endothelial cells have a more smooth and intact cellular membrane in SHR-L+Y group. In conclusion, Losartan combined with folic acid could improve arterial endothelial structure in SHR which has no significant correlation with plasma Hcy.     

Production of IL-6 by T cells from the femoral head of patients with rapidly destructive coxopathy (RDC)

RDC is a syndrome with unknown etiology that causes rapid destruction of a hip joint. We have investigated the production of osteoclast-activating cytokines (IL-6, IL-1α and tumour necrosis factor-alpha (TNF-α)), interferon-gamma (IFN-γ) and IL-8 by T cells in the affected joint. The level of IL-6 produced by the T cell lines (TCL) established from the femoral head was significantly higher than that from patients' or healthy donors' peripheral blood mononuclear cells (PBMC). IL-6 production by the TCL from synovial membrane or from patients' PBMC was also significantly higher than that from healthy donors' PBMC. IL-1α production by the TCL from the femoral head was significantly higher than any of the other groups when all the TCL were used for the analysis. TNF-α production was highest in the TCL from patients' PBMC. The levels of IFN-γ or IL-8 were not significantly different among these four groups. The plasma levels of all these cytokines except for IFN-γ, that was rather lower, in RDC patients were not significantly different from those in osteoarthrosis or trauma patients, or healthy donors. These results suggest that T cells at the affected femoral head, and also synovial membrane to some extent, are involved in bone resorption through the production of IL-6 and probably IL-1α in patients with RDC.     

Pathologic finding of increased expression of interleukin-17 in the synovial tissue of rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a common autoimmune disease of chronic systemic inflammatory disorder that will affect multiple tissues and organs such as skin, heart or lungs; but it principally attacks the joints, producing a nonsuppurative inflammatory and proliferative synovitis that often progresses to major damaging of articular cartilage and joint ankylosis. Although the definite etiology is still unknown, recent studies suggest that T-helper cells (Th17) may play a pivotal role in the pathogenesis of RA. And interleukin-17 (IL-17), which is a cytokine of Th17 cells, may be a key factor in the occurrence of RA. The binding of IL-17 to specific receptor results in the expression of fibroblasts, endothelial and epithelial cells and also synthesis of several major factors such as tumor necrosis factor alpha (TNF-α), IL-1β that result in the structural damage of RA joints. Though some previous studies have shown that IL-17 exists in the synovium of RA, few has definite proof quantitatively by pathology about its existence in synovial membrane. This study comprised of 30 RA patients and 10 healthy control, pathologic study of the synovial membrane showed increased expression of IL-17 in the synovial tissue of RA patients, the intensity is compatible with clinical severity of disease as validated by DAS28 score and disease duration. Northern blot study also confirmed the increased expression of IL-17 in the synovial tissues. This study sheds further light that IL-17 may be a key factor in the pathogenesis of RA and a determinant of disease severity.     

Efficacy of Direct Injection of Etanercept into Knee Joints for Pain in Moderate and Severe Knee Osteoarthritis

Purpose

Osteoarthritic (OA) pain is largely considered to be inflammatory pain. However, during the last stage of knee OA, sensory nerve fibers in the knee are shown to be significantly damaged when the subchondral bone junction is destroyed, and this can induce neuropathic pain. Several authors have reported that tumor necrosis factor-α (TNFα) in a knee joint plays a crucial role in pain modulation. The purpose of the current study was to evaluate the efficacy of etanercept, a TNFα inhibitor, for pain in knee OA.

Materials and Methods

Thirty-nine patients with knee OA and a 2-4 Kellgren-Lawrence grading were evaluated in this prospective study. Patients were divided into two groups; hyaluronic acid (HA) and etanercept injection. All patients received a single injection into the knee. Pain scores were evaluated before and 4 weeks after injection using a visual analogue scale (VAS) and the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and they were compared between the groups.

Results

Before injection, VAS and WOMAC scores were not significantly different between the groups (p>0.05). Significant pain relief was found in the etanercept group at 1 and 2 weeks by VAS, and at 4 weeks by WOMAC score, compared with the HA group (p<0.05). No adverse events were observed in either group.

Conclusion

Direct injection of etanercept into OA knee joints was an effective treatment for pain in moderate and severe OA patients. Furthermore, this finding suggests that TNFα is one factor that induces OA pain.     

Low-Intensity Pulsed Ultrasound Reduces the Inflammatory Activity of Synovitis

The purpose of this study was to examine the effect of low-intensity pulsed ultrasound (LIPUS) on the cell proliferation and growth of synovial membrane cells stimulated with inflammatory cytokines, and to evaluate the effectiveness of LIPUS treatment of synovitis in the knee joints of animal models for rheumatoid arthritis. The rabbit knee synovial membrane cell line, HIG-82, was cultured in medium with or without IL-1β or TNF-α. Four hours after stimulation with the cytokines, the cells received LIPUS or sham exposure. Cell proliferation and growth were then analyzed. Using MRL/lpr mice, the anti-inflammatory effects of LIPUS were also evaluated in vivo. Stimulation with proinflammatory cytokines significantly up-regulated cell proliferation which was significantly down-regulated by LIPUS exposure. In MRL/lpr mice, exposure of knee joints to LIPUS caused a significant reduction of histological damage compared to the control. Histological lesions were significantly reduced in the joints treated with LIPUS for 3 weeks. Cox-2-positive cells in the knee joints treated with LIPUS were markedly decreased compared to the control joints. Therefore, LIPUS stimulation may be a medical treatment for joint inflammatory diseases, such as synovitis.     

miR-144 regulates transforming growth factor-β1 iduced hepatic stellate cell activation in human fibrotic liver

Objectives: Activation of hepatic stellate cells (HSCs) into collagen producing myofibroblasts is critical for pathogenesis of liver fibrosis. Transforming growth factor-β1 (TGF-β1) is one of the main profibrogenic mediators for HSC transdifferentiation. Recent studies have shown effect of microRNAs (miRNAs) on regulating TGF-β1-induced HSC activation during liver fibrosis. Here, we aimed to explore the roles of miR-144 and miR-200c in human liver fibrosis. Methods: Expression of TGF-β1 was detected in 42 fibrotic and 18 normal human liver tissues by quantitative real time polymerase chain reaction (qRT-PCR) and immunohistochemistry, and its correlation with α-smooth muscle actin (α-SMA) was calculated. miR-144 and miR-200c expression level in fibrotic liver tissues were also detected by qRT-PCR. The correlation of TGF-β1 expression with miR-200c and miR-144 in the fibrotic liver was analyzed. Results: The results showed that TGF-β1 expression was much higher in fibrotic liver than that in normal liver tissues (P<0.05). TGF-β1 protein high expressing liver fibrosis showed α-SMA positive cells in the liver parenchyma indicating activated HSCs. Expression of TGF-β1 in fibrotic liver was significantly correlated with α-SMA expression (R=0.633, P<0.001). Furthermore, miR-144 was less expressed in liver fibrosis (P<0.05) and was significantly correlated with expression of TGF-β1 in fibrotic liver tissues (R=-0.442, P<0.01). However, miR-200c did not show significant difference between normal and fibrotic liver (P=0.48) and correlation with TGF-β1 expression (R=0.106, P=0.51). Conclusion: All the results indicate that miR-144 can be a novel regulator of TGF-β1-induced HSC activation during liver fibrosis.     

Attenuation of the Progression of Articular Cartilage Degeneration by Inhibition of TGF-β1 Signaling in a Mouse Model of Osteoarthritis

Transforming growth factor beta 1 (TGF-β1) is implicated in osteoarthritis. We therefore studied the role of TGF-β1 signaling in the development of osteoarthritis in a developmental stage-dependent manner. Three different mouse models were investigated. First, the Tgf-β receptor II (Tgfbr2) was specifically removed from the mature cartilage of joints. Tgfbr2-deficient mice were grown to 12 months of age and were then euthanized for collection of knee and temporomandibular joints. Second, Tgfbr2-deficient mice were subjected to destabilization of the medial meniscus (DMM) surgery. Knee joints were then collected from the mice at 8 and 16 weeks after the surgery. Third, wild-type mice were subjected to DMM at the age of 8 weeks. Immediately after the surgery, these mice were treated with the Tgfbr2 inhibitor losartan for 8 weeks and then euthanized for collection of knee joints. All joints were characterized for evidences of articular cartilage degeneration. Initiation or acceleration of articular cartilage degeneration was not observed by the genetic inactivation of Tgfbr2 in the joints at the age of 12 months. In fact, the removal of Tgfbr2 and treatment with losartan both delayed the progression of articular cartilage degeneration induced by DMM compared with control littermates. Therefore, we conclude that inhibition of Tgf-β1 signaling protects adult knee joints in mice against the development of osteoarthritis.Transforming growth factor beta 1 (TGF-β1) is considered an anabolic factor to articular chondrocytes, based largely on results from in vitro and ex vivo experiments in which TGF-β1 can stimulate chondrocytes to synthesize and release extracellular matrix molecules, including proteoglycans and type II collagen.^{1, 2} In addition, results from other studies indicate that the genetic inactivation of Smad3 or disruption of the interaction of Tgf-β1 with its receptor, Tgf-β receptor type II (Tgfbr2), during early development results in osteoarthritis (OA)-like knee joints in mice.^{3, 4, 5} Moreover, a human genetic study reports that a two-nucleotide deletion, 741-742del AT (nonsense mutation), in SMAD3 causes early-onset OA in a human family.⁶ These investigations indicate that TGF-β1 is, at least, required for the normal development of a joint.The lack of TGF-β1 signaling during early development can cause a normal joint to develop into an osteoarthritic joint. However, observations from studies of adult mice as opposed to developing mice suggest that the increase in the activity of TGF-β1 signaling may initiate and accelerate articular cartilage degeneration in adult joints. First, studies in animal models by Itayem et al^{7, 8} suggest that intra-articular injection of TGF-β1 into adult rat knee joints causes early onset of OA. Second, a study by Bakker et al⁹ reports that the constitutive overexpression of active TGF-β1 in adult mouse knee joints results in OA associated with increase in the production of proteoglycans in articular cartilage and hyperplasia of synovium and chondro-osteophyte formation. Note that the enhanced production of extracellular matrix molecules is not necessarily beneficial or physiologic to adult articular cartilage. For instance, one of the earliest pathologic signs in articular cartilage degeneration is the overproduction of proteoglycans in mouse models of OA.^{10, 11} Thus, the overproduction of the proteoglycans could disrupt the homeostasis of adult articular cartilage. Third, the above-mentioned human genetic study reports that a nucleotide change, 859C>T or 782C>T in SMAD3, increases the level of TGF-β1 and activity of the TGF-β1 signaling pathway in two human families associated with early-onset OA.⁶ In addition, data from a human genetic association study suggest that an increase in the expression of SMAD3 is a risk factor for the development of OA.¹² This is in agreement with the observation from studies showing that the level of TGF-β1 is significantly higher in human osteoarthritic tissues than in healthy articular cartilage.^{13, 14} Fourth, we found that the protein level of Tgf-β1 was significantly increased in the articular chondrocyte of adult knee joints in two mouse models of OA, collagen type XI gene-deficient mice and destabilization of the medial meniscus (DMM).¹⁵ On the basis of results from all of the aforementioned studies, a question remains: what is the exact role of TGF-β1 in the development of OA? We hypothesized that TGF-β1 signaling in the development of OA acts in a developmental stage-dependent manner. In this scenario, TGF-β1 is required for the development of articular cartilage; however, once a joint is formed, TGF-β1 is no longer needed. Therefore, induction of TGF-β1 in an adult joint causes articular cartilage degeneration, which eventually leads to OA.To support our hypothesis, we evaluated the articular cartilage of knee joints for evidence of changes in structural characteristics and protein expression of genes in three different conditions of adult mice. First, Tgfbr2 was specifically removed from the articular cartilage of knee and temporomandibular (TM) joints of mice at the age of 8 weeks. The mice were grown to the age of 12 months, at which point knee and TM joints were collected. Second, adult mice (8 weeks old) without Tgfbr2 in the articular cartilage of their knee joints were subjected to DMM surgery (known to induce OA) and were euthanized at 8 and 16 weeks after DMM for the collection of knee joints. Third, adult wild-type C57BL/6 mice were subjected to DMM and then treated with a Tgfbr2 inhibitor, losartan. The mice were euthanized at 8 weeks after DMM for collection of knee joints. The articular cartilage of joints from the mice and their corresponding controls were analyzed.     

The Relationship between HIF-2α and VEGF with Radiographic Severity in the Primary Osteoarthritic Knee

PurposeThe aim of this study was to determine the relationship of hypoxia-inducible factor-2 (HIF-2α) and vascular endothelial growth factor (VEGF) with radiographic severity in primary osteoarthritis (OA) of the knee. Expression of these two factors in cartilage samples from OA knee joints was examined at mRNA and protein levels.ResultsCartilage degeneration correlated with the radiographic severity grade. OA severity, determined using the Mankin scale, correlated positively with the KL grade (r=0.8790, p<0.01), and HIF-2α and VEGF levels with the radiographic severity of knee OA (r=0.7001, p<0.05; r=0.6647, p<0.05).ConclusionIn OA cartilage, HIF-2α and VEGF mRNA and protein levels were significantly and positively correlated. The expression of both factors correlated positively with the KL grade. HIF-2α and VEGF, therefore, may serve as biochemical markers as well as potential therapeutic targets in knee OA.     

Effect of pulsed and continuous therapeutic ultrasound on healthy skeletal muscle in rats

Ultrasound therapy is used to treat injuries in joints, nerves and tendons. Part of the radiation generated is absorbed by nearby undamaged tissues, such as muscles. The aim was to evaluate histomorphological changes in the healthy gastrocnemius muscle in rats irradiated with continuous ultrasound (CUS) and pulsed ultrasound (PUS). Healthy adult rats were used, separated into two groups: CUS and PUS. Both were irradiated in the gastrocnemius muscle for 10 days: the CUS group in continuous mode (3 MHz, 1.0 W/cm², 1 min/session) and the PUS group in pulsed mode (3 MHz, 1.0 W/cm², 100 Hz, 50% duty cycle, 1 min/session). The contralateral muscles were used as a control. Their histological characteristics were analyzed, and the area and perimeter of the muscle fibers were measured. The connective tissue showed no histological changes. The area of muscle fibers of the irradiated groups was significantly greater (CUS 1325.2±182.1 μm², p=0.0278 and PUS 1019.4±125.3 μm², p=0.0398) than the control, and the CUS area was greater than the PUS (p=0.0383). The perimeter of muscle fibers showed significant differences between the irradiated groups (CUS 148±11.12 μm, p=0.0178 and PUS 129.3±8.83 μm, p=0.0236) compared to the control, as well as differences between CUS and PUS (p=0.0319). The application of ultrasound on healthy muscle produces hypertrophy of the muscle fibers, greater when continuous mode is used. It is advisable to apply pulsed, focused ultrasound therapies with sound heads sufficient for the tissue or zone to be treated, thereby reducing the risk of altering the adjacent healthy tissue.     

Dynamic expression of transforming growth factor-betas (TGF-β) and their type I and type II receptors in the synovial tissue of arthritic rats

A well characterized animal model that shares many characteristic features with rheumatoid arthritis (RA) is collagen-induced arthritis (CIA) in DA rats. Recent studies have demonstrated that TGF-β, a multifunctional cytokine, is an important modulator of the immune response in CIA, and possibly also in RA. In this study we have investigated the expression of the precursor forms of TGF-β1, TGF-β2, TGF-β3, as well as TGF-β type I receptor (TGF-βRI) and TGF-β type II receptor (TGF-βRII) in the synovial tissue of arthritic rats during the course of the disease. By using immunohistochemical techniques, an abundant expression of all three TGF-β isoforms and their receptors was observed in the arthritic synovia, an expression that increased with time after onset of disease. Antibodies to TGF-β1, TGF-β2, TGF-βRI and TGF-βRII stained blood vessels intensively, already at the early onset of inflammation, whereas the synovial lining layer and chondrocytes expressed strong immunoreactivity later on in the inflammatory process. The most intense staining with these antibodies was detected in fibroblasts within fibrotic tissue, in particular at the cartilage–pannus junction. Interestingly, TGF-β3 only stained macrophage-like cells and chondrocytes in the synovia. The data suggest that the abundant expression of TGF-β1, TGF-β2, TGF-β3 as well as TGF-βRI and TGF-βRII in the synovia, is of pathogenic importance in the development of CIA, although the question of how the different TGF-β isoforms may enhance or counteract different arthritogenic events remains open.     

Nox2 contributes to cardiac fibrosis in diabetic cardiomyopathy in a transforming growth factor-β dependent manner

Purpose: This study aimed to investigate the effect of Nox2 on cardiac fibrosis and to elucidate the regulatory mechanism of Nox2 in the development of DCM. Methods: We established normal and insulin-resistant cellular model using neonatal rat cardiac fibroblasts. Then Nox2-specific siRNA were transfected into cardiac fibroblasts with Lipofectamine ® 2000 and crambled siRNA sequence was considered as control. Meanwhile, a part of cells were randomly selected to be treated with or without transforming growth factor-β (TGF-β). Moreover, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were respectively performed to determine the expression level of related molecules, such as Nox2, collagen type I and III (COL I and III) and PI3K/AKT and PKC/Rho signaling pathway-related proteins. Results: TGF-β stimulation significantly increased the expression level of Nox2 both in mRNA and protein levels. Suppression of the Nox2 markedly decreased the expression of COL I and COL III in normal and insulin-resistant cellular model with TGF-β stimulation. Moreover, suppression of the Nox2 significantly decreased the expression of PI3K/AKT and PKC/Rho signaling pathway-related proteins in insulin-resistant cellular model with TGF-β stimulation. However, suppression of Nox2 had no effects on these proteins without TGF-β stimulation. Conclusions: Our finding reveals that Nox2 may promote synthesis of COL I and III via involved in PI3K/AKT and PKC/Rho signaling pathway in a TGF-β dependent manner and consequently promote cardiac fibrosis in the development of DCM.     

Palmitate-induced Regulation of PPARγ via PGC1α: a Mechanism for Lipid Accumulation in the Liver in Nonalcoholic Fatty Liver Disease

The aim was to examine the effect of free fatty acids on the regulation of PPARγ-PGC1α pathway, and the effect of PPARγ/PGC1α in NAFLD. The mRNA and protein expression of PGC1α and phospho/total PPARγ were examined in Huh7 cells after the palmitate/oleate treatment with/without the transfection with siRNA against PGC1a. The palmitate content, mRNA and protein expression of PGC1α and PPARγ in the liver were examined in the control and NAFLD mice. Palmitate (500 μM), but not oleate, increased protein expression of PGC1α and phospho PPARγ (PGC1α, 1.42-fold, P=0.038; phospho PPARγ, 1.56-fold, P=0.022). The palmitate-induced PPARγ mRNA expression was reduced after the transfection (0.46‑fold), and the protein expressions of PGC1α (0.52-fold, P=0.019) and phospho PPARγ (0.43-fold, P=0.011) were suppressed in siRNA-transfected cells. The palmitate (12325.8 ± 1758.9 μg/g vs. 6245.6 ± 1182.7 μg/g, p=0.002), and mRNA expression of PGC1α (11.0 vs. 5.5, p=0.03) and PPARγ (4.3 vs. 2.2, p=0.0001) in the liver were higher in high-triglyceride liver mice (>15.2 mg/g) than in low-triglyceride liver mice (<15.2 mg/g). The protein expressions of both PGC1α and PPARγ were higher in the NAFLD group than in the controls (PGC1α, 1.41-fold, P=0.035; PPARγ, 1.39-fold, P=0.042), and were higher in the high-triglyceride liver group (PGC1α, 1.52-fold, p=0.03; PPARγ, 1.22-fold, p=0.05) than in the low-triglyceride liver group. In conclusion, palmitate appear to up-regulate PPARγ via PGC1α in Huh7 cells, and both PGC1α and PPARγ are up-regulated in the NAFLD mice liver, suggesting an effect on lipid metabolism leading to intrahepatic triglyceride accumulation.     

The differential expression of TGF-β1, ILK and wnt signaling inducing epithelial to mesenchymal transition in human renal fibrogenesis: an immunohistochemical study

Epithelial-to-mesenchymal transition (EMT) is a process for fully differentiated epithelial cells to undergo a phenotypic change to fibroblasts via diverse intracellular signaling pathways. While the pivotal role of fibroblasts in renal fibrosis is widely accepted, their origin remains undefined. In addition, although a large number of studies have provided evidence of EMT in human kidney diseases, specific signaling pathways leading to EMT have not yet been discovered in humans. To evaluate the origin of interstitial fibroblasts and signaling pathways involved in the EMT process, we analyzed the differential expression of EMT-related molecules in paraffin-fixed sections from 19 human fibrotic kidneys and 4 control kidneys. In human fibrotic kidneys, tubular epithelial cells (TECs) with intact tubular basement membrane (TBM) showed loss or down-regulation of an epithelial marker (E-cadherin), de novo expression of mesenchymal markers (vimentin and fibronectin), and significant up-regulation of inducers and mediators controlling the EMT process (transforming growth factor-β1 (TGF-β1), p-Smad2/3, β1-integrin, p38 mitogen-activated protein kinase (MAPK), WNT5B and β-catenin) in the areas of interstitial inflammation and fibrosis, compared with their expression in control kidneys. In conclusion, the type II EMT process in humans is thought to be an adaptive response of TECs to chronic injury and is regulated by interconnections of TGF-β/Smad, integrin/integrin-linked kinase (ILK) and wnt/β-catenin signaling pathways.