Cancer-associated fibroblasts(CAFs)are one of the most abundant stromal cells in the tumor microenvironment which mediate desmoplastic response and are the primary driver for an immunosuppressive microenvironment,lead...Cancer-associated fibroblasts(CAFs)are one of the most abundant stromal cells in the tumor microenvironment which mediate desmoplastic response and are the primary driver for an immunosuppressive microenvironment,leading to the failure of triple-negative breast cancer(TNBC)immunotherapy.Therefore,depleting CAFs may enhance the effect of immunotherapy(such as PD-L1 antibody).Relaxin(RLN)has been demonstrated to significantly improve transforming growth factor-β(TGF-β)induced CAFs activation and tumor immunosuppressive microenvironment.However,the short half-life and systemic vasodilation of RLN limit its in vivo efficacy.Here,plasmid encoding relaxin(pRLN)to locally express RLN was delivered with a new positively charged polymer named polymeric metformin(PolyMet),which could increase gene transfer efficiency significantly and have low toxicity that have been certified by our lab before.In order to improve the stability of pRLN in vivo,this complex was further formed lipid poly-γ-glutamic acid(PGA)/PolyMetpRLN nanoparticle(LPPR).The particle size of LPPR was 205.5±2.9 nm,and the zeta potential was+55.4±1.6 mV.LPPR displayed excellent tumor penetrating efficacy and weaken proliferation of CAFs in 4T1luc/CAFs tumor spheres in vitro.In vivo,it could reverse aberrantly activated CAFs by decreasing the expression of profibrogenic cytokine and remove the physical barrier to reshape the tumor stromal microenvironment,which enabled a 2.2-fold increase in cytotoxic T cell infiltration within the tumor and a decrease in immunosuppressive cells infiltration.Thus,LPPR was observed retarded tumor growth by itself in the 4T1 tumor bearing-mouse,and the reshaped immune microenvironment further led to facilitate antitumor effect when it combined with PD-L1 antibody(aPD-L1).Altogether,this study presented a novel therapeutic approach against tumor stroma using LPPR to achieve a combination regimen with immune checkpoint blockade therapy against the desmoplastic TNBC model.展开更多
BACKGROUND Separation of the pubic symphysis can occur during the peripartum period.Relaxin(RLX)is a hormone primarily secreted by the corpus luteum that can mediate hemodynamic changes during pregnancy as well as loo...BACKGROUND Separation of the pubic symphysis can occur during the peripartum period.Relaxin(RLX)is a hormone primarily secreted by the corpus luteum that can mediate hemodynamic changes during pregnancy as well as loosen the pelvic ligaments.However,it is unknown whether RLX is associated with peripartum pubic symphysis separation and if the association is affected by other factors.AIM To study the association between RLX and peripartum pubic symphysis separation and evaluate other factors that might affect this association.METHODS We performed a cross-sectional study of pregnant women between April 2019 and January 2020.Baseline demographic characteristics,including gestational age,weight,neonatal weight,delivery mode and duration of the first and second stages of labor,were recorded.The clinical symptoms were used as a screening index during pregnancy,and the patients with pubic symphysis and inguinal pain were examined by color Doppler ultrasonography to determine whether there was pubic symphysis separation.Serum RLX concentrations were evaluated 1 d after delivery using an enzyme-linked immunosorbent assay,and pubic symphysis separation was diagnosed based on postpartum X-ray examination.We used an independent-sample t test to analyze the association between serum RLX levels and peripartum pubic symphysis separation.Multivariate regression analysis was used to evaluate whether the association between RLX and peripartum pubic symphysis separation was confounded by other factors,and the association between RLX and the severity of pubic symphysis separation was also assessed.We used Pearson correlation analysis to determine the factors related to RLX levels as well as the correlation between the degree of pubic symphysis separation and activities of daily living(ADL)and pain.RESULTS A total of 54 women were enrolled in the study,with 15 exhibiting(observational group)and 39 not exhibiting(control group)peripartum pubic symphysis separation.There were no statistically significant differences in terms of maternal age,gestational age,pre-pregnancy weight,weight gain during pregnancy,delivery modes,or duration of the first or second stages of labor between the 2 groups.We did,however,note a statistically significant difference in serum RLX concentrations and neonatal weight between the observational and control groups(122.3±0.7μg/mL vs 170.4±42.3μg/mL,P<0.05;3676.000±521.725 g vs 3379.487±402.420 g,P<0.05,respectively).Multivariate regression analyses showed that serum RLX level[odds ratio(OR):1.022)and neonatal weight(OR:1.002)were associated with pubic symphysis separation peripartum.The degree of separation of the pubic symphysis was negatively correlated with ADL and positively correlated with pain.There was no statistically significant association between serum RLX levels and the severity of pubic symphysis separation after adjusting for confounding factors.CONCLUSION Serum RLX levels and neonatal weight were associated with the occurrence,but not the severity,of peripartum pubic symphysis separation.展开更多
AIM To investigate the signaling pathways involved in the relaxin(RLX) effects on ileal preparations from mice through mechanical and electrophysiological experiments.METHODS For mechanical experiments, ileal preparat...AIM To investigate the signaling pathways involved in the relaxin(RLX) effects on ileal preparations from mice through mechanical and electrophysiological experiments.METHODS For mechanical experiments, ileal preparations from female mice were mounted in organ baths containing Krebs-Henseleit solution. The mechanical activity was recorded via force-displacement transducers, which were coupled to a polygraph for continuous recording of isometric tension. Electrophysiological measurements were performed in current-and voltage-clamp conditions by a microelectrode inserted in a single smooth muscle cell(SMC) of the ileal longitudinal layer. Both the membrane passive properties and inward voltage-dependent L-type Ca2+ currents were recorded using suitable solutions and voltage stimulation protocols.RESULTS Mechanical experiments showed that RLX induced a decay of the basal tension and a reduction in amplitude of the spontaneous contractions. The effects of RLX were partially reduced by 1 H-[1,2,4]oxadiazolo[4,3-a ]-quinoxalin-1-one(ODQ) or 9-cyclopentyladenine mesylate(9 CPA), inhibitors of guanylate cyclase(GC) and adenylate cyclase(AC), respectively, and were abolished in the concomitant presence of both drugs. Electrophysiological experiments demonstrated that RLX directly influenced the biophysical properties of ileal SMCs, decreasing the membrane conductance, hyperpolarizing the resting membrane potential, reducing the L-type calcium current amplitude and affecting its kinetics. The voltage dependence of the current activation and inactivation time constant was significantly speeded by RLX. Each electrophysiological effect of RLX was reduced by ODQ or 9 CPA, and abolished in the concomitant presence of both drugs as observed in mechanical experiments. CONCLUSION Our new findings demonstrate that RLX influences ileal muscle through a dual mechanism involving both GC and AC.展开更多
OBJECTIVE To develop ERK1/2 activation assays to detect RXFP3 activation or inhibition by its agonists or antagonists.METHODS Plated HEK-RXFP3,CHO-RXFP3,HEK293 Tand CHO-K1 cells in poly-L-lysine coated well plates.The...OBJECTIVE To develop ERK1/2 activation assays to detect RXFP3 activation or inhibition by its agonists or antagonists.METHODS Plated HEK-RXFP3,CHO-RXFP3,HEK293 Tand CHO-K1 cells in poly-L-lysine coated well plates.The cells were serum starved and treated with either human relaxin-3(H3relaxin)(10nmol·L-1),R3B1-22R(10μmol·L-1)and pertussis toxin(PTX,100ng·mL-1).The cells were lysed and the ERK1/2 activation was determined by SDS-PAGE followed by immunoblotting for phosphorylated ERK1/2(pERK1/2)and total ERK1/2(tERK1/2)for the lysates.RESULTSThe quantification of the data revealed that the peak of ERK1/2activation can be detected precisely at 10 min post stimulation with 10nmol·L-1 H3 relaxin in both HEK-RXFP3 and CHO-RXFP3 cell lines in all three trials compared to the cells treated with vehicle(P<0.05).However,HEK-RXFP3 cells demonstrated a transient activation of ERK1/2and CHORXFP3 cells demonstrated a continuous activation of ERK1/2which was inhibited by the Gi inhibitor,PTX.Activation of ERK1/2was significantly inhibited by pre-treating the cells with RXFP3 antagonist R3B1-22 Rin HEK-RXFP3 cells.ERK1/2 activation was observed neither in wild type HEK293 Tnor in CHO-K1 cells.CONCLUSION The developed assay can detect RXFP3 activation or inhibition by agonists and antagonists via the detection of pERK1/2 in multiple cell lines.This assay will also be useful to detect signaling pathways upstream to ERK1/2 activation mediated by RXFP3.Activation of ERK1/2 in CHO-RXFP3 cells was mediated by Gi proteins at 10 min as well as at 25-35 min time points.展开更多
OBJECTIVE Relaxin-3 is a novel neuropeptide of the relaxin insulin family of peptides.So far,many studies have reported the role of relaxin-3/RXFP3 system in regulating feeding,stress response and cognition.In previou...OBJECTIVE Relaxin-3 is a novel neuropeptide of the relaxin insulin family of peptides.So far,many studies have reported the role of relaxin-3/RXFP3 system in regulating feeding,stress response and cognition.In previous studies using RXFP3 stable cell lines,inhibition of adenylate cyclase by the activation of Gi/o proteins have been reported.Based on this signaling event,we have developed inhibition of forskolin induced cAMP assay to detect RXFP3 activation or inhibition by its agonists or antagonists.METHODS To detect inhibition of adenylate cyclase up on RXFP3 activation by human relaxin-3(H3relaxin),HEK-RXFP3,CHO-RXFP3,SN56 and GT1-7cells were plated in poly-L-lysine coated 24 well plates.The following day,the cells were starved in serum free cell culture media for 6h.Next,cells were treated in triplicate with serum free media(control)and H3 relaxin for 5-15 min.Then,the cells were treated with serum free media with DMSO(control)or forskolin(5-100μmol·L-1)for 15 min at 37℃ with 5%CO2.At the end of the incubation,cell culture media was discarded and the cells were lysed with 0.1mol·L-1 HCl.The cAMP concentration in each lysate was detected by ELISA(Cayman Chemicals).The data from three experiments were analysed using one way ANOVA followed by Bonferroni post hoc test or Dunnett′s post hoc test.RESULTS In CHO-RXFP3 and HEK-RXFP3 cells,10nmol·L-1 of H3 relaxin was able to significantly inhibit the forskolin(5μmol·L-1)induced cAMP levels(P<0.05).In SN56 neuronal like cell line endogenously expressing RXFP3,100nmol·L-1 H3 relaxin was able to significantly reduce forskolin(3μmol·L-1)induced cAMP(P<0.05).However,in wild type HEK293 Tand CHO-K1 cells,10n mol·L-1 H3 relaxin was not able to significantly reduce the forskolin 22(5μmol·L-1)induced cAMP levels.In GT1-7 mouse hypothalamic cells endogenously expressing RXFP3,100nmol·L-1 H3 relaxin and 5or 3μmol·L-1 forskolin,was able toa significantly increase cAMP levels(P<0.05).CONCLUSION Inhibition of forskolin induced cAMP assay can be used to detect Gi/o mediated cAMP inhibition related signaling events due to RXFP3 activation by its agonists in CHO-RXFP3,HEK-RXFP3 and SN56 cell lines.展开更多
AIM: To assess the prognostic value of serum human relaxin 2 (H2 RLN) level in patients with esophageal squamous cell carcinoma (ESCC). METHODS: From October 1998 to September 2009, 146 patients with histopathological...AIM: To assess the prognostic value of serum human relaxin 2 (H2 RLN) level in patients with esophageal squamous cell carcinoma (ESCC). METHODS: From October 1998 to September 2009, 146 patients with histopathologically confirmed ESCC were enrolled in this study. One hundred patients underwent en bloc esophagectomy, and 46 patients with unresectable tumors underwent palliative surgery. Five of the 146 patients died of surgical complications. Serum levels of H2 RLN were measured by enzyme linked immunosorbent assay. The relationship between serum H2 RLN level and each of the clinicopathological parameters was analyzed using the χ2 test. Patients were classified into two groups according to their H2 RLN level (< 0.462 ng/mL vs ≥ 0.462 ng/mL). When any analysis cell had fewer than five cases, the Fisher's exact test was used. The statistical difference between groups A and B in each clinicopathological category was determined by the Student's t test (two-tailed) or analysis of variance. Survival curves were plotted using the Kaplan-Meier method. The statistical difference in survival between the different groups was compared using the log-rank test. Survival correlation with the prognostic factors was further investigated by multivariate analysis using the Cox proportional hazards model with backward stepwise likelihood ratio. RESULTS: ESCC patients tended to have significantly higher serum H2 RLN concentrations (0.48 ± 0.17 ng/ mL, n=141) compared with the healthy control group (0.342 ± 0.12 ng/mL, n=112). There was a significant difference between patients with lymph node involvement (0.74 ± 0.15 ng/mL, n=90), distant metastasis (0.90 ± 0.19 ng/mL, n=32) and those without lymph node involvement (0.45 ± 0.12 ng/mL, n=51), and distant metastasis (0.43 ± 0.14 ng/mL, n=109), respectively (P < 0.01). Patients with high H2 RLN levels (≥ 0.462 ng/mL) had a poorer prognosis than patients with low serum H2 RLN levels (< 0.462 ng/mL; P=0.0056). The H2 RLN level was also correlated with survival and tumor-node-metastasis staging, but not with age, tumor size, gender, lymphovascular invasion or the histological grade of tumors. Cox regression analysis showed that H2 RLN was an independent variable. CONCLUSION: Serum concentrations of H2 RLN are frequently elevated in ESCC patients and are correlated with disease metastasis and survival. Serum concentrations of H2 RLN may be an important prognostic marker in ESCC patients.展开更多
Since its discovery in the 1920's the relaxin peptide hormone family has not only grown in number to now seven members(relaxin-1, relaxin-2, relaxin-3, insulin-like peptide(INSL) 3, INSL4, INSL5 and INSL6), but ev...Since its discovery in the 1920's the relaxin peptide hormone family has not only grown in number to now seven members(relaxin-1, relaxin-2, relaxin-3, insulin-like peptide(INSL) 3, INSL4, INSL5 and INSL6), but ever more effects, suchs as vasodilatory, angiogenic, anti-apoptopic, anti-fibriotic and anti-inflammatory, have been linked to them. While relaxin-2 has mainly been investigated in the context of cardiac protection, most comprehensively in the RELAX-AHF and RELAX AHF2 studies, a small number of studies have furthermore assessed the potential neuroprotective effects of especially relaxin-2 and other members of the relaxin family. In this short review we summarise and discuss recent efforts to utilize relaxin hormones for neuroprotection and point out potential future fields of research and translational applications. While many questions still need to be answered, the promising results of the available studies definitely warrant future well-designed studies on neuroprotection by relaxin peptides.展开更多
AIM To determine the effect of combined serelaxin and rosiglitazone treatment on established hepatic fibrosis.METHODS Hepatic fibrosis was induced in mice by carbon tetrachloride administration for 6 wk, or vehicle al...AIM To determine the effect of combined serelaxin and rosiglitazone treatment on established hepatic fibrosis.METHODS Hepatic fibrosis was induced in mice by carbon tetrachloride administration for 6 wk, or vehicle alone(nonfibrotic mice). For the final 2 wk, mice were treated with rosiglitazone, serelaxin, or both rosiglitazone and serelaxin. Serum liver enzymes and relaxin levels were determined by standard methods. The degree of liver collagen content was determined by histology and immunohistochemistry. Expression of type?Ⅰ?collagen was determined by quantitative PCR. Activation of hepatic stellate cells was assessed by alpha-smoothmuscle actin(SMA) levels. Liver peroxisome proliferator activated receptor-gamma coactivator 1 alpha(PGC1α) was determined by Western blotting.RESULTS Treatment of mice with CCl4 resulted in hepatic fibrosis as evidenced by increased liver enzyme levels(ALT and AST), and increased liver collagen and SMA. Monotherapy with either serelaxin or rosiglitazone for 2 wk was generally without effect. In contrast, the combination of serelaxin and rosiglitazone resulted in significantly improved ALT levels(P < 0.05). Total liver collagen content as determined by Sirius red staining revealed that only combination treatment was effective in reducing total liver collagen(P < 0.05). These results were supported by immunohistochemistry for type?Ⅰ?collagen, in which only combination treatment reduced fibrillar collagen levels(P < 0.05). The level of hepatic stellate cell activation was modestly, but significantly, reduced by serelaxin treatment alone, but combination treatment resulted in significantly lower SMA levels. Finally, while hepatic fibrosis reduced liver PGC1α levels, the combination of serelaxin and rosiglitazone resulted in restoration of PGC1α protein levels.CONCLUSION The combination of serelaxin and rosiglitazone treatment for 2 wk was effective in significantly reducing established hepatic fibrosis, providing a potential new treatment strategy.展开更多
To clarify the role of endogenous relaxin on sperm motility, relaxin in semen was neutralized by anti relaxin antibody in vitro. 22 semen samples were collected from infertility clinic and tested with Hamilton Thorn 2...To clarify the role of endogenous relaxin on sperm motility, relaxin in semen was neutralized by anti relaxin antibody in vitro. 22 semen samples were collected from infertility clinic and tested with Hamilton Thorn 2000 Motility Analyzer to detect sperm motility (%), progressive motility (%), path velocity (micro/sec) and velocity (0～4 grade) at the time of 0, 15, 30 and 60 min respectively. The results showed that sperm motility declined significantly after being incubated with anti relaxin serum. Sperm progressive motility declined more obviously. This experiment revealed that endogenous relaxin could play an important role in the physiological process of maintaining sperm motility, especially progressive motility.展开更多
基金This work was funded by the Medical and Health Science and Technology Program of Zhejiang Province(2021KY813)the National Natural Science Foundation of China(82174095)the National Natural Science Foundation of Zhejiang Province(LZ22H290001).
文摘Cancer-associated fibroblasts(CAFs)are one of the most abundant stromal cells in the tumor microenvironment which mediate desmoplastic response and are the primary driver for an immunosuppressive microenvironment,leading to the failure of triple-negative breast cancer(TNBC)immunotherapy.Therefore,depleting CAFs may enhance the effect of immunotherapy(such as PD-L1 antibody).Relaxin(RLN)has been demonstrated to significantly improve transforming growth factor-β(TGF-β)induced CAFs activation and tumor immunosuppressive microenvironment.However,the short half-life and systemic vasodilation of RLN limit its in vivo efficacy.Here,plasmid encoding relaxin(pRLN)to locally express RLN was delivered with a new positively charged polymer named polymeric metformin(PolyMet),which could increase gene transfer efficiency significantly and have low toxicity that have been certified by our lab before.In order to improve the stability of pRLN in vivo,this complex was further formed lipid poly-γ-glutamic acid(PGA)/PolyMetpRLN nanoparticle(LPPR).The particle size of LPPR was 205.5±2.9 nm,and the zeta potential was+55.4±1.6 mV.LPPR displayed excellent tumor penetrating efficacy and weaken proliferation of CAFs in 4T1luc/CAFs tumor spheres in vitro.In vivo,it could reverse aberrantly activated CAFs by decreasing the expression of profibrogenic cytokine and remove the physical barrier to reshape the tumor stromal microenvironment,which enabled a 2.2-fold increase in cytotoxic T cell infiltration within the tumor and a decrease in immunosuppressive cells infiltration.Thus,LPPR was observed retarded tumor growth by itself in the 4T1 tumor bearing-mouse,and the reshaped immune microenvironment further led to facilitate antitumor effect when it combined with PD-L1 antibody(aPD-L1).Altogether,this study presented a novel therapeutic approach against tumor stroma using LPPR to achieve a combination regimen with immune checkpoint blockade therapy against the desmoplastic TNBC model.
基金The Science and Technology Development Plan of Taian,No.2018NS0203.
文摘BACKGROUND Separation of the pubic symphysis can occur during the peripartum period.Relaxin(RLX)is a hormone primarily secreted by the corpus luteum that can mediate hemodynamic changes during pregnancy as well as loosen the pelvic ligaments.However,it is unknown whether RLX is associated with peripartum pubic symphysis separation and if the association is affected by other factors.AIM To study the association between RLX and peripartum pubic symphysis separation and evaluate other factors that might affect this association.METHODS We performed a cross-sectional study of pregnant women between April 2019 and January 2020.Baseline demographic characteristics,including gestational age,weight,neonatal weight,delivery mode and duration of the first and second stages of labor,were recorded.The clinical symptoms were used as a screening index during pregnancy,and the patients with pubic symphysis and inguinal pain were examined by color Doppler ultrasonography to determine whether there was pubic symphysis separation.Serum RLX concentrations were evaluated 1 d after delivery using an enzyme-linked immunosorbent assay,and pubic symphysis separation was diagnosed based on postpartum X-ray examination.We used an independent-sample t test to analyze the association between serum RLX levels and peripartum pubic symphysis separation.Multivariate regression analysis was used to evaluate whether the association between RLX and peripartum pubic symphysis separation was confounded by other factors,and the association between RLX and the severity of pubic symphysis separation was also assessed.We used Pearson correlation analysis to determine the factors related to RLX levels as well as the correlation between the degree of pubic symphysis separation and activities of daily living(ADL)and pain.RESULTS A total of 54 women were enrolled in the study,with 15 exhibiting(observational group)and 39 not exhibiting(control group)peripartum pubic symphysis separation.There were no statistically significant differences in terms of maternal age,gestational age,pre-pregnancy weight,weight gain during pregnancy,delivery modes,or duration of the first or second stages of labor between the 2 groups.We did,however,note a statistically significant difference in serum RLX concentrations and neonatal weight between the observational and control groups(122.3±0.7μg/mL vs 170.4±42.3μg/mL,P<0.05;3676.000±521.725 g vs 3379.487±402.420 g,P<0.05,respectively).Multivariate regression analyses showed that serum RLX level[odds ratio(OR):1.022)and neonatal weight(OR:1.002)were associated with pubic symphysis separation peripartum.The degree of separation of the pubic symphysis was negatively correlated with ADL and positively correlated with pain.There was no statistically significant association between serum RLX levels and the severity of pubic symphysis separation after adjusting for confounding factors.CONCLUSION Serum RLX levels and neonatal weight were associated with the occurrence,but not the severity,of peripartum pubic symphysis separation.
基金Supported by University of Florence(ex60%) ROBERTA SQUECCORICATEN15(to Squecco R)
文摘AIM To investigate the signaling pathways involved in the relaxin(RLX) effects on ileal preparations from mice through mechanical and electrophysiological experiments.METHODS For mechanical experiments, ileal preparations from female mice were mounted in organ baths containing Krebs-Henseleit solution. The mechanical activity was recorded via force-displacement transducers, which were coupled to a polygraph for continuous recording of isometric tension. Electrophysiological measurements were performed in current-and voltage-clamp conditions by a microelectrode inserted in a single smooth muscle cell(SMC) of the ileal longitudinal layer. Both the membrane passive properties and inward voltage-dependent L-type Ca2+ currents were recorded using suitable solutions and voltage stimulation protocols.RESULTS Mechanical experiments showed that RLX induced a decay of the basal tension and a reduction in amplitude of the spontaneous contractions. The effects of RLX were partially reduced by 1 H-[1,2,4]oxadiazolo[4,3-a ]-quinoxalin-1-one(ODQ) or 9-cyclopentyladenine mesylate(9 CPA), inhibitors of guanylate cyclase(GC) and adenylate cyclase(AC), respectively, and were abolished in the concomitant presence of both drugs. Electrophysiological experiments demonstrated that RLX directly influenced the biophysical properties of ileal SMCs, decreasing the membrane conductance, hyperpolarizing the resting membrane potential, reducing the L-type calcium current amplitude and affecting its kinetics. The voltage dependence of the current activation and inactivation time constant was significantly speeded by RLX. Each electrophysiological effect of RLX was reduced by ODQ or 9 CPA, and abolished in the concomitant presence of both drugs as observed in mechanical experiments. CONCLUSION Our new findings demonstrate that RLX influences ileal muscle through a dual mechanism involving both GC and AC.
基金The project supported by the Biomedical Research Council of Singapore(BMRC 10/1/21/19/645)the National Medical Research Council of Singapore(NMRC/1287/2011)the Ministry of Education,Singapore,Academic Research Fund Tier 1Seed Fund for Basic Science Research(T1-BSRG 2014-03)
文摘OBJECTIVE To develop ERK1/2 activation assays to detect RXFP3 activation or inhibition by its agonists or antagonists.METHODS Plated HEK-RXFP3,CHO-RXFP3,HEK293 Tand CHO-K1 cells in poly-L-lysine coated well plates.The cells were serum starved and treated with either human relaxin-3(H3relaxin)(10nmol·L-1),R3B1-22R(10μmol·L-1)and pertussis toxin(PTX,100ng·mL-1).The cells were lysed and the ERK1/2 activation was determined by SDS-PAGE followed by immunoblotting for phosphorylated ERK1/2(pERK1/2)and total ERK1/2(tERK1/2)for the lysates.RESULTSThe quantification of the data revealed that the peak of ERK1/2activation can be detected precisely at 10 min post stimulation with 10nmol·L-1 H3 relaxin in both HEK-RXFP3 and CHO-RXFP3 cell lines in all three trials compared to the cells treated with vehicle(P<0.05).However,HEK-RXFP3 cells demonstrated a transient activation of ERK1/2and CHORXFP3 cells demonstrated a continuous activation of ERK1/2which was inhibited by the Gi inhibitor,PTX.Activation of ERK1/2was significantly inhibited by pre-treating the cells with RXFP3 antagonist R3B1-22 Rin HEK-RXFP3 cells.ERK1/2 activation was observed neither in wild type HEK293 Tnor in CHO-K1 cells.CONCLUSION The developed assay can detect RXFP3 activation or inhibition by agonists and antagonists via the detection of pERK1/2 in multiple cell lines.This assay will also be useful to detect signaling pathways upstream to ERK1/2 activation mediated by RXFP3.Activation of ERK1/2 in CHO-RXFP3 cells was mediated by Gi proteins at 10 min as well as at 25-35 min time points.
基金The project supported by the Biomedical Research Council of Singapore(BMRC 10/1/21/19/645)National Medical Research Council of Singapore(NMRC/1287/2011)the Ministry of Education,Singapore,Academic Research Fund Tier 1Seed Fund for Basic Science Research(T1-BSRG 2014-03)
文摘OBJECTIVE Relaxin-3 is a novel neuropeptide of the relaxin insulin family of peptides.So far,many studies have reported the role of relaxin-3/RXFP3 system in regulating feeding,stress response and cognition.In previous studies using RXFP3 stable cell lines,inhibition of adenylate cyclase by the activation of Gi/o proteins have been reported.Based on this signaling event,we have developed inhibition of forskolin induced cAMP assay to detect RXFP3 activation or inhibition by its agonists or antagonists.METHODS To detect inhibition of adenylate cyclase up on RXFP3 activation by human relaxin-3(H3relaxin),HEK-RXFP3,CHO-RXFP3,SN56 and GT1-7cells were plated in poly-L-lysine coated 24 well plates.The following day,the cells were starved in serum free cell culture media for 6h.Next,cells were treated in triplicate with serum free media(control)and H3 relaxin for 5-15 min.Then,the cells were treated with serum free media with DMSO(control)or forskolin(5-100μmol·L-1)for 15 min at 37℃ with 5%CO2.At the end of the incubation,cell culture media was discarded and the cells were lysed with 0.1mol·L-1 HCl.The cAMP concentration in each lysate was detected by ELISA(Cayman Chemicals).The data from three experiments were analysed using one way ANOVA followed by Bonferroni post hoc test or Dunnett′s post hoc test.RESULTS In CHO-RXFP3 and HEK-RXFP3 cells,10nmol·L-1 of H3 relaxin was able to significantly inhibit the forskolin(5μmol·L-1)induced cAMP levels(P<0.05).In SN56 neuronal like cell line endogenously expressing RXFP3,100nmol·L-1 H3 relaxin was able to significantly reduce forskolin(3μmol·L-1)induced cAMP(P<0.05).However,in wild type HEK293 Tand CHO-K1 cells,10n mol·L-1 H3 relaxin was not able to significantly reduce the forskolin 22(5μmol·L-1)induced cAMP levels.In GT1-7 mouse hypothalamic cells endogenously expressing RXFP3,100nmol·L-1 H3 relaxin and 5or 3μmol·L-1 forskolin,was able toa significantly increase cAMP levels(P<0.05).CONCLUSION Inhibition of forskolin induced cAMP assay can be used to detect Gi/o mediated cAMP inhibition related signaling events due to RXFP3 activation by its agonists in CHO-RXFP3,HEK-RXFP3 and SN56 cell lines.
文摘AIM: To assess the prognostic value of serum human relaxin 2 (H2 RLN) level in patients with esophageal squamous cell carcinoma (ESCC). METHODS: From October 1998 to September 2009, 146 patients with histopathologically confirmed ESCC were enrolled in this study. One hundred patients underwent en bloc esophagectomy, and 46 patients with unresectable tumors underwent palliative surgery. Five of the 146 patients died of surgical complications. Serum levels of H2 RLN were measured by enzyme linked immunosorbent assay. The relationship between serum H2 RLN level and each of the clinicopathological parameters was analyzed using the χ2 test. Patients were classified into two groups according to their H2 RLN level (< 0.462 ng/mL vs ≥ 0.462 ng/mL). When any analysis cell had fewer than five cases, the Fisher's exact test was used. The statistical difference between groups A and B in each clinicopathological category was determined by the Student's t test (two-tailed) or analysis of variance. Survival curves were plotted using the Kaplan-Meier method. The statistical difference in survival between the different groups was compared using the log-rank test. Survival correlation with the prognostic factors was further investigated by multivariate analysis using the Cox proportional hazards model with backward stepwise likelihood ratio. RESULTS: ESCC patients tended to have significantly higher serum H2 RLN concentrations (0.48 ± 0.17 ng/ mL, n=141) compared with the healthy control group (0.342 ± 0.12 ng/mL, n=112). There was a significant difference between patients with lymph node involvement (0.74 ± 0.15 ng/mL, n=90), distant metastasis (0.90 ± 0.19 ng/mL, n=32) and those without lymph node involvement (0.45 ± 0.12 ng/mL, n=51), and distant metastasis (0.43 ± 0.14 ng/mL, n=109), respectively (P < 0.01). Patients with high H2 RLN levels (≥ 0.462 ng/mL) had a poorer prognosis than patients with low serum H2 RLN levels (< 0.462 ng/mL; P=0.0056). The H2 RLN level was also correlated with survival and tumor-node-metastasis staging, but not with age, tumor size, gender, lymphovascular invasion or the histological grade of tumors. Cox regression analysis showed that H2 RLN was an independent variable. CONCLUSION: Serum concentrations of H2 RLN are frequently elevated in ESCC patients and are correlated with disease metastasis and survival. Serum concentrations of H2 RLN may be an important prognostic marker in ESCC patients.
文摘Since its discovery in the 1920's the relaxin peptide hormone family has not only grown in number to now seven members(relaxin-1, relaxin-2, relaxin-3, insulin-like peptide(INSL) 3, INSL4, INSL5 and INSL6), but ever more effects, suchs as vasodilatory, angiogenic, anti-apoptopic, anti-fibriotic and anti-inflammatory, have been linked to them. While relaxin-2 has mainly been investigated in the context of cardiac protection, most comprehensively in the RELAX-AHF and RELAX AHF2 studies, a small number of studies have furthermore assessed the potential neuroprotective effects of especially relaxin-2 and other members of the relaxin family. In this short review we summarise and discuss recent efforts to utilize relaxin hormones for neuroprotection and point out potential future fields of research and translational applications. While many questions still need to be answered, the promising results of the available studies definitely warrant future well-designed studies on neuroprotection by relaxin peptides.
基金Supported by The United States Department of Veterans Affairs Biomedical Laboratory Research and Development Program,No.BX000849National Institutes of Health,NIAAA,No.R01AA015509
文摘AIM To determine the effect of combined serelaxin and rosiglitazone treatment on established hepatic fibrosis.METHODS Hepatic fibrosis was induced in mice by carbon tetrachloride administration for 6 wk, or vehicle alone(nonfibrotic mice). For the final 2 wk, mice were treated with rosiglitazone, serelaxin, or both rosiglitazone and serelaxin. Serum liver enzymes and relaxin levels were determined by standard methods. The degree of liver collagen content was determined by histology and immunohistochemistry. Expression of type?Ⅰ?collagen was determined by quantitative PCR. Activation of hepatic stellate cells was assessed by alpha-smoothmuscle actin(SMA) levels. Liver peroxisome proliferator activated receptor-gamma coactivator 1 alpha(PGC1α) was determined by Western blotting.RESULTS Treatment of mice with CCl4 resulted in hepatic fibrosis as evidenced by increased liver enzyme levels(ALT and AST), and increased liver collagen and SMA. Monotherapy with either serelaxin or rosiglitazone for 2 wk was generally without effect. In contrast, the combination of serelaxin and rosiglitazone resulted in significantly improved ALT levels(P < 0.05). Total liver collagen content as determined by Sirius red staining revealed that only combination treatment was effective in reducing total liver collagen(P < 0.05). These results were supported by immunohistochemistry for type?Ⅰ?collagen, in which only combination treatment reduced fibrillar collagen levels(P < 0.05). The level of hepatic stellate cell activation was modestly, but significantly, reduced by serelaxin treatment alone, but combination treatment resulted in significantly lower SMA levels. Finally, while hepatic fibrosis reduced liver PGC1α levels, the combination of serelaxin and rosiglitazone resulted in restoration of PGC1α protein levels.CONCLUSION The combination of serelaxin and rosiglitazone treatment for 2 wk was effective in significantly reducing established hepatic fibrosis, providing a potential new treatment strategy.
文摘To clarify the role of endogenous relaxin on sperm motility, relaxin in semen was neutralized by anti relaxin antibody in vitro. 22 semen samples were collected from infertility clinic and tested with Hamilton Thorn 2000 Motility Analyzer to detect sperm motility (%), progressive motility (%), path velocity (micro/sec) and velocity (0～4 grade) at the time of 0, 15, 30 and 60 min respectively. The results showed that sperm motility declined significantly after being incubated with anti relaxin serum. Sperm progressive motility declined more obviously. This experiment revealed that endogenous relaxin could play an important role in the physiological process of maintaining sperm motility, especially progressive motility.
