Matrix Metalloproteinase-9 mRNA Expression Research Articles

Time-course and muscle-specific gene expression of matrix metalloproteinases and inflammatory cytokines in response to acute treadmill exercise in rats.

The extracellular matrix (ECM) of skeletal muscle plays a pivotal role in tissue repair and growth, and its remodeling tightly regulated by matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and inflammatory cytokines. This study aimed to investigate changes in the mRNA expression of MMPs (Mmp-2 and Mmp-14), TIMPs (Timp-1 and Timp-2), and inflammatory cytokines (Il-1β, Tnf-α, and Tgfβ1) in the soleus (SOL) and extensor digitorum longus (EDL) muscles of rats following acute treadmill exercise. Additionally, muscle morphology was examined using hematoxylin and eosin (H&E) staining. Male rats were subjected to acute treadmill exercise at 25m/min for 60min with a %0 slope. The mRNA expression of ECM components and muscle morphology in the SOL and EDL were assessed in both sedentary and exercise groups at various time points (immediately (0) and 1, 3, 6, 12, and 24h post-exercise). Our results revealed a muscle-specific response, with early upregulation of the mRNA expression of Mmp-2, Mmp-14, Timp-1, Timp-2, Il-1β, and Tnf-α observed in the SOL compared to the EDL. A decrease in Tgfβ1 mRNA expression was evident in the SOL at all post-exercise time points. Conversely, Tgfβ1 mRNA expression increased at 0 and 3h post-exercise in the EDL. Histological analysis also revealed earlier cell infiltration in the SOL than in the EDL following acute exercise. Our results highlight how acute exercise modulates ECM components and muscle structure differently in the SOL and EDL muscles, leading to distinct muscle-specific responses.

Anti-rheumatoid arthritis effects of traditional Chinese medicine Fufang Xiaohuoluo pill on collagen-induced arthritis rats and MH7A cells.

Fufang Xiaohuoluo pill (FFXHL) is a commonly used prescription in clinical practice for treating rheumatoid arthritis in China, yet its specific mechanism remains unclear. This study aims to elucidate the pharmacological mechanisms of FFXHL using both in vivo and in vitro experiments. The collagen-induced arthritis (CIA) rat model was established to evaluate FFXHL's therapeutic impact. Parameters that include paw swelling, arthritis scores, and inflammatory markers were examined to assess the anti-inflammatory and analgesic effects of FFXHL. Human fibroblast-like synoviocytes (MH7A cells) is activated by tumour necrosis factor-alpha (TNF-α) were used to explore the anti-inflammatory mechanism on FFXHL. Our findings indicate that FFXHL effectively reduced paw swelling, joint pain, arthritis scores, and synovial pannus hyperplasia. It also lowered serum levels of TNF-α, interleukin-1β (IL1β), and interleukin-6 (IL-6). Immunohistochemical analysis revealed decreased expression of nuclear factor-kappa B (NF-κB) p65 in FFXHL-treated CIA rat joints. In vitro experiments demonstrated FFXHL's ability to decrease protein secretion of IL-1β and IL-6, suppress mRNA expression of matrix metalloproteinases (MMP) -3, -9, and -13, reduce reactive oxygen species (ROS) levels, and inhibit NF-κB p65 translocation in TNF-α stimulated MH7Acells. FFXHL also suppressed protein levels of extracellular signal-regulated kinase (ERK), c-Jun Nterminal kinase (JNK), p38 MAP kinase (p38), protein kinase B (Akt), p65, inhibitor of kappa B kinase α/β (IKKα/β), Toll-like receptor 4 (TLR4), and myeloid differentiation primary response 88 (MyD88) induced by TNF-α in MH7A cells. The findings imply that FFXHL exhibits significant anti-inflammatory and antiarthritic effects in both CIA rat models and TNF-α-induced MH7A cells. The potential mechanism involves the inactivation of TLR4/MyD88, mitogen-activated protein kinases (MAPKs), NF-κB, and Akt pathways by FFXHL.

Effect of Resveratrol on MMP-2 Expression in Scleral Fibroblasts: An In Vitro Study.

To investigate the effects of resveratrol (Res) on human fetal scleral fibroblasts (HFSFs) and its potential mechanism. HFSFs were randomly divided into the Res-treated group and the control group. Following, HFSFs were treated with or without a concentration of 10 μM Res for 48 h. To detect the expression of related genes, reverse transcription quantitative PCR (RT-qPCR) and western blotting were used. The apoptosis rate of different groups was determined using flow cytometry. The mRNA expression of matrix metalloproteinase 2 (MMP-2), Collagen, Type I, Alpha 1 (COL1A1), Janus Kinase 2 (JAK2), and Signal Transducer and Activator of Transcription 3 (STAT3)" was downregulated in the Res-treatment group compared to the control group, according to RT-qPCR. Western blotting revealed that Res therapy reduced the expression of MMP-2, JAK2, P-JAK2, STAT3, P-STAT3, and Bcl-2 associated protein X (Bax) while increasing the expression of COL1A1 and B-cell lymphoma-2 (Bcl-2). Flow cytometry showed that the cell apoptosis rate was significantly lower in HFSFs treated with Res. In conclusion, these findings suggest that Res increases COL1A1 expression while inhibiting MMP-2 and cell apoptosis in HFSFs, possibly through modulation of the JAK2/STAT3 signaling pathway.

Guggulsterone protects against cigarette smoke-induced COPD linked lung inflammation.

Recently, we have shown that guggulsterone is the principal constituent responsible for protective effects of Commiphora wightii against elastase-induced chronic obstructive pulmonary disease (COPD)-linked inflammation/emphysema. Given that cigarette smoke (CS) exposure is a primary risk factor for COPD and beneficial effects of guggulsterone have not been investigated in CS-induced COPD-linked lung inflammation. The present work was designed to validate the potential of guggulsterone in amelioration of COPD-linked lung inflammation by using a CS-based mouse model of the condition. Male BALB/c mice were exposed to 9 cigarettes/day with 1 h interval for 4 days daily. Guggulsterone was administered daily at a dose of 10 mg/kg orally for 4 consecutive days, 1 h before initiation of CS exposure. Mice were subjected to measurement of lung function followed by procurement of bronchoalveolar lavage fluid (BALF)/lung tissue. BALF was analyzed for inflammatory cells and pro-inflammatory cytokines. Lung tissue was subjected to RT-PCR for gene expression analysis. Data showed that CS exposure resulted in a significant increase in total BALF cells, predominantly neutrophils, and macrophages. Interestingly, guggulsterone administration significantly blunted CS-induced inflammation as reflected by reduced neutrophil and macrophage count. Further, the compound inhibited CS-induced gene expression of pro-inflammatory mediators TNF-α/ IL-1β/ G-CSF/and KC in lungs along with the production of pro-inflammatory mediators TNF-α/ IL-1β/ IL-6/ G-CSF/ KC/and MCP-1 in BALF. Further, guggulsterone improved the lung function parameters upon CS exposure. Analysis of mRNA expression of matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 suggests that guggulsterone may restore the fine balance between matrix-degrading proteases and its inhibitor in lung tissue upon CS exposure, which may contribute in the development of emphysema at later stages. Overall, our data show that guggulsterone protects against CS-induced COPD-linked lung inflammation by modulating relevant molecular players. Based on the potential effects of guggulsterone in the amelioration of CS-induced lung inflammation, we speculate that guggulsterone might alter chronic CS-induced emphysema.

Impact of hyperoxia on the phenotype of pulmonary artery smooth muscle cells

Objective: To investigate the influence of varied oxygen (O2) concentration environments on the phenotypic transformation of pulmonary artery smooth muscle cells (PASMC) and the mechanism of pulmonary hypertension. Methods: Primary rat PASMC were isolated and cultured through the process of enzymatic digestion. Following identification, the stable passaged PASMC were subjected to a 6-hour incubation in sealed containers with normal O2 content (group C) and relative O2 content comprising 55% (group H55), 75% (group H75), and 95% (group H95). mRNA and protein expression of α-Actin (α-SMA), smooth muscle 22α (SM22α), osteopontin (OPN), and matrix metalloproteinase-2 (MMP-2) were measured using real-time quantitative PCR and western blot analysis. Results: The H55 group displayed no significant difference from the C group in terms of mRNA and relative protein expression levels for α-SMA, SM22α, OPN, and MMP-2 (all P>0.05). On the other hand, groups H75 and H95 exhibited a reduction in mRNA and relative protein expression of α-SMA and SM22α, along with an increase in mRNA and relative protein expression of OPN and MMP-2 when compared with both the C and H55 groups (all P<0.05). The H95 group showed a higher relative mRNA expression of MMP-2 as compared to the H75 group (P<0.05). Conclusions: Oxygen concentration environments of 75% or higher can serve as the foundation for the pathogenesis of pulmonary hypertension, essentially by inducing a phenotypic transformation in PASMC towards adopting a robust secretory function. This induction is contingent upon the concentration of oxygen present.

Anti-osteoarthritis, Bone Protective and Antiinflammatory Effect of Lusianthridin against Monosodium Iodoacetate Induced Osteoarthritis via Suppression of Inflammatory Pathway.

Osteoarthritis (OA) is characterized by the gradual deterioration and worsening of the knee joint, leading to both pain and deformity. The current research exhibited the anti-osteoarthritis effect of lusianthridin against monosodium iodoacetate (MIA) induced OA in rats. RAW cells were used for the cell viability. The inflammatory cytokines and mediators were estimated in the cell lines after the lipopolysaccharide (LPS) treatment. For the in vivo study, the rats were received the intraperitoneal administration of MIA (3 mg/kg) for the induction of OA. The rats were received the oral administration of lusianthridin (5, 10 and 20 mg/kg) and the body and organ weight estimated. Antioxidant, cytokines, inflammatory and matrix metalloproteinases (MMP) level were also estimated. The mRNA expression of MMP were also estimated. The lusianthridin treatment remarkably suppressed the cell viability. LPS induced RAW cell suppressed the level of nitrate, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), prostaglandin (PGE2), MMP-2 and MMP-9 level. Lusianthridin remarkably altered the level of body weight and organ weight (liver, spleen, renal and heart weight). lusianthridin suppressed the oxidative stress via altered the level of antioxidant parameters. Lusianthridin significantly (p < 0.001) decreased the level of cartilage oligometrix matrix protein (COMP) and c-reactive protein (CRP); cytokines such as TNF-α, IL-1β, IL-6, IL-10; inflammatory parameters include 5- Lipoxygenase (5-LOX), COX-2, leukotriene B4 (LTB4), PGE2; transforming growth factor beta (TGF-β); MMP level like MMP-1, 3, 9, 13, respectively. Lusianthridin significantly suppressed the mRNA expression of MMP. Collectively, the result of the study showed that antiosteoarthritis effect of lusianthridin via suppression of inflammatory parameters.

Chronic Iodine Intake Excess Damages the Structure of Articular Cartilage and Epiphyseal Growth Plate.

This study aimed to explore the influence of excess iodine on the articular cartilage and epiphyseal growth plate in rats. Wistar rats (n = 200) were randomly divided into five groups with 40 rats in each: normal iodine (NI), 5-fold high iodine group (5HI), 10-fold high iodine group (10HI), 50-fold high iodine group (50HI), and 100-fold high iodine group (100HI). The rats were executed in 6 and 12months. 24-h urinary iodine concentration (UIC) was monitored by arsenic-cerium catalytic spectrophotometry. The chemiluminescence method was used to determine the thyroid function. The pathological changes in the epiphyseal plate, articular cartilage, and thickness of the epiphyseal plate were observed. The mRNA expression of collagen II (ColII), collagen X, matrix metalloproteinase-13 (MMP-13), and fibroblast growth factor receptor 1 in articular chondrocytes was detected by RT-PCR. 24-h UIC increased as iodine intake increased. In the 12th month, there was a significant increase in serum sTSH and a decrease in serum FT4 in HI groups, compared to the NI group. There was a decrease in the number of proliferating cells in the epiphyseal plate and an increase in the number of mast cell layers. The chondrocytes appeared disorganized, and the tidal lines were disturbed or even broken. Growth plate thickness decreased with increasing iodine intake. Compared with the NI group, ColII and MMP-13 mRNA expression in chondrocytes in all HI groups significantly increased. Chronic iodine overdose increases the risk of hypothyroidism. Chronic iodine overdose leads to abnormal morphology of epiphyseal growth plates and articular cartilage, increasing the risk of osteoarthritis.

Naringenin attenuates inflammation and apoptosis of osteoarthritic chondrocytes via the TLR4/TRAF6/NF-κB pathway.

Naringenin is a flavonoid extracted from the seed coat of Anacardiaceae plants. Increasing evidence indicates that it has several properties of biological significance, such as anti-infection, sterilization, anti-allergy, antioxidant free radical, and anti-tumor. However, its effect on osteoarthritis has not been elucidated properly. In this study, the treatment of primary chondrocytes with interleukin (IL)-1β was found to increase the secretions of IL-6, tumor necrosis factor (TNF)-α, and cyclooxygenase-2 (COX-2). Further, the mRNA expression of matrix metalloproteinase ((MMP)3, MMP9, and MMP13), the protein expression of Recombinant A Disintegrin And Metalloproteinase With Thrombospondin 5 (ADAMTS5), and cell apoptosis increased; the protein expression of Collagen II decreased. The injury of primary chondrocytes induced by IL-1β was reversed under the intervention of naringenin; this reversal was dose-dependent. The mechanistic study showed that naringenin inhibited the toll-like receptor 4 (TLR4)/TNF receptor-associated factor 6 (TRAF6)/NF-κB pathway in IL-1β-stimulated primary cells, and LPS, a TLR4 activator, reversed this inhibitory effect. In addition, a mouse model of osteoarthritis was established and treated with naringenin. The results revealed that naringenin alleviated the pathological symptoms of osteoarthritis in mice, reduced the expression of TLR4 and TRAF6, and the phosphorylation of NF-κB in knee cartilage tissue. It also inhibited the secretion of inflammatory factors, reduced extracellular matrix degradation, and decreased the protein expression of cleaved caspase3. In conclusion, the findings of this study suggest that naringenin may be a potential option for the treatment of osteoarthritis.

Leptin Induces MMP-1 Expression Through the RhoA/ERK1/2/NF-κB Axis in Human Intervertebral Disc Cartilage Endplate-Derived Stem Cells.

Intervertebral disc (IVD) degeneration, associated with aging, may cause low back pain and disability, with obesity as a significant risk factor. In a prior study, we found a positive correlation between IVD degeneration and levels of matrix metalloproteinase-1 (MMP-1) and leptin. Yet, the interaction between MMP-1 and leptin in IVD degeneration is unclear. Our research seeks to explore leptin's influence on MMP-1 expression and the underlying mechanisms in human intervertebral disc cartilage endplate-derived stem cells, specifically SV40 cells. The mRNA and protein expression in leptin-stimulated SV40 cells were assessed using RT-real-time PCR and Western blotting or ELISA, respectively. We examined leptin-mediated RhoA activation through a GTP-bound RhoA pull-down assay. Furthermore, the phosphorylation levels of mitogen-activated protein kinases and AKT in leptin-stimulated SV40 cells were analyzed using Western blotting. The activation of NF-κB by leptin was investigated by assessing phosphorylation of IKKα/β, IκBα, and NF-κB p65, along with the nuclear translocation of NF-κB p65. To understand the underlying mechanism behind leptin-mediated MMP-1 expression, we employed specific inhibitors. Leptin triggered the mRNA and protein expression of MMP-1 in SV40 cells. In-depth mechanistic investigations uncovered that leptin heightened RhoA activity, promoted ERK1/2 phosphorylation, and increased NF-κB activity. However, leptin did not induce phosphorylation of JNK1/2, p38, or AKT. When we inhibited RhoA, ERK1/2, and NF-κB, it resulted in a decrease in MMP-1 expression. Conversely, inhibition of reactive oxygen species and NADPH oxidase did not yield the same outcome. Additionally, inhibiting RhoA or ERK1/2 led to a reduction in leptin-induced NF-κB activation. Moreover, inhibiting RhoA also decreased leptin-mediated ERK1/2 phosphorylation. These results indicated that leptin induced MMP-1 expression in SV40 cells through the RhoA/ERK1/2/NF-κB axis. This study provided the pathogenic role of leptin and suggested the potential therapeutic target for IVD degeneration.

Identification of Molecular Mechanisms Responsible for the MMP-9-1562C/T Dependent Differential Regulation of Matrix Metalloproteinase-9 Expression in Human Neuron-like Cells.

The MMP-9-1562C/T polymorphism exerts an impact on the occurrence and progression of numerous disorders affecting the central nervous system. Using luciferase assays and Q-RT-PCR technique, we have discovered a distinct allele-specific influence of the MMP-9-1562C/T polymorphism on the MMP-9 (Extracellular Matrix Metalloproteinase-9) promoter activity and the expression of MMP-9 mRNA in human neurons derived from SH-SY5Y cells. Subsequently, by employing a pull-down assay paired with mass spectrometry analysis, EMSA (Electromobility Shift Assay), and EMSA supershift techniques, as well as DsiRNA-dependent gene silencing, we have elucidated the mechanism responsible for the allele-specific impact of the MMP-9-1562C/T polymorphism on the transcriptional regulation of the MMP-9 gene. We have discovered that the activity of the MMP-9 promoter and the expression of MMP-9 mRNA in human neurons are regulated in a manner that is specific to the MMP-9-1562C/T allele, with a stronger upregulation being attributed to the C allele. Furthermore, we have demonstrated that the allele-specific action of the MMP-9-1562C/T polymorphism on the neuronal MMP-9 expression is related to HDAC1 (Histone deacetylase 1) and ZNF384 (Zinc Finger Protein 384) transcriptional regulators. We show that HDAC1 and ZNF384 bind to the C and the T alleles differently, forming different regulatory complexes in vitro. Moreover, our data demonstrate that HDAC1 and ZNF384 downregulate MMP-9 gene promoter activity and mRNA expression in human neurons acting mostly via the T allele.

Effects of prenatal mobile phone radiation exposure on MMP9 expression: Implications for inflammation, oxidative stress, and sensory-motor impairment after neonatal hypoxia- ischemia in rats

ObjectiveNon-ionizing radiofrequency radiation, which finds application in various sectors such as industry, commerce, medicine, and particularly in mobile phone technology, has emerged as a topic of significant concern during pregnancy. The aim of this study was to investigate the effect of cell phone radio-frequency (RF) radiation during pregnancy on the Matrix metalloproteinase 2 (MMP-2) and (MMP-9) 9 expressions after neonatal hypoxia-ischemia (HI) in rats. Materials and methodsTwo groups were formed by randomly assigning female Wistar rats: Group 1 consisted of female rats that were not exposed to RF radiation during pregnancy, while Group 2 comprised female rats that were exposed to RF radiation during pregnancy. After delivery, male offspring were divided into four groups including: (a) SHAM, (b) Exposure (EXP), (c) hypoxia-ischemia (HI), (d) HI/Exposure (HI/EXP). Seven days after HI induction, neurobehavioral tests were performed, and then brain tissue was taken from the skull to measure MMP-2 and MMP-9 expressions, inflammation, oxidative stress, infarct volume and cerebral edema. ResultsMMP-9 mRNA expression in the HI/EXP group was significantly higher than the HI, SHAM and EXP groups. MMP-2 mRNA expression levels in the HI group were significantly higher than Sham and the EXP groups.TNF-α and Total oxidant capacity (TOC) levels in the HI/EXP group were significantly higher than HI, EXP and SHAM groups. Total antioxidant capacity (TAC) level in the HI/EXP group were significantly lower than HI, EXP and SHAM groups. Cerebral edema and infarct volume in the HI/EXP group were significantly greater than the HI group. Sensory-motor function was significantly weaker in HI/EXP as compared HI group. ConclusionOur findings indicate that during pregnancy, exposure to mobile phone RF radiation intensifies damage from HI in rat pups by elevating MMP-9 activity.

Synthesis and secretion of Nerve Growth Factor is regulated by Nitric Oxide in bladder cells in vitro under a hyperglycemic environment

Urine samples of female patients with overactive bladder (OAB) are characterized by low levels of nerve growth factor (NGF) and elevated concentrations of nitric oxide (NO) compared to healthy controls. We therefore examined how NO might regulate NGF synthesis using rat bladder smooth muscle (SMCs) and urothelial (UROs) cells in culture. In UROs, incubation in hyperglycemic conditions to mimic insulin insensitivity present in the OAB cohort increased secretion of NO and concomitantly decreased NGF, except when the NO synthase inhibitor, l-NAME (1 mM) was present. Sodium nitroprusside (SNP) (300 μM, 24 h), a NO generator, decreased NGF levels and decreased cyclic GMP (cGMP) content, a process validated by the cGMP synthase inhibitor ODQ (100 μM). Alternatively, SNP increased mRNA of both NGF and matrix metalloproteinase-9 (MMP-9). MMP-9 knockout of UROs by Crispr-Cas9 potently decreased the effect of SNP on NGF, implying a dependent role of NO on MMP-9. On the other hand, matrix metalloproteinase-7 (MMP-7) activity was increased by SNP, which taken together with increase in NGF mRNA, suggests a compensatory mechanism. In SMCs, hyperglycemic conditions had the same effect on extracellular content of NO and NGF than in UROs. SNP also decreased NGF secretion but increased cGMP content. Stable permeable analogs of cGMP 8-(4-Chlorophenylthio)-cGMP (1 mM) and N2,2′-O-Dibutyryl-cGMP (3 mM) inhibited NGF release. NGF and MMP-9 mRNA expression was unchanged by SNP. Deletion of MMP-9 in SMCs by Crispr-Cas9 did not alter the effect of SNP. Finally, SNP decreased MMP-7 activity, diminishing the conversion of proNGF to NGF. These results demonstrate that enhanced NO secretion triggered by high glucose decreases NGF secretion through pathways unique for each cell type that involve cGMP and proteases MMP-7 and MMP-9. These results might help to explain our observations from the urine from patients with OAB associated with metabolic syndrome.

Effect of acetylalkannin from Arnebia euchroma on proliferation, migration, and invasion of human melanoma A375 cells

This study aimed to explore the effect and mechanism of acetylalkannin from Arnebia euchroma on the proliferation, migration, and invasion of human melanoma A375 cells. A375 cells were divided into a blank group, and low-, medium-, and high-dose acetylalkannin groups(0.5, 1.0, and 2.0 μmol·L~(-1)). The MTT assay was used to detect cell proliferation. Cell scratch and transwell migration assays were used to detect cell migration ability, and the transwell invasion assay was used to detect cell invasion ability. Western blot was used to detect the protein expression of migration and invasion-related N-cadherin, vimentin, matrix metalloproteina-se-9(MMP-9), and Wnt/β-catenin pathway-related Wnt1, Axin2, glycogen synthase kinase-3β(GSK-3β), phosphorylated GSK-3β(p-GSK-3β), β-catenin, cell cycle protein D_1(cyclin D_1), and p21. Real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR) was used to detect the mRNA expression of E-cadherin, matrix metalloproteinase-2(MMP-2), N-cadherin, vimentin, β-catenin, snail-1, and CD44. MTT results showed that the cell inhibition rates in the acetylalkannin groups significantly increased as compared with that in the blank group(P&lt;0.01). The results of cell scratch and transwell assays showed that compared with the blank group, the acetylalkannin groups showed reduced cell migration and invasion, and migration and invasion rates(P&lt;0.05, P&lt;0.01) and weakened horizontal and vertical migration and invasion abilities. Western blot results showed that compared with the blank group, the high-dose acetylalkannin group showed increased expression of Axin2 protein(P&lt;0.05), and decreased expression of N-cadherin, vimentin, MMP-9, Wnt1, p-GSK-3β, β-catenin, cyclin D_1, and p21 proteins(P&lt;0.05, P&lt;0.01). The expression of GSK-3β protein did not change significantly. PCR results showed that the overall trend of MMP-2, N-cadherin, vimentin, β-catenin, snail-1, and CD44 mRNA expression was down-regulated(P&lt;0.01), and the expression of E-cadherin mRNA increased(P&lt;0.01). Acetylalkannin can inhibit the proliferation, migration, and invasion of human melanoma A375 cells, and its mechanism of action may be related to the regulation of Wnt/β-catenin signaling pathway.

Effects of Brimonidine, Omidenepag Isopropyl, and Ripasudil Ophthalmic Solutions to Protect against H2O2-Induced Oxidative Stress in Human Trabecular Meshwork Cells

Purpose We investigated whether hydrogen peroxide (H2O2)-induced oxidative stress causes human trabecular meshwork (HTM) cell dysfunction observed in open angle glaucoma (OAG) in vitro, and the effects of topical glaucoma medications on oxidative stress in HTM cells. Methods We used commercially available ophthalmic solutions of brimonidine, omidenepag isopropyl, and ripasudil in the study. HTM cells were exposed to H2O2 for 1 h, with or without glaucoma medications. We assessed cell viability and senescence via WST-1 and senescence-associated-β-galactosidase (SA-β-Gal) activity assays. After exposure to H2O2 and glaucoma medications, we evaluated changes in markers of fibrosis and stress by using real-time quantitative polymerase chain reaction (qPCR) to measure the mRNA levels of collagen type I alpha 1 chain (COL1A1), fibronectin, alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP-2), endoplasmic reticulum stress markers of C/EBP homologous protein (CHOP), 78-kDa glucose-regulated protein (GRP78), and splicing X-box binding protein-1 (sXBP-1). Results HTM cell viability decreased and SA-β-Gal activity increased significantly after exposure to H2O2. Treatment with three ophthalmic solutions attenuated these changes. Real-time qPCR revealed that H2O2 upregulated the mRNA levels of COL1A1, fibronectin, α-SMA, CHOP, GRP78, and sXBP-1, whereas it downregulated MMP-2 mRNA expression significantly. Brimonidine suppressed the upregulation of stress markers CHOP and GRP78. Additionally, omidenepag isopropyl and ripasudil decreased the upregulation of COL1A1 and sXBP-1. Furthermore, ripasudil significantly suppressed fibrotic markers fibronectin and α-SMA, compared with the other two medications. Conclusion In vitro, H2O2 treatment of HTM cells induced characteristic changes of OAG, such as fibrosis changes and the upregulation of stress markers. These glaucomatous changes were attenuated by additional treatments with brimonidine, omidenepag isopropyl, and ripasudil ophthalmic solutions.

Sleep deprivation disturbs uterine contractility and structure in pregnant rats: role of matrix metalloproteinase 9 and transforming growth factor-β.

Sleep deprivation (SD) during pregnancy can impact the delivery procedure, with prolongation of the labor duration. Matrix metalloproteinase-9 (MMP9) and transforming growth factor-β (TGF-β) are regulators of uterine remodeling. Their dysregulation is vital for abnormal placentation and uterine enlargement in complicated pregnancies. Therefore, this study aims to explore the outcome of SD throughout pregnancy on ex vivo uterine contractility, MMP9 and TGF-β, and uterine microscopic structure. A total of 24 pregnant rats were divided into two groups. From the first day of pregnancy, animals were exposed to partial SD/6h/day. Uterine in vitro contractile responses to oxytocin, acetylcholine, and nifedipine were assessed. Additionally, uterine levels of superoxide dismutase and malondialdehyde and uterine mRNA expression of MMP9, TGF-β, and apoptotic biomarkers were analyzed. The results showed that SD significantly reduced uterine contractile responses to oxytocin and acetylcholine, while it augmented the relaxing effect of nifedipine. In addition, it significantly increased oxidative stress status, MMP9, TGF-β, and apoptotic biomarkers' mRNA expression. All were accompanied by degeneration of endometrial glands, vacuolization with apoptotic nuclei, and increased area% of collagen fibers. Finally, increased uterine MMP9 and TGF-β mRNA expression during SD clarified their potential role in modulating uterine contractility and structure.

All-trans retinoic acid regulates the expression of MMP-2 and TGF-β2 via RDH5 in retinal pigment epithelium cells.

To investigate the effect of all-trans retinoic acid (ATRA) on retinol dehydrogenase 5 (RDH5), matrix metalloproteinase-2 (MMP-2) and transforming growth factor-β2 (TGF-β2) transcription levels, and the effect of RDH5 on MMP-2 and TGF-β2 in retinal pigment epithelium (RPE) cells. After adult RPE cell line-19 (ARPE-19 cells) intervened with gradient concentrations of ATRA (0-20 µmol/L) for 24h, flow cytometry was used to detect the proliferation and apoptosis of cells in each group, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect RDH5, MMP-2 and TGF-β2 mRNA expression. Then, after ARPE-19 cells transfected with three different siRNA targets for 48h, the RDH5 knockdown efficiency of each group and expression of MMP-2 and TGF-β2 mRNA within them was detected by qRT-PCR. Flow cytometry results showed that ATRA could inhibit the proliferation of RPE cells and promote the apoptosis of RPE cells, and the difference of apoptosis was statistically significant when the ATRA concentration exceeded 5 µmol/L and compared with the normal control group (P=0.027 and P=0.031, respectively). qRT-PCR results showed that ATRA could significantly inhibit the expression level of RDH5 mRNA (P<0.001) and promote the expression of MMP-2 and TGF-β2 mRNA (P=0.03 and P<0.001, respectively) in a dose-dependent manner, especially when treated with 5 µmol/L ATRA. The knockdown efficiency of RDH5 siRNA varies with different targets, among which RDH5 siRNA-435 had the highest knockdown efficiency, i.e., more than 50% lower than that of the negative control group (P=0.02). When RDH5 was knocked down for 48h, the results of qRT-PCR showed that the expressions of MMP-2 and TGF-β2 mRNA were significantly up-regulated (P<0.001). ATRA inhibits the expression of RDH5 and promotes MMP-2 and TGF-β2, and further RDH5 knockdown significantly upregulates MMP-2 and TGF-β2. These findings suggest that RDH5 may be involved in an epithelial-mesenchymal transition of RPE cells mediated by ATRA.

Effects of firocoxib, trimethoprim sulfamethoxazole and altrenogest on inflammation and foal survival after administration to mares with experimentally induced placentitis

Bacterial placentitis is a leading cause of pregnancy loss in mares. Infection, inflammation and prostaglandin production induce preterm delivery leading to poor foal survival. Treatment strategies for placentitis that control infection and inhibit inflammation are vital. The goals of this study were: 1. Assess the anti-inflammatory effects of firocoxib, trimethoprim sulfamethoxazole (TMS) and altrenogest administered to mares with placentitis; 2. Determine the efficacy of firocoxib, TMS and altrenogest for improving foal outcome in mares with placentitis. We hypothesized that this treatment would significantly decrease pro-inflammatory mediators in fetal fluids and tissues when compared to untreated, infected mares and would also improve foal survival. Placentitis was induced in 13 pregnant mares (270-300 days gestation; via intracervical inoculum of Streptococcus zooepidemicus). Mares were randomly assigned to: infected/untreated (UNTREATED; n=6); or infected/treated groups (TREATED; n=7; administered firocoxib, TMS, and altrenogest from the onset of clinical signs of placentitis until abortion/delivery). Fetal fluids, membranes and tissue samples were obtained at abortion/delivery for quantification of TNF-α, IL-1β, IL-10, IL-6, IL-8, PGE2, PGF2α, and matrix metalloproteinases (MMP) -1, -3, -9 using ELISA (fluids) and real-time PCR (tissues). P < 0.05 was considered significant. Gestation length and foal survival were significantly higher in TREATED (mean days post-infection = 40; 7/7 live foals) than UNTREATED (mean days post-infection = 14; 2/6 live foals). Administration of firocoxib, TMS andaltrenogest resulted in significantly lower amniotic fluid concentrations of IL-1β, IL-6, PGE2 and PGF2α in TREATED than UNTREATED. Amniotic fluid IL-10 concentration tended (P < 0.1) to be lower in TREATED than UNTREATED. Cytokine and prostaglandin concentrations in allantoic fluid were not different between groups except PGE2 which tended (P < 0.1) to be lower in TREATED than UNTREATED. The relative quantity of cytokine and MMP mRNA expression in fetal membranes and foal tissues (non-survivors) was log-transformed and estimated marginal means determined. Fetal membranes from TREATED had significantly lower mRNA expression for IL-8 and TNFα compared to membranes from UNTREATED. mRNA expression of IL-1β, IL-6 and MMP3 tended (P < 0.1) to be lower in membranes from TREATED than UNTREATED. mRNA expression of IL-1β, IL-8, IL-10, TNFα, MMP1, 3 and 9 were significantly different between fetal membrane sites. In non-surviving foals, IL-8 mRNA expression was significantly lower in liver than lung while MMP3 expression was significantly higher in liver than lung or spleen. These data provide physiologic evidence that administration of firocoxib, TMS and altrenogest to mares with placentitis has broad anti-inflammatory effects and may improve foal survival. Research funding: Grayson Jockey Club Research Foundation; Merial Animal Health; Kentucky Thoroughbred Breeders Association, University of Florida, University of Georgia.

Human gingival tissue‐derived mesenchymal stem cells inhibit proliferation and invasion of rheumatoid fibroblast‐like synoviocytes via the CD39/CD73 signaling pathway

AbstractBackgroundGingiva‐derived mesenchymal stem cells (GMSCs) suppress immune and inflammatory responses in experimental arthritis models. Here, we determined whether GMSCs suppress the proliferation and invasion of rheumatoid arthritis fibroblast‐like synoviocytes (RA FLSs).MethodsSurface markers of GMSCs were analyzed by flow cytometry. mRNA expression of matrix metalloproteinases and pro‐inflammatory cytokine in RA FLSs was measured by quantitative polymerase chain reaction (PCR). RA FLS proliferation was analyzed by a 5‐ethynyl‐2′‐deoxyuridine assay. To explore the molecular mechanisms, we assessed the effect of GMSCs on RA FLS proliferation by adding indoleamine‐2,3‐dioxygenase (IDO), CD39, or CD73 inhibitors. The invasion was analyzed by a transwell assay. The anti‐inflammatory effects of GMSCs were assessed in a type II collagen‐induced arthritis (CIA) mouse model.ResultsCompared with human dermal fibroblast, GMSCs displayed a higher expression of CD39. Interleukin (IL)‐8, IL‐6, MMP‐1, MMP‐3, MMP‐13, and monocyte chemoattractant protein‐1 mRNA were all decreased in RA FLSs after incubation with GMSCs. GMSCs significantly reduced RA FLS proliferation in a dose‐dependent manner in vitro, which was partly dependent on CD39/CD73 signaling. GMSCs significantly impeded the invasive capacity of RA FLSs in a dose‐dependent manner in vitro. GMSCs infusion delayed arthritis onset and reduced arthritis scores in the CIA model.ConclusionGMSCs are effective to inhibit the aggressive behavior of RA FLSs and treat experimental arthritis, implying their therapeutic potential in RA patients.

Therapeutic Effect of Acupotomy at Sanheyang for Cartilage Collagen Damage in Moderate Knee Osteoarthritis: A Rabbit Model.

Acupotomy based on the meridian-sinew theory of traditional Chinese medicine has benefits in treating knee osteoarthritis (KOA). The current study aims to prove that acupotomy at the sinew points of Sanheyang protect the knee joint and alleviate the progression of moderate KOA by evaluating KOA symptoms, cartilage structure, and analyzing the changes of cytokines in rabbit cartilage. The model used was mono-iodoacetate-induced moderate KOA in the rabbit's right leg. Rabbits were divided into the model group, the acupotomy group, and the control group, with each group receiving two parts of treatment for 2 weeks and 4 weeks. We evaluated pain in the knee joint and range of motion. The articular cartilage sections were stained with Safranin O/Fast Green and Masson. We used immunohistochemistry and real-time PCR to detect the protein and mRNA expressions of collagen prototype II (COL-II), matrix metalloproteinase 13 (MMP13), and integrin-β1 (ITG-β1). Compared with the model group, the acupotomy group had higher body weight, lower pain score, higher range of motion, lower Mankin score, and significantly lower protein and mRNA expression of MMP13. After 4 weeks of treatment, Col-II expression in the acupotomy group was significantly higher than that in the model group and the expression of ITG-β1 in the model group was abnormally increased. Acupotomy at Sanheyang improved the pain symptoms and range of joint motion in rabbits with moderate KOA, and could protect Col-II by regulating MMP13, which may be related to ITG-β1-mediated mechanical force transmission, thus reducing the damage to cartilage structure and delaying the progression of moderate KOA.

Expression of HMGB1-TLR4 in Placentas from Preeclamptic Pregnancies and Its Effect on Proliferation and Invasion of HTR-8/SVneo Cells

Objectives: Placental inflammation possibly underlies preeclampsia (PE) pathogenesis. This study aimed to investigate the expression of the high mobility box group 1 (HMGB1)-toll-like receptor 4 (TLR4) signalling pathway in preeclamptic placentas and determine whether HMGB1 regulates the biological behaviour of trophoblasts in vitro. Design: Placental biopsies were taken from 30 preeclamptic patients and 30 normotensive controls. In vitro experiments were carried out in HTR-8/SVneo human trophoblast cells. Participants/Materials, Setting, Methods: HMGB1, TLR4, and nuclear factor kappa B (NF-κB) mRNA and protein were quantified to compare their expression in human placentas from preeclamptic and normotensive pregnancies. HTR-8/SVneo cells were stimulated with HMGB1 (50–400 μg/L) for 6–48 h, and proliferation and invasion of HTR-8/SVneo cells were measured via Cell Counting Kit-8 and transwell assays. HTR-8/SVneo cells were also transfected with HMGB1 and TLR4 small interfering RNA (siRNA) to investigate the effect of knocking down these proteins. The mRNA and protein expression of TLR4, NF-κB, and matrix metalloproteinase 9 (MMP-9) were determined using quantitative real-time PCR and Western blotting, respectively. Data were analysed with either a t-test or one-way analysis of variance. Results: The mRNA and protein levels of HMGB1, TLR4, and NF-κB were significantly higher in the placentas from preeclamptic pregnancies than from normal pregnancies (p < 0.05). HMGB1 stimulation (at concentrations up to 200 μg/L) of HTR-8/SVneo cells significantly increased invasion and proliferation over time. However, at an HMGB1 stimulation concentration of 400 μg/L, the invasion and proliferation ability of HTR-8/SVneo cells decreased. Compared to controls, mRNA and protein expression levels of TLR4, NF-κB, and MMP-9 increased (mRNA level fold change: 1.460, 1.921, 1.667; protein level fold change: 1.600, 1.750, 2.047) when stimulated with HMGB1 (p < 0.05) but decreased when HMGB1 was knocked down (p < 0.05). TLR4 siRNA transfection combined with HMGB1 stimulation reduced the mRNA (fold change: 0.451) and protein (fold change: 0.289) expression of TLR4 (p < 0.05), while NF-κB and MMP-9 were unaffected (p > 0.05). Limitations: Only one trophoblast cell line was used in this study, and the findings were not confirmed in animal studies. Conclusions: This study explored the pathogenesis of PE from two aspects: inflammation and trophoblast invasion. The overexpression of HMGB1 in placentas from preeclamptic pregnancies suggests this protein may be involved in PE pathogenesis. In vitro, HMGB1 was found to regulate the proliferation and invasion of HTR-8/SVneo cells by activating the TLR4-NF-κB-MMP-9 pathway. These findings have implications for targeting HMGB1 could be a therapeutic strategy for treating PE. In the future, we will further verify this in vivo and in other trophoblast cell lines, further exploring the molecular interactions of the pathway.