Understanding the complexity of Tityus serrulatus venom: A focus on high molecular weight components

Tityus serrulatus scorpion is responsible for a significant number of envenomings in Brazil, ranging from mild to severe, and in some cases, leading to fatalities. While supportive care is the primary treatment modality, moderate and severe cases require antivenom administration despite potential limitations and adverse effects. The remarkable proliferation of T. serrulatus scorpions, attributed to their biology and asexual reproduction, contributes to a high incidence of envenomation. T. serrulatus scorpion venom predominantly consists of short proteins acting as neurotoxins (α and ß), that primarily target ion channels. Nevertheless, high molecular weight compounds, including metalloproteases, serine proteases, phospholipases, and hyaluronidases, are also present in the venom. These compounds play a crucial role in envenomation, influencing the severity of symptoms and the spread of venom. This review endeavors to comprehensively understand the T. serrulatus scorpion venom by elucidating the primary high molecular weight compounds and exploring their potential contributions to envenomation. Understanding these compounds' mechanisms of action can aid in developing more effective treatments and prevention strategies, ultimately mitigating the impact of scorpion envenomation on public health in Brazil.

Diagnostic value of cysteine rich 61, glyoxalase Ⅰ and microRNA-155 in endometrial carcinoma / 中国医师进修杂志

Objective:To investigate the diagnostic value of cysteine rich 61 (Cyr61), glyoxalase Ⅰ (GLO -1) and microRNA-155(miR-155) in endometrial carcinoma.Methods:Eighty-five patients with endometrial cancer treated in Lu Southwest Hospital from June 2017 to March 2020 were selected as the observation group, including 15 cases of recurrence and 70 cases of non-recurrence. In addition, 85 patients with benign uterine lesions were selected as the control group. The levels of serum Cyr61, GLO-1 and miR-155 were compared between the two groups, the correlation between the levels of serum Cyr61, GLO-1 and miR-155 and clinicopathological features were analyzed, the receiver operating characteristic (ROC) curve was drawn, the diagnostic value of the levels of serum Cyr61, GLO-1 and miR-155 in endometrial cancer were evaluated, and the relationship between the levels of serum Cyr61, GLO-1 and miR-155 and the recurrence of endometrial cancer were analyzed.Results:The levels of serum Cyr61, GLO-1 and miR-155 in the observation group were higher than those in the control group: (294.74 ± 78.41) μg/L vs. (156.82 ± 50.62) μg/L, (96.27 ± 19.85) pmol/L vs. (79.83 ± 15.69) pmol/L, 6.82 ± 2.27 vs. 2.57 ± 0.78, the differences were statistically significant ( P<0.05). The levels of serum Cyr61, GLO-1 and miR-155 in patients with endometrial cancer were positively correlated with clinical stage, myometrial invasion and lymph node metastasis ( P<0.05). The area under the curve(AUC) of serum Cyr61, GLO-1 and miR-155 in the combined diagnosis of endometrial cancer was 0.906. The levels of serum Cyr61, GLO-1 and miR-155 in recurrence patients were higher than those non-recurrence patients : (358.21 ± 89.63) μg/L vs. (281.14 ± 75.29) μg/L, (109.89 ± 20.14) pmol/L vs. (93.35 ± 16.37)pmol/L, 8.04 ± 2.51 vs. 6.56 ± 2.17, the differences were statistically significant ( P<0.05). The levels of serum Cyr61, GLO-1 and miR-155 were the risk factors of recurrence in patients with endometrial cancer ( P< 0.05). Conclusions:The levels of serum Cyr61, GLO-1 and miR-155 in patients with endometrial cancer are significantly increased, which are related to clinical stage, degree of invasion, lymph node metastasis and recurrence. Detecting their levels can be used to diagnose endometrial cancer and predict recurrence, so as to guide clinical treatment.

Protective effect of rapamycin on cognitive function in lupus mice by regulating Cysteine rich 61 and autophagy in hippocampus / 中华风湿病学杂志

Objective:To investigate the effects of rapamycin (RAPA) on the cognitive function of lupus mice by regulating Cysteine rich 61 (Cyr61) and autophagy.Methods:MRL/lpr lupus mice were randomly divided into lupus group and rapamycin + lupus group, wild-type C57BL/6 mice were randomly divided into normal control group and rapamycin group with six mice in each group, RAPA + lupus group and rapamycin group were intraperitoneally injected with RAPA (2.0 mg/kg). The lupus group and the normal control group were injected with equal amounts of dimethyl sulfoxide (DMSO). Morris water maze was used to observe the cognitive function of mice. Western blotting was used to detect the expression of Cyr61, Programmed cell death-1 (Beclin-1), Microtubule associated protein 1 light chain3B (LC3B). Hematoxylin-eosin (HE) staining was used to observe the pathological changes in the hippocampus. The changes of neurons and bodies in hippocampus were observed by Nissl staining. The localization and expression of Cyr61 and LC3B in hippocampus were detected by immunofluorescence staining. One-way analysis of variance (ANOVA) was used between groups, and LSD-T test was used for pairwise comparison.Results:Western blotting results showed thatthe protein expression of Cyr61 (0.99±0.15) was significantly increased ( P=0.011), and the protein expression of Beclin-1 (0.64±0.04) and LC3B(0.54±0.05) was significantly decreased in lupus group ( P=0.025, P= 0.008) when compared with normal control group (0.73±0.08, 0.81±0.12, 0.80±0.03). The expressions of Cyr61 (0.75±0.05, 0.75±0.08), Beclin-1 (0.84±0.08, 0.92±0.04) and LC3B (0.93±0.16, 0.76±0.08) in rapamycin group and rapamycin + lupus group were not significantly changed ( P>0.05). Compared with rapamycin group, the protein expression of Cyr61 (0.99±0.15) was significantly increased ( P=0.016), and Beclin-1 (0.64±0.04), LC3B (0.54±0.05) was significantly decreased in lupus group ( P=0.013, P=0.001). The expressions of Cyr61 (0.75± 0.08), Beclin-1 (0.92±0.04) and LC3B (0.76±0.08) were not significantly changed in rapamycin+lupus group ( P=0.999, P=0.241, P=0.062). Compared with lupus group, the expression of Cyr61 (0.75±0.08) protein in rapamycin+lupus group was significantly decreased ( P=0.016), and the expression of Beclin-1 (0.92±0.04) and LC3B(0.76±0.08) protein were significantly increased ( P=0.002, P=0.017). Immunofluorescence results showed that Cyr61 and LC3B were mainly expressed in the cytoplasm of hippocampal neurons, and the quantitative detection results were consistent with western blot results, the differences were statistically significant ( P=0.025, P=0.032). HE staining showed that the levels and number of cells in the hippocampus of mice with lupus were reduced, and the arrangement was sparse, and the nuclei were hyperchromatic, showing nuclear pyknosis and migration. The results of Nissl staining showed that there were relatively fewer Nissl bodies, loose arrangement of neurons and vacuolar areas in some cells, which were improved after RAPA treatment in lupus mice. Conclusion:RAPA can protect the cognitive function of lupus mice by inhibiting the expression of Cyr61 in hippocampus and promoting autophagy.

Therapeutic effects of the extract of Sancao Formula, a Chinese herbal compound, on imiquimod-induced psoriasis via cysteine-rich protein 61 / 中西医结合学报

OBJECTIVE@#Psoriasis is a common chronic inflammatory skin disease that is prone to recurrence, and the proinflammatory factor, cysteine-rich protein 61 (Cyr61), is important in its pathophysiology. Long-term clinical practice has shown that Sancao Formula (SC), a Chinese herbal compound, is effective in the treatment of psoriasis, but the precise mechanism remains unknown. In this study, we investigate the mechanism by which SC extract alleviates imiquimod (IMQ)-induced psoriasis.@*METHODS@#The expression of Cyr61 in psoriatic lesions and normal healthy skin was detected using immunohistochemical analysis to investigate the biological role of Cyr61 in models of psoriatic inflammation. A psoriatic mouse model was established by topical application of IMQ, and the effect of topical application of SC extract was evaluated using the psoriasis area and severity index (PASI) score, hematoxylin-eosin staining, and histopathological features of the skin. Next, a HaCaT cell inflammation model was established using interferon-γ (IFN-γ), and the effect of SC extract on the mRNA and protein levels of Cyr61 and intercellular cell adhesion molecule-1 (ICAM-1) was confirmed using Western blot and quantitative real-time polymerase chain reaction analyses.@*RESULTS@#Immunohistochemical staining showed that the expression of Cyr61 in psoriatic lesions was higher than that in normal skin samples (78.26% vs 41.18%, P < 0.05), and the number of Cyr61-positive cells in psoriatic lesions was also significantly higher than in normal skin (18.66 ± 2.51 vs 4.33 ± 1.52, P < 0.05). Treatment in mice with IMQ-induced psoriasis showed that SC extract could significantly improve the inflammatory phenotype, PASI score (10.875 ± 0.744 vs 3.875 ± 0.582, P < 0.05), and pathological features compared with those in IMQ model group; SC treatment was also associated with decreased levels of Cyr61 and ICAM-1. In the IFN-γ-induced inflammatory cell model, the mRNA and protein levels of Cyr61 and ICAM-1 were upregulated, while the SC extract downregulated the levels of Cyr61 and ICAM-1.@*CONCLUSION@#The results provide a theoretical basis for the involvement of Cyr61 in the pathogenesis of psoriasis, and suggest that SC should be used to target Cyr61 for the prevention of psoriasis recurrence.

Relationship of CRIM1 and epithelial-mesenchymal transition in hepatocellular carcinoma / 国际外科学杂志

Objective To study the expression and the clinical significance of cysteine rich transmembrane BMP regulator 1 (CRIM1) in hepatocellular carcinoma (HCC) and discuss the association between CRIM1 and epithelial-mesenchymal transition (EMT).Methods The cases were came from the Subei People's Hospital Affilated Hospital of Yangzhou University from January 2013 to December 2017.CRIM1 and EMT related proteins (E-cadherin,Vimentin) in parts of HCC tissues and their paired peritumoural tissues were tested by Western blotting.The gray value was test by t test.The observation indicators:(1) expression of CRIM1 protein and EMT-related protein (E-cadherin,Vimentin) in liver cancer tissues and paracancerous tissues.(2) The relationship between CRIM1 protein expression and clinicopathological factors in patients with liver cancer.The expression ofCRIM1 in HCC tissues and adjacent tissues was detected by immunohistochemistry(IHC),which was divided into high expression group and low expression group according to the histochemical score,and the relation between the expression of CRIM1 and the clinicopathological factors of the patients was analyzed by chi-square test and Spearman correlation analysis.Finally,the relation between CRIM1 and overall survival of HCC patients was analyzed by Kaplan Meier Plotter database.Results The expression of CRIM1 in tumor and matched paratumor specimens were 0.15 ± 0.03,0.8 ± 0.04,and E-cadherinin tumor and matched paratumor specimenswere 0.20 ±0.05,0.56 ± 0.06,their expression in paracancerous tissues was higher than HCC tissues (t =14.21,4.69,P ＜ 0.05),while the expression of Vimentin in tumor and matched paratumor specimens were 0.74 ± 0.08,0.45 ± 0.06,the expression in tumor tissues were significantly higher than adjacent tissues (t =2.87,P ＜ 0.05).The expression of CRIM1 in HCC tissues was further verified by immunohistochemistry,which shows that CRIM1 was overexpressed in paracancerous tissues.In 114 patients,46 cases of CRIM1 protein were highly expressed in liver cancer tissues,and 68 cases of CRIM1 protein were low expressed.The expression of CRIM1 obviously related with the level of αt-fetoprotein (AFP),tumor size and symptom(r =-0.43,-0.34,-0.24,x2 =9.381,5.248,8.117,P ＜ 0.05).However,other clinicopathological features were not correlated with CRIM 1 expression,including age,tumour differentiation,tumor number.Finally,the overall survival was different between CRIM1 high expression and low expression according to the Kaplan Meier Plotter database.Conclusions The expression of CRIM1 is negatively correlated with the EMT process in HCC.CRIM1 might be a potential molecular marker for prognosis.

Stretch-induced Expression of CYR61 Increases the Secretion of IL-8 in A549 Cells via the NF-κβ/Iκβ Pathway / 华中科技大学学报（医学）（英德文版）

Mechanical ventilation (MV) with large tidal volumes can increase lung alveolar permeability and initiate inflammatory responses,resulting in ventilator-induced lung injury (VILI).The mechanisms of the injurious effects of MV and the genetic susceptibility remain unclear.VILI-related genes such as cysteine-rich angiogenic inducer 61 (Cyr61)have been demonstrated to play a detrimental role in the aggressive ventilation strategies.In the present study,we investigated the involvement of Cyr61 in the VIM and the underlying mechanism.A549 cells were exposed to cyclic stretch of varying durations and then the mRNA and protein levels of Cyr61 were measured by real-time PCR and Western blotting,respectively.Additionally,after exposure ofA549 cells to cyclic stretch for 5 min to 1 h,the expression levels of nuclear factor kappaB (NF-κB) and IL-8 were detected by ELISA and Western blotting.Thereafter,Cyr61 expression was depressed in A549 cells with the siRNA pGenesill.1-Cyr61-3 before the cyclic stretch,and IL-8 secretion and the activation of NF-κB pathways were probed by ELISA and Western blotting,respectively.Moreover,a NF-κB inhibitor (PDTC) and an activator (TNF) were used before mechanical stretch.Realtime PCR and ELISA were performed to detect the mRNA and protein of IL-8,respectively.The results showed that the mechanical cyclic stretch led to increased Cyr61 expression at mRNA and protein levels in A549 cells.Additionally,cyclic stretch also mobilized NF-κB from the cytoplasm to the nucleus and increased IL-8 secretion in A549 cells.The inhibition of Cyr61 blocked the NF-κB activation and IL-8 secretion in response to cyclic stretch.Inhibition of NF-κB attenuated the mRNA and protein expression of IL-8 in A549 cells transfected with Cyr61 siRNA.It was suggested that Cyr61/NF-κB signaling pathway mediates the upregulation of IL-8 in response to cyclic stretch in A594 cells.These findings support the hypothesis that Cyr61 plays a critical role in acute lung inflammation triggered by mechanical strain.

Stretch-induced Expression of CYR61 Increases the Secretion of IL-8 in A549 Cells via the NF-κβ/Iκβ Pathway / 华中科技大学学报（医学）（英德文版）

Mechanical ventilation (MV) with large tidal volumes can increase lung alveolar permeability and initiate inflammatory responses,resulting in ventilator-induced lung injury (VILI).The mechanisms of the injurious effects of MV and the genetic susceptibility remain unclear.VILI-related genes such as cysteine-rich angiogenic inducer 61 (Cyr61)have been demonstrated to play a detrimental role in the aggressive ventilation strategies.In the present study,we investigated the involvement of Cyr61 in the VIM and the underlying mechanism.A549 cells were exposed to cyclic stretch of varying durations and then the mRNA and protein levels of Cyr61 were measured by real-time PCR and Western blotting,respectively.Additionally,after exposure ofA549 cells to cyclic stretch for 5 min to 1 h,the expression levels of nuclear factor kappaB (NF-κB) and IL-8 were detected by ELISA and Western blotting.Thereafter,Cyr61 expression was depressed in A549 cells with the siRNA pGenesill.1-Cyr61-3 before the cyclic stretch,and IL-8 secretion and the activation of NF-κB pathways were probed by ELISA and Western blotting,respectively.Moreover,a NF-κB inhibitor (PDTC) and an activator (TNF) were used before mechanical stretch.Realtime PCR and ELISA were performed to detect the mRNA and protein of IL-8,respectively.The results showed that the mechanical cyclic stretch led to increased Cyr61 expression at mRNA and protein levels in A549 cells.Additionally,cyclic stretch also mobilized NF-κB from the cytoplasm to the nucleus and increased IL-8 secretion in A549 cells.The inhibition of Cyr61 blocked the NF-κB activation and IL-8 secretion in response to cyclic stretch.Inhibition of NF-κB attenuated the mRNA and protein expression of IL-8 in A549 cells transfected with Cyr61 siRNA.It was suggested that Cyr61/NF-κB signaling pathway mediates the upregulation of IL-8 in response to cyclic stretch in A594 cells.These findings support the hypothesis that Cyr61 plays a critical role in acute lung inflammation triggered by mechanical strain.

Effect and mechanism of cysteine-rich protein 61 on transforming growth factor-β1-activated renal fibroblasts / 中华肾脏病杂志

Objective To observe the expression of cysteine-rich protein 61 (Cyr61) in transforming growth factor-β1 (TGF-β1)-activated renal fibroblasts (NRK-49F),and to explore its effect and mechanism.Methods (1) NRK-49F cells were activated by TGF-β1 with different concentrations (0.0,0.5,1.0,2.0,5.0 μg/L).Western blotting was used to detect the expression of Cyr61 protein,and CCK-8 assay was used to test the proliferative activity of NRK-49F cells.(2) NRK-49F cells with low expression and over expression of Cyr61 were established by plasmid transfection.The cells were divided into control group (null vector transfection),over-expression group and lowexpression group.The proliferation was discovered by CCK-8 assay after 24,48 and 72 h.Further,5.0 μg/L TGF-β1 activated these three groups.The proliferation was also discovered by CCK-8 assay and the cell cycle was analyzed by flow cytometry.The mRNA expressions of fibrosis markers (Collα1,Col3αl,MMP9,MMP13) and factors of cell senescence signal pathway (p53,p21,Rb,p16) were ascertained by real time PCR,and the protein expressions of Col3 and MMP9 were detected by Western blotting.Results (1) Compared with 0.0 μg/L TGF-β1 group,the proliferation of NRK-49F cells was enhanced in 0.5,1.0,2.0 and 5.0 μg/L TGF-β1 groups (all P ＜ 0.05),while the expression of Cyr61 protein was decreased in 1.0 μg/L group and increased in 5.0 μg/L group (all P ＜0.05).(2) The proliferation of over-expression group was lower than that of control group after 24,48and 72 h (all P＜ 0.05),which was in a time-dependent manner.(3) Compared with control group activated by TGF-β1,the over-expression group expressed less fibrosis factors (Col1α1 and Col3α1)and more anti-fibrosis factors (MMP9 and MMP13) with decreased proliferation (all P ＜ 0.05).Simultaneously,the proportion of cells bogged down in G1 phases,as well as the expressions of p53,p21 and Rb mRNA increased (all P ＜ 0.05).The above effects of low-expression group were just opposite to over-expression group.Moreover,there was no significant difference in the expression of p16 gene among the three groups (P ＞ 0.05).Conclusions Cyr61 can curb the proliferation and fibrotic phenotypes of fibroblasts,thereafter slowing down the process of renal fibrosis.The p53/p21/Rb interrelated cell senescence signal pathway may be involved in the anti-fibrosis process.

MicroRNA-27a-mediated repression of cysteine-rich secretory protein 2 translation in asthenoteratozoospermic patients / 亚洲男科学杂志(英文版)

Cysteine-rich secretory protein 2 (CRISP2) is an important protein in spermatozoa that plays roles in modulating sperm flagellar motility, the acrosome reaction, and gamete fusion. Spermatozoa lacking CRISP2 exhibit low sperm motility and abnormal morphology. However, the molecular mechanisms underlying the reduction of CRISP2 in asthenoteratozoospermia (ATZ) remain unknown. In this study, low expression of CRISP2 protein rather than its mRNA was observed in the ejaculated spermatozoa from ATZ patients as compared with normozoospermic males. Subsequently, bioinformatic prediction, luciferase reporter assays, and microRNA-27a (MIR-27a) transfection experiments revealed that MIR-27a specifically targets CRISP2 by binding to its 3' untranslated region (3'-UTR), suppressing CRISP2 expression posttranscriptionally. Further evidence was provided by the clinical observation of high MIR-27a expression in ejaculated spermatozoa from ATZ patients and a negative correlation between MIR-27a expression and CRISP2 protein expression. Finally, a retrospective follow-up study supported that both high MIR-27a expression and low CRISP2 protein expression were associated with low progressive sperm motility, abnormal morphology, and infertility. This study demonstrates a novel mechanism responsible for reduced CRISP2 expression in ATZ, which may offer a potential therapeutic target for treating male infertility, or for male contraception.

Value of serum CRISPLD2 levels for the diagnosis and prognosis evaluation of sepsis patients / 中华危重病急救医学

Objective To investigate the value of cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) in diagnosis and prognosis in patients with sepsis.Methods Clinical data of patients admitted to intensive care unit (ICU) of the First Hospital of Hebei Medical University from December 2014 to December 2016 were retrospectively analyzed. According to the severity of sepsis, the patients were divided into three groups: sepsis patients, severe sepsis patients and septic shock patients, and 100 healthy persons were enrolled as control group. Levels of serum CRISPLD2, procalcitonin (PCT) and C-reactive protein (CRP), acute physiology and chronic health evaluation Ⅱ (APACHEⅡ) score, sequential organ failure assessment (SOFA) score, and 28-day prognosis were recorded. Analysis of the correlation between CRISPLD2 and PCT, CRP, APACHEⅡscore, SOFA score was done. The receiver operating characteristic (ROC) curve was plotted for the CRISPLD2 value for the diagnosis and prognosis in patients with sepsis.Results A total of 115 patients with sepsis were enrolled in this study, including 52 sepsis, 48 severe sepsis, and 15 septic shock; 29 patients died after 28 days, 28-days mortality rate was 25.2%. There was no significant difference in CRISPLD2 between sepsis and healthy control group (mg/L: 204.1±74.5 vs. 211.3±12.0, P > 0.05); the level of CRISPLD2 in septic shock group was significantly lower than that in sepsis group and severe sepsis group (mg/L: 139.0±55.0 vs. 240.2±89.6, 233.0±8.9, bothP 216.0 mg/L, the sensitivity was 96.7%, and the specificity was 92.6%, which power lied between PCT and CRP. The AUC of CRISPLD2 for prognosis was significantly lower than that of PCT [0.617 (0.507-0.727) vs. 0.786 (0.668-0.903),P <0.01]; when the cut-off value of CRISPLD2 was 103.5 mg/L, the sensitivity was 100%, and the specificity was 25.6%. Conclusion CRISPLD2 is a potential biomarker in sepsis, but cannot predict the prognosis of patients with sepsis.

The expression and significance of cysteine-rich 61, connective tissue growth factor, vascular endothelial growth factor and microvascular density marked by CD105 in muscle tissue of patients with dermatomyosits and polymyosits / 中华风湿病学杂志

Objective By detecting vascular cysteine-rich 61(Cyr61) related factor,connective tissue growth factor (CTGF),vascular endothelial growth factor (VEGF) and CD105 markers of microvascular density (MVD) of muscle tissue in patients with PM/DM,the role and significance of the expression of Cyr61,CTGF,VEGF and CD105 in the process of vascular lesions of dermatomyosits (DM) and polymyosits (PM) were discussed.Methods The expression of Cyr61,CTGF,VEGF and CD105 markers of micro vascular density (MVD) were detected in 10 cases of DM,10 cases of PM and 20 controls by using immunohistochemical Envision two step method.Data were analyzed using Statistical Product and Service Solutions (SPSS) statistical software.Fisher's exact probability analysis and Spearman correlation analysis were conducted.Results Compared with the control group,Cyr61,CTGF,VEGF positive expression rate in muscle tissue of patients with DM and PM group were significantly different (P＜0.01),the positive expression rates of Cyr61,CTGF,VEGF in DM group and PM group were 90％,70％,90％,80％,80％,70％,and the control group (5％,10％,5％) respectively.In the muscle tissue of patients with DM and PM group,CD105 markers of capillaries could be seen,and MVD in DM and PM group were higher than that in the control group,the difference was statistically significant (F=8.103,P=0.001).Cyr61,CTGF and VEGF protein expression levels in muscle tissueof patients with DM and PM were positively correlated with MVD.Conclusion The muscle tissue of PM/DMpatients may have new blood vessels formation.Cyr61,CTGF,VEGF may be involved in the formation of newblood vessels in the PM/DM muscle tissue.The results of this study suggest that microvascular lesion plays animportant role in the immune pathogenesis of inflammatory myopathy such as PM/DM.

Effect and mechanism of cysteine-rich protein 61 on transforming growth factor-β1-activated renal fibroblasts / 中华肾脏病杂志

Objective To observe the expression of cysteine-rich protein 61 (Cyr61) in transforming growth factor-β1 (TGF-β1)-activated renal fibroblasts (NRK-49F),and to explore its effect and mechanism.Methods (1) NRK-49F cells were activated by TGF-β1 with different concentrations (0.0,0.5,1.0,2.0,5.0 μg/L).Western blotting was used to detect the expression of Cyr61 protein,and CCK-8 assay was used to test the proliferative activity of NRK-49F cells.(2) NRK-49F cells with low expression and over expression of Cyr61 were established by plasmid transfection.The cells were divided into control group (null vector transfection),over-expression group and lowexpression group.The proliferation was discovered by CCK-8 assay after 24,48 and 72 h.Further,5.0 μg/L TGF-β1 activated these three groups.The proliferation was also discovered by CCK-8 assay and the cell cycle was analyzed by flow cytometry.The mRNA expressions of fibrosis markers (Collα1,Col3αl,MMP9,MMP13) and factors of cell senescence signal pathway (p53,p21,Rb,p16) were ascertained by real time PCR,and the protein expressions of Col3 and MMP9 were detected by Western blotting.Results (1) Compared with 0.0 μg/L TGF-β1 group,the proliferation of NRK-49F cells was enhanced in 0.5,1.0,2.0 and 5.0 μg/L TGF-β1 groups (all P ＜ 0.05),while the expression of Cyr61 protein was decreased in 1.0 μg/L group and increased in 5.0 μg/L group (all P ＜0.05).(2) The proliferation of over-expression group was lower than that of control group after 24,48and 72 h (all P＜ 0.05),which was in a time-dependent manner.(3) Compared with control group activated by TGF-β1,the over-expression group expressed less fibrosis factors (Col1α1 and Col3α1)and more anti-fibrosis factors (MMP9 and MMP13) with decreased proliferation (all P ＜ 0.05).Simultaneously,the proportion of cells bogged down in G1 phases,as well as the expressions of p53,p21 and Rb mRNA increased (all P ＜ 0.05).The above effects of low-expression group were just opposite to over-expression group.Moreover,there was no significant difference in the expression of p16 gene among the three groups (P ＞ 0.05).Conclusions Cyr61 can curb the proliferation and fibrotic phenotypes of fibroblasts,thereafter slowing down the process of renal fibrosis.The p53/p21/Rb interrelated cell senescence signal pathway may be involved in the anti-fibrosis process.

Role of cysteine-rich protein 61 in ocular neovascular disease / 中华实验眼科杂志

Cysteine-rich protein 61 (CYR61) is a secreted protein.It consists of four different structural domains and plays an important role in all kinds of cell life activities,such as proliferation,migration,adhesion,angiogenesis,inflammation regulation,embryogenesis and cartilage formation,by the means of controlling variety of cytokines and signaling pathways.Meanwhile,it also has been widely concerned because of its critical function in neovascularization.In this paper,we present the research progress in CYR61 and its application in ocular fundus neovascular disease,such as diabetic retinopathy,retinopathy of prematurity and wet age-related macular degeneration,in the structure of review,so as to provide a new perspective on the pathogenesis and therapy of ocular fundus neovascular disease.

Extracellular Calcium-Binding Peptide-Modified Ceramics Stimulate Regeneration of Calvarial Bone Defects

Secreted protein, acidic, cysteine-rich (SPARC)-related modular calcium binding 1 (SMOC1) has been implicated in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs). In this study, we found that a peptide (16 amino acids in length), which is located in the extracellular calcium (EC) binding domain of SMOC1, stimulated osteogenic differentiation of human BMSCs in vitro and calvarial bone regeneration in vivo. Treatment of BMSCs with SMOC1-EC peptide significantly stimulated their mineralization in a dose-dependent manner without changing their rate of proliferation. The expression of osteogenic differentiation marker genes, including type 1 collagen and osteocalcin, also increased in a dose-dependent manner. To examine the effect of the SMOC1-EC peptide on bone formation in vivo, the peptide was covalently immobilized onto hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) particles. X-ray photoelectron spectroscopy analysis showed that the peptide was successfully immobilized onto the surface of HA/β-TCP. Implantation of the SMOC1-EC peptide-immobilized HA/β-TCP particles into mouse calvarial defects and subsequent analyses using microcomputed tomography and histology showed significant bone regeneration compared with that of calvarial defects implanted with unmodified HA/β-TCP particles. Collectively, our data suggest that a peptide derived from the EC domain of SMOC1 induces osteogenic differentiation of human BMSCs in vitro and efficiently enhances bone regeneration in vivo.

Dexamethasone Inhibits TGF-β1-Induced Cell Migration by Regulating the ERK and AKT Pathways in Human Colon Cancer Cells Via CYR61 / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: One of the features in cancer development is the migration of cancer cells to form metastatic lesions. CYR61 protein promotes migration and the epithelial-mesenchymal transition in several cancer cell types. Evidence suggests that CYR61 and dexamethasone are relevant to colorectal cancer. However, relationships between them and colorectal cancer are still unclear. Understanding the molecular mechanism of colorectal cancer progression related with CYR61 and dexamethasone, which is widely used for combination chemotherapy, is necessary for improved therapy. MATERIALS AND METHODS: We used colorectal cancer cells, HCT116, co-treated with transforming growth factor β1 (TGF-β1) and dexamethasone to examine the inhibitory migration effect of dexamethasone by migratory assay. Alternatively, both migratory pathways, expression of AKT and ERK, and the target factor CYR61 was also tested by co-treatment with TGF-β1 and dexamethasone. RESULTS: We report that dexamethasone significantly inhibited TGF-β1-induced cell migration, without affecting cell proliferation. Importantly, we observed that TGF-β1 promoted the epithelial-mesenchymal transition process and that dexamethasone co-treatment abolished this effect. ERK and AKT signaling pathways were found to mediate TGF-β1-induced migration, which was inhibited by dexamethasone. In addition, TGF-β1 treatment induced CYR61 expression whereas dexamethasone reduced it. These observations were compatible with the modulation of migration observed following treatment of HCT116 cells with human recombinant CYR61 and anti-CYR61 antibody. Our results also indicated that TGF-β1 enhanced collagen I and reduced matrix metalloproteinase 1 expression, which was reversed by dexamethasone treatment. CONCLUSION: These findings suggested that dexamethasone inhibits AKT and ERK phosphorylation, leading to decreased CYR61 expression, which in turn blocks TGF-β1-induced migration.

The role of Cyr61 in tumors and its clinical application / 中华检验医学杂志

Cyr61/CCN1 is a secreted extracellular matrix ( ECM) protein, which has been shown to regulate a multitude of cellular responses.Many researches indicate that Cyr61 plays important roles in oncogenesis and development of tumor and is considered to be a novel potential oncogene.This review would provide a comprehensive summary about the roles of Cyr61 in the diagnosis and treatment of tumor.

Inhibition of CCN1 siRNA on retinal endothelial cells / 中华实验眼科杂志

Background Cysteine-rich 61 (Cyr61)/CCN1 has been reported to stimulate retinal neovascularization (RNV) in retinopathy of prematurity (ROP).However, whether CCN1 small interfering RNA (CCN1 siRNA) can inhibit or cure ROP has not been extensively investigated.Objective This study was to investigate the regulation effect of CCN1 specific siRNA expression vector on retinal endothelial cells.Methods Rhesus choroid-retinal vascular endothelial cells (RF/6A) were cultured under the normoxic (normoxia control group) and hypoxic condition (1％ O2,5％ CO2 with 94％ N2) in vitro, and then lipofectamineTM 2000 (LF2000) vector plasmid with or without CCN1 siRNA was transiently transfected in the hypoxic-cultured cells as the CCN1 siRNA transfected group and hypoxic control group, respectively.Reverse transcription PCR was employed to detect the expression of CCN1 siRNA plasmid 24 hours after transfection.The vatality of the cells was assayed by cell counting kit-8 (CCK-8) 0,24,48,72 and 96 hours after cultured.Twenty-four hours after cultured,the apoptosis of the cells was evaluated by flow cytometry, and the expressions of CCN1 and vascular endothelial growth factor (VEGF) proteins were detected by immunofluorescence technique and Western blot assay.Results The expression band of CCN1 siRNA was detected in the cells 24 hours after transfection of CCN1 siRNA.CCK-8 assay showed that RF/6A cells were significantly increased over time, and the proliferating value (absorbancy) of the cells was significantly reduced in the CCN1 siRNA transfected group compared with in the normoxia control group and hypoxic control group (Fgroup =198.45, P＜0.05;Ftime =39.26, P＜ 0.05).The apoptosis rates of the cells were (68.9± 1.1) ％ , (18.9±1.3)％ and (39.6± 1.8)％ in the CCN1 siRNA transfected group, normoxia control group and hypoxic control group,and the apoptosis rates of the CCN1 siRNA transfected group were evidently higher than those of the normoxia control group and hypoxic control group (t =2.93 ,t=2.56 ,both at P＜0.05).CCN1 and VEGF proteins were weakly expressed in the normoxia control group and strongly expressed in the hypoxic control group,however,their expression intensity was evidently weakened in the CCN1 siRNA transfected group.The related expression levels of CCN1 and VEGF proteins in the CCN1 siRNA transfected group were significantly lower than those in the hypoxic control group (both at P＜0.05).Conclusions RNA interference targeting CCN1 can inhibit proliferation and promote apoptosis of RF/6A cells.CCN1 siRNA can arrest RNV probably by downregulating the expression levels of CCN1 and VEGF in the cells.

A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide

Background: The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin. Methods: The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps.It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a+. Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer. Results: Both expression systems pQE40 and pET28a+ expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitroconditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly. Conclusions: The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.

A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide

Background:The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin.Methods:The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps.It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a+. Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer.Results:Both expression systems pQE40 and pET28a+ expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitroconditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly.Conclusions:The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.(AU)

The protection effect of cysteine rich-protein 61 on renal tubular epithelial cells against apoptosis induced by hypoxia / 中华肾脏病杂志

Objective To observe the expression of cysteine rich-protein 61 (Cyr61) on renal tubular cells,to explore its effects against hypoxic induced kidney injury and the underlying mechanisms.Methods A stably Cyr61 expressed tubular cell line Cyr61-HK2 was established based on HK2 cells and recombinant Cyr61-lentivirus.BrdU incorporation assay was used for cell proliferation.The apoptosis of cells was analyzed by flow cytometry with Annexin V and propidiumiodide staining.Western bloting was used to detect the protein expression of BAD,Akt and ERK.Results (1) Cyr61-HK2 cells displayed more proliferation ability than HK2 cells.(2) Under hypoxia condition,the apoptosis of both HK2 and Cyr61-HK2 cells increased,but the apoptosis of Cyr61-HK2 cells was lesser than HK2 cells.(3) The expression of Cyr61 led to the phosphorylation of BAD,Akt and ERK on 0 h,0.5 h,1 h (all P ＜ 0.05).Conclusion The expression of Cyr61 can promote cell proliferation and dampen cell apoptosis induced by hypoxia,which may be involved in the Akt/ERK signal pathway.