MMP-9 mRNA Expression Research Articles

PEDF upregulated in Ngfr-positive cells suppresses proliferation of pulmonary artery smooth muscle cells

Abstract Background Pulmonary arterial hypertension (PAH) is a disease with poor prognosis that causes right heart failure due to progressive pulmonary artery remodeling. Although existing pulmonary vasodilators contribute to pulmonary artery reverse remodeling by reducing share stress, the development of therapeutic agents that directly target pulmonary vascular remodeling is desired. Nerve growth factor receptor (Ngfr) is involved in the inflammatory reaction and repair process of damaged tissues. We have previously reported that Ngfr-positive cells are increased in the peripheral blood (PB) of PAH patients and are associated with disease progression. We also found that Ngfr inhibited the pathogenetic progression of pulmonary hypertension in a model of hypoxia-induced pulmonary hypertension. However, it remains unclear how Ngfr positive cells are involved in the pathology of PAH. Purpose In this study, we investigate how Ngfr-positive cells affect the pathogenesis of PAH. Methods & Results To evaluate the localization of Ngfr-positive cells in lung tissue, WT mice (C57BL/6) transplanted with bone marrow (BM) of GFP-Tg mice were kept under hypoxia for 3 weeks to create hypoxia-induced pulmonary hypertension (PH) model. Immunostaining of lung tissue sections indicated that double positive cells (Ngfr+GFP+) were present in the interstitial tissue around the small pulmonary artery. To examine the cellular characteristics of Ngfr-positive cells, RNA sequencing of Ngfr-positive and Ngfr-negative cells in PB was performed. Significantly 78 genes were upregulated in Ngfr-positive cells, including 11 secreted proteins. Among these, we focused on pigment epithelium-derived factor (PEDF), a secreted protein involved in angiogenesis. PEDF mRNA expression was decreased in the lung of Ngfr-KO mice compared to WT mice. We next examined the effect of PEDF on pulmonary artery smooth muscle cells (PASMCs). Platelet-derived growth factor (PDGF)-induced cell proliferation of PASMC was inhibited by PEDF stimulation. Furthermore, PEDF stimulation significantly inhibited PDGF-induced mRNA expressions of TNF and MMP9 in PASMCs. Conclusion In this study, we show that PEDF expression is upregulated in Ngfr-positive cells and that PEDF inhibits PDGF-induced hPASMC proliferation, suggesting that NGFR-positive cells may suppress pulmonary vascular remodeling through PEDF-mediated paracrine effects. PEDF might be a new therapeutic target for PH.

Histamine and Th2 cytokines independently and synergistically upregulate MMP12 expression in human M2 macrophages

Beyond Th2 cells and various immune cells, M2 macrophages have been identified in lesional skin of atopic dermatitis (AD) indicating their involvement in the disease’s underlying mechanisms. MMP12, a matrix-degrading enzyme, which is predominantly produced by macrophages, is increased in skin lesions of AD patients. In this study we investigated the expression of MMP12 mRNA in lesional AD skin at single cell level through RNA sequencing (scRNA-seq) and the expression of MMP12 in M2 macrophages from healthy individuals and AD patients in response to Th2 cytokines and histamine using quantitative PCR and ELISA. Additionally, we analyzed macrophages from dupilumab-treated AD patients using the same methods to assess the influence of Th2 cytokines on MMP12 expression ex-vivo. ScRNA-seq identified macrophages as the primary producers of MMP12 in lesional AD skin. In-vitro, both MMP12 mRNA and protein expression were significantly increased in monocytes during differentiation to M2 macrophages in the presence of histamine, of Th2 cytokines or of Th2 cytokines in combination with histamine. In M2 macrophages obtained from dupilumab-treated AD patients, the upregulation of MMP12 expression by IL-4 and IL-13 was attenuated. Our findings unveil a novel mechanism whereby Th2 cytokines and histamine regulate MMP12 expression, potentially impacting skin barrier homeostasis in AD.

Parthenolide Inhibits Tumor Cell Growth and Metastasis in Melanoma A2058 Cells.

Skin melanoma is a potentially lethal cancer and ranks as the 17th most common cancer worldwide. Overcoming resistance to advanced-stage melanoma is a significant challenge in its treatment. Parthenolide (PAR) is recognized as a potent anticancer small molecule, yet its potential in treating melanoma is poorly investigated. Our objective was to investigate the apoptotic and anti-metastatic properties of PAR against the A2058 melanoma cells in vitro. This study employed various assays, such as cytotoxicity, apoptosis, cell cycle analysis, reactive oxygen species (ROS) production, mRNA expressions, western blotting, gelatin zymography, and scratch assay. The synergy between PAR and dacarbazine, a chemotherapy drug for treating skin cancer, was also assessed. Our study revealed that PAR significantly reduced the viability of A2058 cancer cells, demonstrating greater potency against cancer cells compared to normal L929 cells (IC50: 20 μM vs. 27 μM after 24h). PAR increased ROS production, elevated mRNA expression of pro-apoptotic Bax and NME1 genes, and decreased expression of the MITF gene. PAR induced apoptosis and cell cycle arrest in A2058 cells, as evidenced by the increased proportion of cells in the late apoptotic phase and sub-G1 cell cycle arrest. MMP-2 and MMP-9 mRNA and protein expressions, gelatinase activity, and the migration of A2058 cells were also decreased by PAR, suggesting its potential to suppress cancer cell invasion. These results, along with the synergic effect with dacarbazine, indicated that PAR may have the potential to be a therapeutic drug for melanoma by triggering apoptosis and suppressing invasion and migration.

Gelatinase MMP Expression Is Correlated with Muscle Fiber Growth in Maiwa Yak Gluteus Maximus.

Yak (Bos grunniens) is the only large mammal species in the Qinghai-Tibet Plateau. The most of the studies in yak remain confined for the main contributor of meat, which requires a good understanding of muscle growth. Matrix metalloproteinases-2 (MMP-2) and MMP-9 are widely expressed in mammal tissues they mainly degrade collagen in the extracellular matrix for muscle development. However, the influence of MMPs on yak muscle remains unclear. Hence, we assessed the expression of MMP-2, MMP-9, and related factors with ages in Maiwa yak for study the correlation between MMPs expression and yak muscle growth. The mRNA expression of MMP-2, MMP-9, MMP-14, and collagen III increased with age, except collagen I by quantitative real-time PCR. Moreover, muscle fiber diameter increased with age, whereas the density decreased, which showed that fiber grew thicker with age using hematoxylin-eosin staining. Interestingly, MMP and collagen expression significantly decreased with age using western blotting. Pearson correlation method showed that both mRNA and protein expression of MMP-14 and collagen were strongly correlated with muscle fiber growth, but MMP-2 protein and MMP-9 mRNA expression were moderately correlated with muscle fiber growth. Overall, the expression of MMPs and collagen significantly changed with age, which means that MMPs and their function related genes could correlate with Maiwa yak muscle fiber growth.

Yiqi Yangxue formula inhibits cartilage degeneration in knee osteoarthritis by regulating LncRNA-UFC1/miR-34a/MMP-13 axis

Ethnopharmacological relevanceKnee osteoarthritis (KOA) is a prevalent and disabling clinical condition affecting joint structures worldwide. The Yiqi Yangxue formula (YQYXF) is frequently prescribed in clinical settings for the treatment of KOA. Existing research has primarily focused on alterations in drug metabolism, with limited investigation into the epigenetic effects of YQYXF, particularly in relation to non-coding RNA. Aim of the studyExploring the effects of YQYXF on critical factors of long chain non-coding RNA UFC1/miR-34a/matrix metalloproteinase-13 (MMP-13) axis and their interrelationships. MethodsUHPLC-QE-MS technology was used to identify the YQYXF ingredients in rat serum. KEGG and GO analysis were performed on the targets of blood components acting on KOA using a database. Simultaneously, a protein interaction network was constructed using target proteins and metabolites to identify the core components and key pathways of YQYXF. The KOA rat model was established using an improved Hulth method. SPF SD rats were randomly divided into normal group, sham surgery group, model group, celecoxib capsules group (18 mg/kg), YQYXF low, medium and high dose groups (4.6g/kg, 9.2g/kg, 18.4g/kg). Observe the synovial and cartilage tissues of rats using pathological methods. RT-PCR was used to detect the levels of UFC1, miR-34a, and MMP-13 in cartilage. Immunohistochemistry was used to detect the levels of MMP-13 and ADAMTS-5 in cartilage. ELISA method was used to detect the levels of MMP-13 and ADAMTS-5 in serum. In addition, we further validated the regulation of crucial factor expression levels of UFC1/miR-34a/MMP-13 axis in rat chondrocytes and degenerative chondrocytes of KOA patients by YQYXF, providing a basis for its treatment of KOA. ResultsThe compounds that YQYXF enters the bloodstream mainly contain flavonoids and phenylpropanoid compounds. The core components that act on OA include quercetin, fisetin, and demethylweldelolactone. The main target pathways are the IL-17 signaling pathway, lipid and atherosclerosis, cellular sensitivity, inflammatory mediator regulation of TRP channels, TNF signaling pathway, relaxin signaling pathway and C-type lectin receptor signaling pathway. YQYXF inhibited the expression of miR-34a and MMP-13 mRNA, and reduced the protein levels of MMP-13 and ADAMTS-5. In vitro studies have confirmed that 20% YQYXF serum promoted UFC1 and reduce miR-34a levels. In addition, miR-34a in sh-UFC1+10% YQYXF serum and sh-UFC1+20% YQYXF serum groups significantly decreased compared to the sh-UFC1 group. ConclusionThe anti-KOA cartilage degeneration effect of YQYXF might be related to inhibiting cell apoptosis and promoting cell proliferation, which regulated the lncRNA-UFC1/miR-34a/MMP-13 axis.

Discovery of a novel thiophene carboxamide analogue as a highly potent and selective sphingomyelin synthase 2 inhibitor for dry eye disease therapy

Dry eye disease (DED) is a prevalent and intractable ocular disease induced by a variety of causes. Elevated sphingomyelin (SM) levels and pro-inflammatory cytokines were detected on the ocular surface of DED patients, particularly in the meibomian glands. Sphingomyelin synthase 2 (SMS2), one of the proteins involved in SM synthesis, would light a novel way of developing a DED therapy strategy. Herein, we report the design and optimization of a series of novel thiophene carboxamide derivatives to afford 14l with an improved highly potent inhibitory activity on SM synthesis (IC50, SMS2 = 28 nmol/L). Moreover, 14l exhibited a notable protective effect of anti-inflammation and anti-apoptosis on human corneal epithelial cells (HCEC) under TNF-α-hyperosmotic stress conditions in vitro, with an acceptable ocular specific distribution (corneas and meibomian glands) and pharmacokinetics (PK) profiles (t1/2, cornea = 1.11 h; t1/2, meibomian glands = 4.32 h) in rats. Furthermore, 14l alleviated the dry eye symptoms including corneal fluorescein staining scores and tear secretion in a dose-dependent manner in mice. Mechanically, 14l reduced the mRNA expression of Tnf-α, Il-1β and Mmp-9 in corneas, as well as the proportion of very long chain SM in meibomian glands. Our findings provide a new strategy for DED therapy based on selective SMS2 inhibitors.

Protective Effects of Sesame Glycoproteins on Ultraviolet-Induced Skin Aging: In Vitro and In Vivo Studies.

Ultraviolet (UV) radiation is a primary factor in skin photoaging, leading to wrinkles, reduced elasticity, and pigmentation changes due to damage to cellular DNA, proteins, and lipids. Glycoproteins from sesame cake (SPE) have potential protective effects against UV-induced skin aging. This study investigated the anti-photoaging effects of SPE on UV-induced damage in human keratinocyte HaCaT cells and SKH-1 hairless mice. SPE was evaluated for its ability to mitigate UV-induced damage in HaCaT cells by assessing MMP-1 protein and mRNA expression levels, as well as the activity of transcription factors AP-1 and NF-κB. The phosphorylation of AKT and MAPK pathways was also analyzed. In vivo, SKH-1 hairless mice were exposed to UV radiation, and the effects of SPE on wrinkle formation and skin structure were assessed by measuring wrinkle length, area, and volume. SPE significantly inhibited UV-induced MMP-1 protein and mRNA expression in HaCaT cells, indicating suppression of AP-1 and NF-κB transcription factors involved in MMP-1 production. Additionally, SPE reduced UV-induced phosphorylation of AKT and MAPK pathways. In SKH-1 hairless mice, SPE treatment led to significant reductions in wrinkle length, area, and volume, preserving skin structure in UV-exposed mice. The findings demonstrate that SPE has protective effects against UV-induced photoaging by inhibiting key molecular pathways associated with skin aging. SPE shows promise as a natural anti-photoaging agent, providing a foundation for future skincare product development. Further studies are warranted to explore the molecular mechanisms in detail and to validate these effects through clinical trials.

Molecular Dissection of the Arsenic-Induced Leukocyte Incursion into the Inflamed Thymus and Spleen and Its Amelioration by Co-supplementation of L-Ascorbic Acid and α-Tocopherol.

Arsenic, a surreptitious presence in our environment, perpetuates a persistent global menace with its deleterious impacts. It possesses the capability to trigger substantial immunosuppression by instigating inflammation in critical organs like the thymus and spleen. L-Ascorbic acid (L-AA) exhibits robust immunoregulatory prowess by orchestrating the epigenetic terrain through TET and JHDM pathways. Conversely, α-tocopherol (α-T) demonstrates the capacity to dampen the production of pro-inflammatory cytokines by modulating the PI3K-Akt axis. Given these insights, this inquiry embarks on exploring the mitigative potential of L-AA and α-T co-supplementation at the transcriptome level within leukocytes under arsenic exposure. Concurrently, the research endeavours to unravel the potent anti-inflammatory effects of administering α-T and L-AA, alleviating inflammation within the spleen and thymus amidst arsenic-induced insult and delving deeply into their immunomodulatory mechanisms. The rats were randomly allocated into eight distinct groups for subsequent experimentation: (I) the control group was administered solely with distilled water as the vehicle (control); (II) NaAsO2-treated group (As); (III) NaAsO2 treated along with L-ascorbic acid and α-tocopherol supplemented group (As + L-AA + α-T); (IV) L-ascorbic acid and α-tocopherol supplemented group (L-AA + α-T); (V) NaAsO2 treated along with L-ascorbic acid supplemented group (As + L-AA); (VI) only L-ascorbic acid supplemented group (L-AA); (VII) NaAsO2 treated along with α-tocopherol supplemented group (As + α-T); (VIII) only α-tocopherol supplemented group (α-T). Rats treated with NaAsO2 exhibited an increased neutrophil count in their bloodstream, as revealed by a comprehensive transcriptomic analysis showcasing heightened expressions of ItgaM, MMP9, and Itga4 within circulating leukocytes under arsenic exposure. Concurrently, arsenic heightened the expression of pro-inflammatory cytokines within the thymus and spleen. This elevated cytokine activity promoted the upregulation of ICAM-1 on vascular endothelial cells, facilitating the infiltration of Ly6g + leukocytes into the afflicted thymus and spleen. Remarkably, the combination of L-AA acid and α-T demonstrated substantial therapeutic efficacy, adeptly reducing the influx of Ly6g + leukocytes into these immune sites and subsequent reduction of excessive collagen deposition. The dynamic duo of L-AA and α-T achieved this amelioration by suppressing the expression of ItgaM, MMP9, and Itga4 mRNA within circulating leukocytes and moderating tissue levels of pro-inflammatory cytokines in arsenic-exposed thymus and spleen.

Activin A inhibits the migration of human lung adenocarcinoma A549 cells induced by EGF

Activin A, a member of the transforming growth factor β (TGF-β) superfamily, is involved in tumorigenesis and tumor progression. However, it remains unclear whether activin A can affect the migration of lung adenocarcinoma (LUAD) cells. In this study, the results of differentially expressed genes (DEGs) identification revealed that lung adenocarcinoma tissues exhibited lower expression of activin βA mRNA, but higher expression of epidermal growth factor (EGF) and MMP9 mRNA compared to nontumor tissues. Moreover, we found that activin A inhibited human LUAD A549 cell proliferation promoted by EGF. Additionally, EGF induced A549 cell migration in microfluidic device, while activin A attenuated EGF actions. Simultaneously, EGF increased the levels of migration-related proteins, but activin A played the opposite role. Furthermore, the study revealed that EGF upregulated the ratio of p-ERK/ERK in A549 cells, which was weakened by activin A, and A549 cell migration regulated by activin A was not related to calcium signaling. In addition, the inhibitory effect of activin A on EGF-induced A549 cell migration was attenuated by the ERK inhibitor FR180204. These findings demonstrate that activin A effectively hinders the migration of A549 cells induced by EGF through ERK1/2 signaling, suggesting that targeting activin A may hold promise in the treatment of EGF-dependent LUAD growth and metastasis.

The Lian-Dou-Qing-Mai Formula activates the PPARγ-LXRα-ABCA1/ABCG1 pathway by regulating IL-10, leading to the promotion of cholesterol efflux and a reduction in atherosclerotic plaques.

To observe the effect of the Lian-Dou-Qing-Mai (LDQM) formula on lipid metabolism in mice and explore its mechanism from the perspective of regulating the PPARγ/LXRα/ABCA1 signaling pathway. THP-1 cells were induced to transform into foam cells with ox-LDL. Atherosclerosis (AS) models were constructed using a high-fat diet in ApoE-/- mice. Detection kits were used to evaluate triglyceride (TG) and total cholesterol (TC) content; TNF-α, MCP-1, MMP-9, TMP-1, PPARγ, LXRα, ABCA1, and ABCG1 mRNA and protein expression were identified using real-time PCR and western blot. Interleukin-10 (IL-10) blood levels were measured using an enzyme-linked immunosorbent assay (ELISA), and aortic plaque development and lipid deposition were seen using hematoxylin and eosin (HE) and oil red O staining, respectively. In the cell model, LDQM could inhibit the formation of THP-1 macrophage-derived foam cells and the expression of inflammatory factors, promote macrophage cholesterol efflux, increase the expression of IL-10, and activate the PPARγ-LXRα-ABCA1/ABCG1 pathway. Additional IL-10 treatment further promotes LDQM-induced cholesterol efflux in THP-1 cells; in In vivo models, LDQM inhibited the area of atherosclerotic lesions, aortic lipid deposition, and inflammation levels in ApoE-/- mice through IL-10, and activated the expression level of the PPARγ-LXRα-ABCA1/ABCG1 pathway. LDQM may affect the PPARγ/LXRα/ABCA1 signaling pathway through IL-10, regulate lipid metabolism, reduce serum inflammatory expression and lipid deposition, and improve the formation of atheroplaques.

Anti-inflammatory and DNA Repair Effects of Astragaloside IV on PC12 Cells Damaged by Lipopolysaccharide.

This study aimed to establish a neural cell injury model in vitro by stimulating PC12 cells with lipopolysaccharide (LPS) and to examine the effects of astragaloside IV on key targets using high-throughput sequence technology and bioinformatics analyses. PC12 cells in the logarithmic growth phase were treated with LPS at final concentrations of 0.25, 0.5, 0.75, 1, and 1.25 mg/mL for 24 h. Cell morphology was evaluated, and cell survival rates were calculated. A neurocyte inflammatory model was established with LPS treatment, which reached a 50% cell survival rate. PC12 cells were treated with 0.01, 0.1, 1, 10, or 100 µmol/L astragaloside IV for 24 h. The concentration of astragaloside IV that did not affect the cell survival rate was selected as the treatment group for subsequent experiments. NOS activity was detected by colorimetry; the expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1β, TLR4, NOS and COX-2 mRNA and protein were detected by RT-qPCR and Western blotting. The differentially expressed genes (DEGs) between the groups were screened using a second-generation sequence (fold change>2, P<0.05) with the following KEGG enrichment analysis, RT-qPCR and Western blotting were used to detect the mRNA and protein expression of DEGs related to the IL-17 pathway in different groups of PC12 cells. The viability of PC12 cells was not altered by treatment with 0.01, 0.1, or 1 µmol/L astragaloside IV for 24 h (P>0.05). However, after treatment with 0.5, 0.75, 1, or 1.25 mg/mL LPS for 24 h, the viability steadily decreased (P<0.01). The mRNA and protein expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1β, TLR4, NOS, and COX-2 were significantly increased after PC12 cells were treated with 1 mg/mL LPS for 24 h (P<0.01); however, these changes were reversed when PC12 cells were pretreated with 0.01, 0.1, or 1 µmol/L astragaloside IV in PC12 cells and then treated with 1 mg/mL LPS for 24 h (P<0.05). Second-generation sequencing revealed that 1026 genes were upregulated, while 1287 genes were downregulated. The DEGs were associated with autophagy, TNF-α, interleukin-17, MAPK, P53, Toll-like receptor, and NOD-like receptor signaling pathways. Furthermore, PC12 cells treated with a 1 mg/mL LPS for 24 h exhibited increased mRNA and protein expression of CCL2, CCL11, CCL7, MMP3, and MMP10, which are associated with the IL-17 pathway. RT-qPCR and Western blotting analyses confirmed that the DEGs listed above corresponded to the sequence assay results. LPS can damage PC12 cells and cause inflammatory reactions in nerve cells and DNA damage. astragaloside IV plays an anti-inflammatory and DNA damage protective role and inhibits the IL-17 signaling pathway to exert a neuroprotective effect in vitro.

MiR-21 attenuated inflammation targeting MyD88 in human chondrocytes stimulated with Hyaluronan oligosaccharides

Inflammation is the body's response to injuries, which depends on numerous regulatory factors. Among them, miRNAs have gained much attention for their role in regulating inflammatory gene expression at multiple levels. In particular, miR-21 is up-regulated during the inflammatory response and reported to be involved in the resolution of inflammation by down-regulating pro-inflammatory mediators, including MyD88. Herein, we evaluated the regulatory effects of miR-21 on the TLR-4/MyD88 pathway in an in vitro model of 6-mer HA oligosaccharides-induced inflammation in human chondrocytes.The exposition of chondrocytes to 6-mer HA induced the activation of the TLR4/MyD88 pathway, which culminates in NF-kB activation. Changes in miR-21, TLR-4, MyD88, NLRP3 inflammasome, IL-29, Caspase1, MMP-9, iNOS, and COX-2 mRNA expression of 6-mer HA-stimulated chondrocytes were examined by qRT-PCR. Protein amounts of TLR-4, MyD88, NLRP3 inflammasome, p-ERK1/2, p-AKT, IL-29, caspase1, MMP-9, p-NK-kB p65 subunit, and IKB-a have been evaluated by ELISA kits. NO and PGE2 levels have been assayed by colorimetric and ELISA kits, respectively.HA oligosaccharides induced a significant increase in the expression of the above parameters, including NF-kB activity. The use of a miR-21 mimic attenuated MyD88 expression levels and the downstream effectors. On the contrary, treatment with a miR-21 inhibitor induced opposite effects. Interestingly, the use of a MyD88 siRNA confirmed MyD88 as the target of miR-21 action.Our results suggest that miR-21 expression could increase in an attempt to reduce the inflammatory response, targeting MyD88.

Establishment of a Rabbit Model of Adjacent Intervertebral Disk Degeneration After Lumbar Fusion and Fixation and Evaluation of Autophagy Factor Expression in Nucleus Pulposus Cells.

The objectives of this research were to establish an animal model of adjacent segment degeneration (ASD) bordering lumbar fusion and to investigate the expression of autophagy factors in nucleus pulposus cells of adjacent intervertebral disks. Twenty-four adult New Zealand white rabbits were enrolled and divided into two groups: group A (n=12) and group B (n=12). Posterolateral fusion and fixation were performed after intervertebral disk degeneration occurred in group A, and the rabbits were monitored for 6 months. Group B was the control group and did not undergo fusion surgery. These rabbits were monitored for 6 months. Real-time quantitative polymerase chain reaction and immunohistochemistry were performed to detect the mRNA and protein expressions of PTEN-induced kinase 1 (PINK1), Parkin, ADAMTS-4, and MMP-3. An external database, the GEO database, was used to examine the expression of these genes and analyze them for differential expression. After lumbar fusion in rabbits, the animal model of ASD exhibited gradual degeneration of adjacent intervertebral disks over time. Group A displayed significantly higher mRNA and protein expressions of PINK1 and MMP-3 but lower expression of ADAMTS-4 compared with group B (P<.05). The results analyzed in the GEO database showed that the expression of PINK1 was higher in group A than in group B, while the expression of ADAMTS-4 was lower in group A than in group B. After posterolateral lumbar fusion in rabbits, the animal ASD model showed gradual deterioration of adjacent intervertebral disks with prolonged follow-up. The findings indicate the important role of autophagy in the apoptosis of nucleus pulposus cells in adjacent intervertebral disks. [Orthopedics. 2024;47(4):e167-e173.].

Sepsis-induced inflammatory demyelination in medullary visceral zone and cholinergic anti-inflammatory pathway: Insights from a Rat's model study

BackgroundOur previous studies have demonstrated that the activated Cholinergic Anti-inflammatory Pathway (CAP) effectively suppresses systemic inflammation and immunity in early sepsis. Some parameters of Heart Rate Variability (HRV) could be used to reflect the regulatory activity of CAP. However, in the early stages of severe sepsis of some patients, the inflammatory storm can still result in multiple organs dysfunction and even death, suggesting they lose CAP's modulation ability. Since CAP is part of the vagus nerve and is directly innervated by the Medullary Visceral Zone (MVZ), we can reasonably concluded that pathological changes induced by MVZ's neuroinflammation should be responsible for CAP's dysfunction in modulating systemic inflammation in early sepsis. MethodsWe conducted two independent septic experiments, the sepsis model rats were prepared by cecum ligation and puncture (CLP) method. In the first experiment, A total of 64 adult male Sprague-Dawley rats were included. Under the condition of sepsis and CAP's pharmacological activation or blockade, we investigated the MVZ's pathological changes, the functional state of key neurons including catecholaminergic and cholinergic neurons, key genes' expression such as Oligodendrocyte Transcription Factor 2 (Olig-2) mRNA, glial fibrillary acidic protein (GFAP) mRNA, and matrix metalloprotein (MMP) −9 mRNA, and CAP's activities reflected by HRV. The second experiment involved in 56 rats, through central anti-inflammation by feeding with 10 mg/ml minocycline sucrose solution as the only water source, or right vagus transection excepting for central anti-inflammation as a mean of the CAP's functional cancel, we confirmed that the neuroinflammation in MVZ affected systemic inflammation through CAP in sepsis. ResultsIn the first experiment, cholinergic and catecholaminergic neurons showed significant apoptosis with reduced expressions of TH, but the expression of CHAT remained relatively unaffected in MVZ in sepsis. HRV parameters representing the tone of the vagus nerve, such as SDNN, RMSSD, HF, SD1, and SD2, did not show significant differences among the three Septic Groups, although they all decreased significantly compared to the Control Group. The expressions of GFAP mRNA and MMP-9 mRNA were up-regulated, while the expression of Olig-2 mRNA was down-regulated in the Septic Groups. Intervention of CAP had a significant effect on cholinergic and catecholaminergic neurons' apoptosis, as well as the expressions of TH/CHAT and these key genes, but had little effect on HRV in sepsis. In the second experiment, the levels of TNF-α, IL-6, in serum and MVZ were significantly increased in sepsis. Central anti-inflammatory treatment reversed these changes. However, right vagotomy abolished the central anti-inflammatory effect. ConclusionsOur study uncovered that MVZ's neuroinflammation may play a crucial role in the uncontrolled systemic inflammation through inflammatory demyelination in MVZ, which disrupts CAP's modulation on the systemic inflammation in early sepsis.

Contribution of Matrix Metalloproteinase-2 and Matrix Metalloproteinase-9 to Upper Tract Urothelial Cancer Risk in Taiwan.

Matrix metalloproteinase (MMP)-2 and -9, which degrade type IV collagen, are linked to cancer invasion and metastasis. Gene polymorphisms in MMP-2 and MMP-9 can influence their function, impacting cancer development and progression. This study analyzed the association between polymorphisms MMP-2 rs243865 (C-1306T), rs2285053 (C-735T), and MMP-9 rs3918242 (C-1562T) with serum concentrations of these enzymes in upper tract urothelial cancer (UTUC) patients. We conducted a case-control study with 218 UTUC patients and 580 healthy individuals in Taiwan. Genotyping was performed using PCR/RFLP on DNA from blood samples, and MMP-2 and MMP-9 serum levels and mRNA expressions in 30 UTUC patients were measured using ELISA and real-time PCR. Statistical analysis showed that MMP-2 rs2285053 and MMP-9 rs3918242 genotypes were differently distributed between UTUC patients and controls (p = 0.0199 and 0.0020). The MMP-2 rs2285053 TT genotype was associated with higher UTUC risk compared to the CC genotype (OR = 2.20, p = 0.0190). Similarly, MMP-9 rs3918242 CT and TT genotypes were linked to increased UTUC risk (OR = 1.51 and 2.92, p = 0.0272 and 0.0054). In UTUC patients, TT carriers of MMP-2 rs2285053 and MMP-9 rs3918242 showed higher mRNA and protein levels (p < 0.01). These findings suggest that MMP-2 rs2285053 and MMP-9 rs3918242 genotypes are significant markers for UTUC risk and metastasis in Taiwan.

Mechanism of Huangqin Qingre Chubi Capsules regulating p53/p21 signaling pathway to delay chondrocyte senescence in osteoarthritic rats

This study aims to investigate the mechanism of Huangqin Qingre Chubi Capsules(HQC) in delaying chondrocyte senescence of osteoarthritic(OA) rats by regulating the p53/p21 signaling pathway. Rheumatic fever paralysis models of OA rats were induced based on monosodiun iodoacetate(MIA) combined with external rheumatic fever environmental stimuli and divided into normal(Con) group, OA model(MIA) group, OA model+rheumatic fever stimulation model(MIA-M) group, MIA-M+HQC low-dose(MIA-M+HQC-L) group, medium-dose(MIA-M+HQC-M) group, and high-dose(MIA-M+HQC-H) group, and MIA-M+glucosamine(MIA-M+GS) group. The models were successfully prepared and administered by gavage for 30 d. The pathological changes of cartilage were observed by hematoxylin-eosin(HE) and Senna O solid green(SO) staining. The expression of interleukin(IL)-1β and IL-6 was detected by enzyme-linked immunosorbent assay(ELISA). Flow cytometry(FCM) was used to detect apoptosis and cell cycle. The mRNA expression of MMP13, ADAMTS-5, COLⅡ, and TGF-β was detected by RT-qPCR. The protein expression of p53/p21, p16, Bax, and Bcl-2 was detected by Western blot. The articular cartilage surface of rats in the Con group was smooth, and the tide line was smooth. The cartilage layer of MIA and MIA-M groups was obviously damaged, and the cartilage matrix was reduced. The above conditions were more severe in the MIA-M group. The cartilage surface of the HQC high-dose group and MIA-M+GS group was basically intact with clear delamination. Compared with the MIA-M+HQC-H group, Mankin's score was higher in the HQC low-dose and medium-dose groups, and the change was not obvious in the MIA-M+GS group. Compared with the Con group, the proportion of chondrocytes G_1 was elevated in the MIA and MIA-M groups, and the proportion of the S phase and G_2 phase was significantly decreased. In addition, the apoptosis rate was increased. Compared with MIA-M, HQC groups inhibited apoptosis and promoted cell proliferation in a concentration-dependent manner. Compared with the MIA-M+HQC-H group, the effect was more significant in the HQC high-dose group than in the HQC medium-low dose, while it was not significant in the MIA-M+GS group. Compared with the Con group, IL-1β and IL-6 were elevated in the MIA and MIA-M groups, and mRNA levels of MMP13 and ADAMTS-5 were elevated. p53, p21, p16, and Bax protein were elevated, and mRNA levels of COLⅡ and TGF-β were decreased. Compared with the MIA-M group, IL-1β and IL-6 decreased after drug interventions of HQC and GS, and mRNA levels of MMP13 and ADAMTS-5, as well as protein levels of p53, p21, Bax, and p16 decreased. In addition, Bcl-2 increased. The improvement of these indexes was significantly better in the MIA-M+HQC-H group than in the HQC low-dose and medium-dose groups, and the difference with the MIA-M+GS group was not significant. HQC delayed MIA-induced chondrocyte senescence in OA rats, inhibited inflammatory response and extracellular matrix(ECM) degradation, and its mechanism may be related to the inhibition of the p53/p21 pathway.

Integrating network pharmacology and experimental evaluation to explore the complementary therapeutic effect and mechanism of melatonin in periodontitis

ObjectiveTo explore the potential targets for melatonin in the treatment of periodontitis through network pharmacologic analysis and experimental validation via in vivo animal models and in vitro cellular experiments. Materials and methodsIn this study, we first screened melatonin targets from Pharm Mapper for putative targets, Drug Bank, and TCMSP databases for known targets. Then, disease database was searched and screened for differential expressed genes associated with periodontitis. The intersection of disease and melatonin-related genes yielded potential target genes of melatonin treatment for periodontitis. These target genes were further investigated by protein-protein interaction network and GO/KEGG enrichment analysis. In addition, the interactions between melatonin and key target genes were interrogated by molecular docking simulations. Then, we performed animal studies to validate the therapeutic effect of melatonin by injecting melatonin into the peritoneal cavity of ligation-induced periodontitis (LIP) mice. The effects of melatonin on the predicted target proteins were also analyzed using Western blot and immunofluorescence techniques. Finally, we constructed an in vitro cellular model and validated the direct effect of melatonin on the predicted targets by using qPCR. ResultsWe identified 8 potential target genes by network pharmacology analysis. Enrichment analysis suggests that melatonin may treat periodontitis by inhibiting the expression of three potential targets (MPO, MMP8, and MMP9). Molecular docking results showed that melatonin could effectively bind to MMP8 and MMP9. Subsequently, melatonin was further validated in a mouse LIP model to inhibit the expression of MPO, MMP8, and MMP9 in the periodontal tissue. Finally, we verified the direct effect of melatonin on the mRNA expression of MPO, MMP8, and MMP9 in an in vitro cellular model. ConclusionsThrough a combination of network pharmacology and experimental validation, this study provides a more comprehensive understanding of the mechanism of melatonin to treat periodontitis. Our study suggests that MPO, MMP8, and MMP9 as key target genes of melatonin to treat periodontitis. These findings present a more comprehensive basis for further investigation into the mechanisms of pharmacological treatment of periodontitis by melatonin.

Tumor-matched and unmatched cancer associated fibroblasts exhibit differential effect on proliferation and FMOD and MMP9 gene expression in head and neck squamous cell carcinoma cells when cocultured in spheroids

BackgroundCancer-associated fibroblasts (CAFs) are the major cellular component of the tumor microenvironment and are known to affect tumor growth and response to various treatments. This study was undertaken to investigate the crosstalk between tumor-matched or unmatched CAFs and head and neck squamous cell carcinoma (HNSCC) cells regarding tumor growth and treatment response.MethodsThree HNSCC cell lines (LK0412, LK0902 and LK0923), were cocultured in 2D or in 3D with their tumor-matched CAFs, site matched CAFs from other tumors or normal oral fibroblasts (NOFs). Cell proliferation was assessed as the amount of Ki67 positive cells/ spheroid area in formalin-fixed- paraffin-embedded 3D spheroids stained with Ki67 antibody. Viability after seven days of cisplatin treatment was measured with CellTiter-Glo 3D Viability Assay. The mRNA expression of CAF-associated markers (ACTA2, COL1A2, FAP, PDGFRα, PDGFRβ, PDPN, POSTN and S100A4) in CAFs before and after coculture with tumor cells as well as mRNA expression of CAF-induced genes (MMP1, MMP9 and FMOD) in tumor cells separated from CAFs after co-culture was measured with RT-qPCR. The expression of selected protein biomarkers was validated with immunohistochemistry based on previous mRNA expression results.ResultsThe proliferation of the LK0412 and LK0902 tumor spheroids varied significantly when cocultured with different CAFs and NOFs as shown by Ki-67 positive cells. RT‒qPCR analysis revealed different molecular profile of the analyzed HNSCC-derived CAFs concerning the expression of CAF-associated markers. The interaction between CAFs and HNSCC cells was more pronounced after coculture with unmatched CAFs as shown by changes in mRNA expression pattern of CAF-specific markers. Additionally, the unmatched CAFs significantly upregulated the mRNA expression of MMP1, MMP9 and FMOD in tumor cells compared to tumor-matched CAFs.ConclusionOur results indicate that tumor-matched CAFs are unique for each tumor and affect the proliferation and the gene/protein expression of tumor cells in a distinct manner. The interaction between tumor unmatched CAFs and HNSCC cells in the tumor spheroids is associated with significant changes in the mRNA expression of CAF-specific markers and significant increases in FMOD and MMP9 in tumor cells compared to when cocultured with tumor-matched CAFs. Taken together, our results show how important the selection of CAFs is to get a reliable in vitro model that mimics the patients’ tumor.

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.

Kartogenin Induces Chondrogenesis in Cartilage Progenitor Cells and Attenuates Cell Hypertrophy in Marrow-Derived Stromal Cells.

Kartogenin (KGN) is a synthetic small molecule that stimulates chondrogenic cellular differentiation by activating smad-4/5 pathways. KGN has been proposed as a feasible alternative to expensive biologic growth factors, such as transforming growth factor β, which remain under strict regulatory scrutiny when it comes to use in patients. This study reports the previously unexplored effects of KGN stimulation on cartilage- derived mesenchymal progenitor cells (CPCs), which have been shown to be effective in applications of cell-based musculoskeletal tissue regeneration. Our findings demonstrate that KGN treatment significantly increased markers of chondrogenesis, SOX9 and COL2 following 3-10 days of treatment in human CPCs. KGN treatment also resulted in a significant dose-dependent increase in GAG production in CPCs. The same efficacy was not observed in human marrow-derived stromal cells (BM-MSCs); however, KGN significantly reduced mRNA expression of cell hypertrophy markers, COL10 and MMP13, in BM-MSCs. Parallel to these mRNA expression results, KGN led to a significant decrease in protein levels of MMP-13 both at 0-5 days and 5-10 days following KGN treatment. In conclusion, this study demonstrates that KGN can boost the chondrogenicity of CPCs and inhibit hypertrophic terminal differentiation of BM-MSCs.