[EMP3 inhibits the formation of heterotypic cell-in-cell structures in hepatocellular carcinoma].

Heterotypic cell-in-cell (heCIC) structures represent a unique intercellular interaction where tumor cells internalize immune cells to enhance the killing efficiency of immune cells. However, the mechanism of heCIC structure formation remains to be fully elucidated. In this study, we explored the role of epithelial membrane protein 3 (EMP3), a PMP-22/EMP/MP20 protein family member highly expressed in the patients with hepatocellular carcinoma and poor prognosis, in the formation of the heCIC structure formed by natural killer cells and hepatocellular carcinoma cells. The analysis of monoclonal hepatocellular carcinoma cell lines revealed that EMP3 presented low expression in the cells with high capability to form heCIC structure and high expression in those with low capability. Knocking down the expression of EMP3 by gene editing promoted the formation of heCIC structures, while overexpression of EMP3 significantly inhibited this process. Additionally, the expression of factors involved in the heCIC structure formation suggested that EMP3 inhibited the formation of heCIC structures by modulating the adhesion ability and cytoskeleton of tumor cells. The findings lay a foundation for enhancing the heCIC-mediated tumor immunotherapy by targeting EMP3.

Biochemical indicators, cell apoptosis, and metabolomic analyses of the low-temperature stress response and cold tolerance mechanisms in Litopenaeus vannamei.

The cold tolerance of Litopenaeus vannamei is important for breeding in specific areas. To explore the cold tolerance mechanism of L. vannamei, this study analyzed biochemical indicators, cell apoptosis, and metabolomic responses in cold-tolerant (Lv-T) and common (Lv-C) L. vannamei under low-temperature stress (18 °C and 10 °C). TUNEL analysis showed a significant increase in apoptosis of hepatopancreatic duct cells in L. vannamei under low-temperature stress. Biochemical analysis showed that Lv-T had significantly increased levels of superoxide dismutase (SOD) and triglycerides (TG), while alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH-L), and uric acid (UA) levels were significantly decreased compared to Lv-C (p < 0.05). Metabolomic analysis displayed significant increases in metabolites such as LysoPC (P-16:0), 11beta-Hydroxy-3,20-dioxopregn-4-en-21-oic acid, and Pirbuterol, while metabolites such as 4-Hydroxystachydrine, Oxolan-3-one, and 3-Methyldioxyindole were significantly decreased in Lv-T compared to Lv-C. The differentially regulated metabolites were mainly enriched in pathways such as Protein digestion and absorption, Central carbon metabolism in cancer and ABC transporters. Our study indicate that low temperature induces damage to the hepatopancreatic duct of shrimp, thereby affecting its metabolic function. The cold resistance mechanism of Lv-T L. vannamei may be due to the enhancement of antioxidant enzymes and lipid metabolism.

The candidate proteins associated with keratoconus: A meta-analysis and bioinformatic analysis.

PURPOSE: Keratoconus (KC) is a multifactorial disorder. This study aimed to conduct a systematic meta-analysis to exclusively explore the candidate proteins associated with KC pathogenesis. METHODS: Relevant literature published in the last ten years in Pubmed, Web of Science, Cochrane, and Embase databases were searched. Protein expression data were presented as the standard mean difference (SMD) and 95% confidence intervals (CI). The meta-analysis is registered on PROSPERO, registration number CRD42022332442 and was conducted in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses statement (PRISMA). GO and KEGG enrichment analysis were performed, as well as the miRNAs and chemicals targeting the candidate proteins were predicted. PPI was analyzed to screen the hub proteins, and their expression was verified by RT-qPCR. RESULTS: A total of 21 studies were included in the meta-analysis, involving 346 normal eyes and 493 KC eyes. 18 deregulated proteins with significant SMD values were subjected to further analysis. In which, 7 proteins were up-regulated in KC compared with normal controls, including IL6 (SMD 1.54, 95%CI [0.85, 2.24]), IL1B (SMD 2.07, 95%CI [0.98, 3.16]), TNF (SMD 2.1, 95%CI [0.24, 3.96]), and MMP9 (SMD 1.96, 95%CI [0.68, 3.24]). While 11 proteins were down-regulated in KC including LOX (SMD 2.54, 95%CI [-4.51, -0.57]). GO and KEGG analysis showed that the deregulated proteins were involved in inflammation, extracellular matrix (ECM) remodeling, and apoptosis. MMP9, IL6, LOX, TNF, and IL1B were regarded as hub proteins according to the PPI analysis, and their transcription changes in stromal fibroblasts of KC were consistent with the results of the meta-analysis. Moreover, 10 miRNAs and two natural polyphenols interacting with hub proteins were identified. CONCLUSION: This study obtained 18 candidate proteins and demonstrated altered cytokine profiles, ECM remodeling, and apoptosis in KC patients through meta-analysis and bioinformatic analysis. It will provide biomarkers for further understanding of KC pathogenesis, and potential therapeutic targets for the drug treatment of KC.

Efficacy of Diosmin in Reducing Lower-Extremity Swelling and Pain After Total Knee Arthroplasty: A Randomized, Controlled Multicenter Trial.

BACKGROUND: Many patients experience lower-extremity swelling following total knee arthroplasty (TKA), which impedes recovery. Diosmin is a semisynthetic flavonoid that is often utilized to treat swelling and pain caused by chronic venous insufficiency. We aimed to evaluate the efficacy and safety of diosmin in reducing lower-extremity swelling and pain as well as in improving functional outcomes following TKA. METHODS: This study was designed as a randomized, controlled multicenter trial and conducted in 13 university-affiliated tertiary hospitals. A total of 330 patients undergoing TKA were randomized to either receive or not receive diosmin postoperatively. The diosmin group received 0.9 g of diosmin twice per day for 14 consecutive days starting on the day after surgery, whereas the control group received neither diosmin nor a placebo postoperatively. The primary outcome was lower-extremity swelling 1, 2, 3, and 14 days postoperatively. The secondary outcomes were postoperative pain assessed with use of a visual analogue scale, Hospital for Special Surgery score, range of knee motion, levels of the inflammatory biomarkers C-reactive protein and interleukin-6, and complications. RESULTS: At all postoperative time points, diosmin was associated with significantly less swelling of the calf, thigh, and upper pole of the patella as well as with significantly lower pain scores during motion. However, no significant differences in postoperative pain scores at rest, Hospital for Special Surgery scores, range of motion, levels of inflammatory biomarkers, or complication rates were found between the diosmin and control groups. CONCLUSIONS: The use of diosmin after TKA reduced lower-extremity swelling and pain during motion and was not associated with an increased incidence of short-term complications involving the outcomes studied. However, further studies are needed to continue exploring the efficacy and safety of diosmin use in TKA. LEVEL OF EVIDENCE: Therapeutic Level I . See Instructions for Authors for a complete description of levels of evidence.

[Rac1 promotes the formation of heterotypic cell-in-cell structure].

Heterotypic cell-in-cell structures (heCICs) are closely related to tumor development and progression, and have become a new frontier in life science research. Ras-related C3 botulinum toxin substrate 1 (Rac1) belongs to the classic Rho GTPase, which plays a key role in regulating the cytoskeleton and cell movement. To investigate the role and mechanism of Rac1 in the formation of heCICs, tumor cells and immune killer cells were labeled with cell-tracker, respectively, to establish the heCICs model. Upon treatment with the Rac1 inhibitor NSC23766, the formation of heCICs between tumor and immune cells was significantly reduced. The plasmid pQCXIP-Rac1-EGFP constructed by gene cloning was packaged into pseudoviruses that subsequently infect tumor cells to make cell lines stably expressing Rac1. As a result, the formation of heCICs was significantly increased upon Rac1 overexpression. These results demonstrated a promotive role of Rac1 in heCICs formation, which may facilitate treating cell-in-cell related diseases, such as tumors, by targeting Rac1.

Effects of early pregnancy on the complement system in the ovine thymus.

The complement system is crucial for the innate immune system, and complement activation is related to abnormal pregnancy in mice and humans. It is hypothesized that the complement system participates in maternal thymic immune regulation during early pregnancy in sheep. In this study, maternal thymuses were sampled on day 16 of the estrous cycle, and days 13, 16 and 25 of gestation in sheep. Quantitative real-time PCR, Western blot and immunohistochemistry analyses were used to analyze the expression of the complement components C1q, C1r, C1s, C2, C3, C4a, C5b and C9 in the maternal thymus. The results revealed that the mRNA and protein expression of C1r, C1s, C2, C3 and C4a was inhibited by early pregnancy, and the pregnancy recognition signal induced upregulation of C1q, C5b and C9 expression at day 16 of gestation. Furthermore, C3 protein was mostly located in epithelial reticular cells and thymic corpuscles, which may be involved in immune regulation. In summary, early pregnancy inhibits the complement system in the maternal thymus, which may be essential for the maternal immune regulation and successful pregnancy in sheep.

Nxhl Controls Angiogenesis by Targeting VE-PTP Through Interaction With Nucleolin.

Precise regulation of angiogenesis is required for organ development, wound repair, and tumor progression. Here, we identified a novel gene, nxhl (New XingHuo light), that is conserved in vertebrates and that plays a crucial role in vascular integrity and angiogenesis. Bioinformatic analysis uncovered its essential roles in development based on co-expression with several key developmental genes. Knockdown of nxhl in zebrafish causes global and pericardial edema, loss of blood circulation, and vascular defects characterized by both reduced vascularization in intersegmental vessels and decreased sprouting in the caudal vein plexus. The nxhl gene also affects human endothelial cell behavior in vitro. We found that nxhl functions in part by targeting VE-PTP through interaction with NCL (nucleolin). Loss of ptprb (a VE-PTP ortholo) in zebrafish resulted in defects similar to nxhl knockdown. Moreover, nxhl deficiency attenuates tumor invasion and proteins (including VE-PTP and NCL) associated with angiogenesis and EMT. These findings illustrate that nxhl can regulate angiogenesis via a novel nxhl-NCL-VE-PTP axis, providing a new therapeutic target for modulating vascular formation and function, especially for cancer treatment.

Early pregnancy affects expression of Toll-like receptor signaling members in ovine spleen.

Toll-like receptors (TLRs) are involved to the maternal immune tolerance. The spleen is essential for adaptive immune reactions. However, it is unclear that early pregnancy regulates TLR-mediated signalings in the maternal spleen. The purpose of this study was to investigate the effects of early pregnancy on expression of TLR signaling members in the ovine spleen. Ovine spleens were collected at day 16 of the estrous cycle, and at days 13, 16 and 25 of pregnancy (n = 6 for each group). Real-time quantitative PCR, western blot and immunohistochemistry analysis were used to detect TLR signaling members, including TLR2, TLR3, TLR4, TLR5, TLR7, TLR9, myeloid differentiation primary-response protein 88 (MyD88), tumor necrosis factor receptor associated factor 6 (TRAF6) and interleukin-1-receptor-associated kinase 1 (IRAK1). The results showed that expression levels of TLR2, TLR4 and IRAK1 were downregulated, but expression levels of TLR3, TLR5, TLR7, TLR9, TRAF6 and MyD88 were increased during early pregnancy. In addition, MyD88 protein was located in the capsule, trabeculae and splenic cords of the maternal spleen. This paper reports for the first time that early pregnancy has effects on TLR signaling pathways in the ovine spleen, which is beneficial for understanding the maternal immune tolerance during early pregnancy.

Toll-like receptor signaling is changed in ovine lymph node during early pregnancy.

Toll-like receptors (TLRs) participate in regulation of adaptive immune responses, and lymph nodes play key roles in the initiation of immune responses. There is a tolerance to the allogenic fetus during pregnancy, but it is unclear that expression of TLR signaling is in ovine lymph node during early pregnancy. In this study, lymph nodes were sampled from day 16 of nonpregnant ewes and days 13, 16, and 25 of pregnant ewes, and the expressions of TLR family (TLR2, TLR3, TLR4, TLR5 and TLR9), adaptor proteins, including myeloid differentiation primary-response protein 88 (MyD88), tumor necrosis factor receptor associated factor 6 (TRAF6), and interleukin-1-receptor-associated kinase 1 (IRAK1), were analyzed through real-time quantitative polymerase chain reaction, Western blot, and immunohistochemistry analysis. The results showed that mRNA and protein levels of TLR2, TLR3, TLR4, TRAF6, and MyD88 were upregulated in the maternal lymph node, but TLR5, TLR9, and IRAK1 were downregulated during early pregnancy. In addition, MyD88 protein was located in the subcapsular sinus and lymph sinuses. Therefore, it is suggested that early pregnancy induces changes in TLR signaling in maternal lymph node, which may be involved in regulation of maternal immune responses in sheep.

Single-Cell Ribonucleic Acid Sequencing Clarifies Cold Tolerance Mechanisms in the Pacific White Shrimp (Litopenaeus Vannamei).

To characterize the cold tolerance mechanism of the Pacific white shrimp (Litopenaeus vannamei), we performed single-cell RNA sequencing (scRNA-seq) of ∼5185 hepatopancreas cells from cold-tolerant (Lv-T) and common (Lv-C) L. vannamei at preferred and low temperatures (28°C and 10°C, respectively). The cells fell into 10 clusters and 4 cell types: embryonic, resorptive, blister-like, and fibrillar. We identified differentially expressed genes between Lv-T and Lv-C, which were mainly associated with the terms "immune system," "cytoskeleton," "antioxidant system," "digestive enzyme," and "detoxification," as well as the pathways "metabolic pathways of oxidative phosphorylation," "metabolism of xenobiotics by cytochrome P450," "chemical carcinogenesis," "drug metabolism-cytochrome P450," and "fatty acid metabolism." Reconstruction of fibrillar cell trajectories showed that, under low temperature stress, hepatopancreas cells had two distinct fates, cell fate 1 and cell fate 2. Cell fate 1 was mainly involved in signal transduction and sensory organ development. Cell fate 2 was mainly involved in metabolic processes. This study preliminarily clarifies the molecular mechanisms underlying cold tolerance in L. vannamei, which will be useful for the breeding of shrimp with greater cold tolerance.

Myelodysplastic Syndrome with Complex Chromosomal Abnormalities: A Case Report and Literature Review.

OBJECTIVE: Karyotype is the most important diagnostic and prognostic parameter in myelodys-plastic syndrome (MDS). Here, we describe a novel case of MDS with complex chromosomal abnormalities. CASE PRESENTATION: A 55-year-old Chinese female was admitted to the hospital for facial edema and a loss of appetite. Bone marrow aspiration showed the blast cell count 3.6%. Erythrocyte hyperplasia was active, megaloblastoid change was observed, and a wide variability of nuclear numbers, as well as variability of size and shape was present. Bone marrow chromosomal analyses showed 45~48, X, -X, -4, t (5;8) (q13;q22), add (7) (q11), add (13) (p11), -14, del (16) (p13), add (19) (q13), -20, i(21)(q10),+4~6mar [cp15]/46,XX[5]. The patient was diagnosed with MDS with WPSS of the high risk group. IPSS was medium risk-2. IPSS-R was categorized as the extremely high risk group. CONCLUSION: The prognosis and treatment of MDS with complex chromosomal abnormalities are still uncertain, and further studies are needed.

Iontophoresis-assisted accelerated riboflavin/ultraviolet A scleral cross-linking: A potential treatment for pathologic myopia.

Scleral collagen cross-linking is one of the most promising treatments to control the pathologic process of myopia. However, the exact procedure and its impact on animal models of myopia are still to be explored. We modified the scleral riboflavin/ultraviolet A (UVA) cross-linking procedure with an iontophoresis-assisted drug delivery system and an accelerated UVA irradiation (10 mW/cm2, 9 min) and applied this treatment to an animal model of myopia. Ninety-six New Zealand White rabbits developed relatively stable myopia by visual deprivation and then underwent the modified scleral cross-linking surgery. All the statistics and sample collection were obtained from 4 postoperative time points (1-day, 10-day, 1-month and 3-month groups). We found that the ultimate stress, Young's modulus and physiological Young's modulus of treated myopia sclera were significantly increased and maintained in 4 groups. The abnormal elongation of the myopic eye was effectively controlled 1 month after the treatment and even almost halted 3 months after the treatment. The histochemical assay revealed no notable post-surgery damage or apoptosis in the retina and choroid. Vigorous collagen synthesis was observed in scleral fibroblasts of the treated samples but were rarely observed in the untreated ones under electron microscopy. Furthermore, the remarkable difference in collagen gene expression and protein content between treated and untreated samples also indicated that an alteration in collagen metabolism may be triggered by the treatment. The effectiveness and safety exploration suggested that the modified scleral cross-linking procedure may be a potential method to control the pathologic process of myopia.

Induction of MMP­1 and ­3 by cyclical mechanical stretch is mediated by IL­6 in cultured fibroblasts of keratoconus.

In order to understand the effect of mechanical stretch on corneal extracellular matrix remodeling, human keratoconus fibroblasts (HKCFBs) were subjected to cyclic stretch in vitro and the expression of matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases (TIMPs), and inflammatory cytokines were evaluated. HKCFBs were seeded into a flexible membrane base and subjected to a cyclic stretch regimen of 10% equibiaxial stretch at a stretching frequency of 1 Hz for 6 h using a Flexcell tension unit. An antibody directed against interleukin­6 (IL­6 Ab) was used to investigate the roles of IL­6 on mechanical stretch mediated regulation of MMP in HKCFBs. Culture supernatants were assayed using an enzyme­linked immunosorbent assay for MMP­1 and ­3, TIMP­1 and ­2, and IL­6. Total RNA from the cells was extracted, and quantitative polymerase chain reaction was used to determine mRNA for MMP­1 and ­3, TIMP­1 and ­2, and IL­6. In stretched cells, levels of MMP­1 and ­3 demonstrated an increase compared with unstretched cells, but levels of TIMP­1, and ­2 revealed a decrease. Mechanical stretch significantly increased the mRNA expression and protein synthesis of IL­6 compared with unstretched cells. IL­6 induced MMP­1 and ­3 expression, whereas no significant effects were observed in levels of TIMP­1 and ­2 compared with the untreated control groups. Additionally, the IL­6 Ab markedly inhibited the stretch­induced increase in MMP­1 and ­3 in culture supernatants in a dose­dependent manner. No significant differences in TIMP­1 and ­2 protein levels were detected between stretched cells treated with IL­6 Ab and stretched cells without IL­6 Ab treatment. These results indicate that cyclical mechanical stretch augments IL­6 production and MMP expression, and reduces levels of TIMP in HKCFBs. Thus, it is suggested that IL­6 mediates the stretch­induced MMP expression.

Effects of ablation depth and repair time on the corneal elastic modulus after laser in situ keratomileusis.

BACKGROUND: The biomechanical properties of the cornea should be taken into account in the refractive procedure in order to perform refractive surgery more accurately. The effects of the ablation depth and repair time on the elastic modulus of the rabbit cornea after laser in situ keratomileusis (LASIK) are still unclear. METHODS: In this study, LASIK was performed on New Zealand rabbits with different ablation depth (only typical LASIK flaps were created; residual stroma bed was 50 or 30% of the whole cornea thickness respectively). The animals without any treatment were served as normal controls. The corneal thickness was measured by ultrasonic pachymetry before animals were humanly killed after 7 or 28 days post-operatively. The corneal elastic modulus was measured by uniaxial tensile testing. A mathematical procedure considering the actual geometrics of the cornea was created to analyze the corneal elastic modulus. RESULTS: There were no obvious differences among all groups in the elastic modulus on after 7 days post-operatively. However, after 28th days post-operatively, there was a significant increase in the elastic modulus with 50 and 30% residual stroma bed; only the elastic modulus of the cornea with 30% residual stroma bed was significantly higher than that of 7 days. CONCLUSIONS: Changes in elastic modulus after LASIK suggest that this biomechanical effect may correlate with the ablation depth and repair time.

Combined effect of TNF-α and cyclic stretching on gene and protein expression associated with mineral metabolism in cementoblasts.

OBJECTIVE: This work aims to investigate how the combination of TNF-α and cyclic stretching affects the expression of gene and protein associated with mineral metabolism in cementoblasts in vitro. DESIGN: Cementoblasts were cyclically stretched using the Flexcell tension system 4000 in the presence of 10 ng/ml TNF-α. Subsequently, the gene and protein expression CAP, Col I, BSP, OPG, RANKL and RUNX2 were detected using RT- PCR and ELISA/Western immunoblotting methods, respectively. RESULTS: Cyclic stretching alone enhanced CAP, Col I, OPG, RANKL and RUNX2 expression in an amplitude manner, while decreased BSP expression. Expression of all these proteins was attenuated in the presence of TNF-α whether the cells were exposed to cyclic stretching or not. The ratio of RANKL/OPG was increased at any stimulation. CONCLUSION: This results suggest that TNF-α affected the regulation of gene and protein expression induced by mechanical stimulation in cementoblasts. This may suppress anabolism and promote catabolism of cementum. It suggests that inflammatory cytokine may impair the cementum remodeling under mechanical stimulation.

Combined effects of interleukin-1ß and cyclic stretching on metalloproteinase expression in corneal fibroblasts in vitro.

BACKGROUND: Corneal tensile strain increases if the cornea becomes thin or if intraocular pressure increases. However, the effects of mechanical stress on extracellular matrix (ECM) remodelling in the corneal repair process and the corneal anomalies are unknown. METHODS: In this study, the combined effects of interleukin-1ß (IL-1ß) on matrix metalloproteinases (MMPs) in corneal fibroblasts under cyclic stretching were investigated in vitro. Cultured rabbit corneal fibroblasts were subjected to 5, 10 or 15 % cyclic equibiaxial stretching at 0.1 Hz for 36 h in the presence of IL-1ß. Conditioned medium was harvested for the analysis of MMP2 and MMP9 protein production using the gelatin zymography and western blot techniques. RESULTS AND CONCLUSIONS: Cyclic equibiaxial stretching changed the cell morphology by increasing the contractility of F-actin fibres. IL-1ß alone induced the expression of MMP9 and increased the production of MMP2, and 5 % stretching alone decreased the production of MMP2, which indicates that a low stretching magnitude can reduce ECM degradation. In the presence of IL-1ß, 5 and 10 % stretching increased the production of MMP2, whereas 15 % stretching increased the production of MMP9. These results indicate that MMP expression is enhanced by cyclic mechanical stimulation in the presence of IL-1ß, which is expected to contribute to corneal ECM degradation, leading to the development of post-refractive surgery keratectasia.

Induction of matrix metalloproteinase-1 by tumor necrosis factor-α is mediated by interleukin-6 in cultured fibroblasts of keratoconus.

Inflammatory molecules and matrix metalloproteinase (MMPs) have been found over-expressed in the tear film of patients with keratoconus. However, the mechanistic link between inflammatory molecules and MMPs in the pathogenesis of keratoconus remains still elusive. Therefore, we investigated the effect of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) on MMP-1 expression and used IL-6 antibody (IL-6 Ab) to examine the role of IL-6 on TNF-α mediated regulation of MMP-1 in fibroblasts of normal cornea and keratoconus. Real-time polymerase chain reaction, Enzyme-linked immunosorbent assay, and Western blot data demonstrated that MMP-1 and IL-6 were expressed in fibroblasts of normal cornea and keratoconus. Levels of MMP-1 and IL-6 were significantly higher in keratoconus than normal cornea. TNF-α treatment led to a significant increase in IL-6 levels. IL-6 treatment induced MMP-1 synthesis in normal cornea and keratoconus. TNF-α increased MMP-1 expression in a dose- and time-dependent manner and this response was completely inhibited by the IL-6 Ab. In conclusion, these results indicate that fibroblasts of keratoconus shows increased levels of IL-6 and MMP-1 gene and protein expression and IL-6 mediates the TNF-α-induced MMP-1 expression.

[Effects of Tumor Necrosis Factor Alpha on the Expression of Matrix Metalloproteinases and Tissue Inhibitors of Matrix Metalloproteinases in Keratoconus Fibroblasts].

The aim of this article is to study the effect of tumor necrosis factor alpha(TNF-α)on the expression of matrix metalloproteinases(MMPs)and tissue inhibitors of matrix metalloproteinases(TIMPs)in keratoconus fibroblasts in vitro.Normal cornea and keratoconus fibroblasts were extracted using enzyme digestion method and were cultured in the medium containing TNF-α(0,10 and 100ng/mL).The expression of MMPs proteins in the supernatant of corneal fibroblasts and the expression of TIMPs in the normal cornea and keratoconus fibroblasts were detected by Western blot and real-time quantitative polymerase chain reaction respectively.The active form of MMP1 could be detected in the supernatant of keratoconus fibroblasts and upregulated by TNF-α.TNF-αcould increase the protein expression of MMP2,MMP3,MMP9 in the supernatant of keratoconus fibroblasts and decrease the gene expression of TIMP1,TIMP2 in keratoconus fibroblasts.The increased MMPs and the decreased TIMPs can increase the degradation of the extracellular matrix.TNF-αmay play an important role in the occurrence and development of keratoconus by regulating the expression of MMPs/TIMPs.

Expression of MMP-2, MT1-MMP, and TIMP-2 by cultured rabbit corneal fibroblasts under mechanical stretch.

Refractive surgery not only leads to tissue injury but also evokes mechanical stress increase of the cornea. How the mechanical stress affects the corneal matrix remodeling, specifically, matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases; TIMPs) is not well understood. In this study, cultured rabbit corneal fibroblasts in vitro were subjected to regimen of 5%, 10%, or 15% equibiaxial stretch at 0.1 Hz for 3 or 24 h. MMP-2 protein level was measured by gelatin zymography and Western blotting. MMP-2, membrane type 1 MMP (MT1-MMP), and TIMP-2 mRNA levels were quantified by real-time quantitative PCR. Extracellular regulated protein kinase (ERK) phosphorylation protein levels were assessed by Western blotting. Our results showed that a 15% stretch resulted in increases in MMP-2 protein, MMP-2 mRNA, and MT1-MMP mRNA levels, but a decrease in TIMP-2 mRNA level. However, a 5% stretch caused decreases in MMP-2 protein and mRNA level, but an increase in TIMP-2 mRNA level, and no change in MT1-MMP mRNA level. A 15% stretch also caused a significant increase in ERK1/2 phosphorylation. Inhibition of the mitogenactivated protein kinase (MEK) pathway with PD98059 attenuated stretch-induced increase in MMP-2 production and ERK activity. These results suggest that small-magnitude stretching may promote corneal matrix synthetic events, whereas large-magnitude stretching promotes corneal matrix degradation by changing the balance between MMPs and TIMPs in corneal fibroblasts. Large-magnitude stretch-induced increase in pro-MMP-2 production was in an ERK-dependent manner.