Objective:To investigate the regulatory effect of miR-146a overexpression on corneal inflammatory response in a mouse model of dry eye,and to analyze its relationship with the IRAK1/TRAF6/NF-кB signaling pathway.Meth...Objective:To investigate the regulatory effect of miR-146a overexpression on corneal inflammatory response in a mouse model of dry eye,and to analyze its relationship with the IRAK1/TRAF6/NF-кB signaling pathway.Methods:A total of 50 SPF-grade BALB/c mice were randomly divided into five groups,with10 mice in each group.Except for the control group,the other four groups were treated with 0.2%benzalkonium chloride(BAC)solution in both eyes to construct a dry eye model.After successful modeling,the control group and model group received NC agomir;the miR antagonist group received miR-146a antagomir;the miR agonist group received miR-146a agomir;and the pathway agonist group received miR-146a agomir+NF-κB activator 2.After four weeks of treatment,the expressions levels of miR-146a,inflammatory factors,and IRAK1/TRAF6/NF-κB signaling pathway proteins were observed and compared among the five groups.Results:After four weeks of treatment,there was a statistically significant difference in the relative expression of miR-146a in the five groups(F=61.058,P<0.001),which was significantly higher in the miR agonist group than in the other four groups.After4 weeks of treatment,there were statistically significant differences in the expression levels of IL-1β,IL-6,IL-8 and TNF-αin the five groups(F=84.757,103.658,55.477,46.762;P<0.001).After four weeks of treatment,there were statistically significant differences in the protein expression levels of IRAK1,TRAF6,NF-κB and IκBαin the five groups(F=62.975,77.173,67.108,29.381;P<0.001),except for the control group,the expression levels of IRAK1,TRAF6 and NF-κB proteins in the miR agonist group were significantly lower than those in the other three groups,and the expression levels of IκBαprotein were significantly higher than those in the other three groups.Conclusion:Overexpression of miR-146a can negatively regulate corneal inflammatory response in dry eye mice through the IRAK1/TRAF6/NF-κB signaling pathway,which can provide new insights for the clinical treatment of dry eye disease.展开更多
Objective:The objective of this study was to investigate the alterations and potential implications of the Osteoprotegerin(OPG)/Receptor Activator of Nuclear Factor-kappa B Ligand(RANKL)/Receptor Activator of Nuclear ...Objective:The objective of this study was to investigate the alterations and potential implications of the Osteoprotegerin(OPG)/Receptor Activator of Nuclear Factor-kappa B Ligand(RANKL)/Receptor Activator of Nuclear Factor-kappa B(RANK)signaling pathway factors in a murine model of sepsis-associated acute kidney injury(SA-AKI).This research aimed to offer novel insights into the mechanistic exploration of SA-AKI.Methods:The SA-AKI model group(CLP group)was established through cecal ligation and puncture surgery(CLP),while the control group consisted of sham-operated animals(Sham group)subjected only to laparotomy without cecal ligation and puncture.Blood samples were collected 24 h post-surgery,and murine kidney tissues were harvested upon euthanasia.Serum levels of Serum Creatinine(Scr)and Blood Urea Nitrogen(BUN)were quantified using assay kits.Furthermore,serum levels of interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α),and interleukin-1 beta(IL-1β)were assessed through enzyme-linked immunosorbent assay(ELISA).Renal tissue pathological alterations were examined employing hematoxylin-eosin staining(HE),and the mRNA and protein levels of OPG,RANKL,and RANK in murine kidney tissues were determined via reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and Western blotting.Results:Comparative analysis revealed that,in comparison to the Sham group,the CLP group demonstrated a significant elevation in the levels of Scr,BUN,IL-6,TNF-α,and IL-1β,with statistically significant disparities(all P<0.05).Histopathological examination of the CLP group's kidneys unveiled glomerular congestion,edema,partial ischemic wrinkling,enlargement of interstitial spaces,the presence of necrotic epithelial cells in select renal tubules,tubular luminal dilation,varying degrees of interstitial edema,and infiltration by a limited number of inflammatory cells.In parallel,relative to the Sham group,the CLP group exhibited substantial upregulation in mRNA expression of OPG and RANK in renal tissues,while RANKL mRNA expression experienced marked downregulation,with statistically significant distinctions(all P<0.05).Moreover,in comparison with the Sham group,the CLP group demonstrated an elevation in protein expression of OPG and RANK in kidney tissues,whereas RANKL protein expression displayed significant downregulation,with statistically significant differences(all P<0.05).Conclusion:In a murine sepsis model,augmented expression of OPG and RANK,coupled with diminished RANKL expression,suggests the potential involvement of the OPG/RANKL/RANK signaling pathway in the pathophysiological progression of SA-AKI.展开更多
Objective:To investigate the association of Micro-rna(miR)-146a-5p expression with preeclampsia,and further explore the potential mechanism involved.Methods:Compared with the blank control group,the expressions of miR...Objective:To investigate the association of Micro-rna(miR)-146a-5p expression with preeclampsia,and further explore the potential mechanism involved.Methods:Compared with the blank control group,the expressions of miR-146a-5p and TRAF6 were detected in lipopolysaccharide(LPS)-induced JEG-3 cells.Chorionic carcinoma cell JEG-3 in vitro culture are divided into control,miR-146a-5p mimic+lipopolysaccharide(lps),miR-146a-5p mimic and miR-146a-5p inhibitor groups.qRT-PCR analysis were used to detect the mRNA of miR-146a-5p,IL-1β,IL-6,IL-8 and TNF-α.Western blot assays were carried out to determine the protein expression of TRAF6/NF-кB pathway related proteins.Results:1.miR-146a expression in miR-146a mimic group were significantly higher than the other three groups(P<0.05).2.Compared with the control group,the expression level of miR-146a-5p in JEG-3 cells induced by LPS was significantly increased,and the expression level of TRAF6 was significantly reduced(P<0.05).3.Compared with the control group,the mRNA expression levels of IL-1β,IL-6,IL-8,and TNF-αdecreased significantly after using miR-146a mimic(P<0.05).After adding miR-146a inhibitor,the mRNA expression levels of IL-1β,IL-6,IL-8,and TNF-αwere significantly increased(P<0.05).However,compared with the mimic+LPS group,the difference was not statistically significant(all P>0.05).The results of Western Blot showed that the expression of TRAF6 and NF-κB protein in JEG-3 cells decreased significantly after adding miR-146a mimic and increased after adding miR-146a inhibitor.Conclusion:MiR-146-5p can affect the inflammation response of Maternal-fetal interface by inhibiting TRAF6/NF-кB signaling pathway in preeclampsia.展开更多
Background: Intramuscular fat(IMF) content is a vital parameter for assessing pork quality. Increasing evidence has shown that microRNAs(miRNAs) play an important role in regulating porcine IMF deposition. Here, a nov...Background: Intramuscular fat(IMF) content is a vital parameter for assessing pork quality. Increasing evidence has shown that microRNAs(miRNAs) play an important role in regulating porcine IMF deposition. Here, a novel miRNA implicated in porcine IMF adipogenesis was found, and its effect and regulatory mechanism were further explored with respect to intramuscular preadipocyte proliferation and differentiation.Results: By porcine adipose tissue miRNA sequencing analysis, we found that miR-146a-5p is a potential regulator of porcine IMF adipogenesis. Further studies showed that miR-146a-5p mimics inhibited porcine intramuscular preadipocyte proliferation and differentiation, while the miR-146a-5p inhibitor promoted cell proliferation and adipogenic differentiation. Mechanistically, miR-146a-5p suppressed cell proliferation by directly targeting SMAD family member 4(SMAD4) to attenuate TGF-β signaling. Moreover, miR-146a-5p inhibited the differentiation of intramuscular preadipocytes by targeting TNF receptor-associated factor 6(TRAF6) to weaken the AKT/mTORC1 signaling downstream of the TRAF6 pathway.Conclusions: MiR-146a-5p targets SMAD4 and TRAF6 to inhibit porcine intramuscular adipogenesis by attenuating TGF-β and AKT/mTORC1 signaling, respectively. These findings provide a novel miRNA biomarker for regulating intramuscular adipogenesis to promote pork quality.展开更多
Although different types of drugs are available for postmenopausal osteoporosis,the limitations of the current therapies including drug resistances and adverse effects require identification of novel anti-osteoporosis...Although different types of drugs are available for postmenopausal osteoporosis,the limitations of the current therapies including drug resistances and adverse effects require identification of novel anti-osteoporosis agents.Here,we defined that norlichexanthone(NOR),a natural product,is a ligand of estrogen receptor-alpha(ERα)and revealed its therapeutic potential for postmenopausal osteoporosis.We used mammalian-one hybrid assay to screen for ERαmodulators from crude extracts of several plant endophytes.As a result,NOR purified from the extract of endophyte ARL-13 was identified as a selective ERαmodulator.NOR directly bound to ERαwith an affinity in nanomolar range,revealing that it is a natural ligand of ERα.NOR induced osteoblast formation in MC3T3-E1 precursor cells.Conversely,NOR inhibited receptor activator of nuclear factor-kappa B ligand(RANKL)-induced osteoclast formation in both RAW264.7 macrophages and mouse primary monocytes.Mechanistically,NOR inhibited RANKL-induced association of ERαand TRAF6 to prevent ERα-mediated TRAF6 activation via Lys63-linked ubiquitination.Importantly,NOR exhibited potent anti-osteoporosis efficacy in an ovariectomized mouse model.Comparing to estrogen,NOR was of much less capability in stimulating endometrial hyperplasia and promoting mammalian cancer cell proliferation.Taken together,our study identified NOR as a natural and high affinity ligand of ERαwith substantial anti-osteoporosis but less estrogenic activity.展开更多
Cyathulae Radix,a traditional Chinese medicine and a common vegetable,boasts a history spanning millennia.It enhances bone density,boosts metabolism,and effectively alleviates osteoporosis-induced pain.Despite its his...Cyathulae Radix,a traditional Chinese medicine and a common vegetable,boasts a history spanning millennia.It enhances bone density,boosts metabolism,and effectively alleviates osteoporosis-induced pain.Despite its historical use,the molecular mechanisms behind Cyathulae Radix’s impact on osteoporosis remain unexplored.In this study,we investigated the effects and mechanisms of Cyathulae Radix ethanol extract(CEE)in inhibiting osteoporosis and osteoclastogenesis.Eight-week-old female mice underwent ovariectomy and were treated with CEE for eight weeks.Micro-computed tomography(micro-CT)assessed histomorphometric parameters,bone tissue staining observed distal femur histomorphology,and three-point bending tests evaluated tibia mechanical properties.Enzyme-linked immunosorbent assay(ELISA)measured serum estradiol(E2),receptor activator for nuclear factor B ligand(RANKL),and osteoprotegerin(OPG)levels.Osteoclastogenesis-related markers were analyzed via Western blotting(WB)and quantitative real-time polymerase chain reaction(qRT-PCR).Additionally,CEE effects on RANKL-induced osteoclast formation and bone resorption were investigated in vitro using tartrate-resistant acid phosphatase(TRAP)staining,qRT-PCR,and WB assay.Compared with the ovariectomy(OVX)group,CEE treatment enhanced trabecular bone density,maximal load-bearing capacity,and various histomorphometric parameters.Serum E2 and OPG levels significantly increased,while Receptor activator of nuclear factor-κB(RANK)decreased in the CEE group.CEE downregulated matrix metallopeptidase 9(MMP-9),Cathepsin K(CTSK),and TRAP gene and protein expression.In bone marrow macrophages(BMMs),CEE reduced mature osteoclasts,bone resorption pit areas,and MMP-9,CTSK,and TRAP expression during osteoclast differentiation.Compared with DMSO treatment,CEE markedly inhibited RANK,TNF receptor associated factor 6(TRAF6),Proto-oncogene c-Fos(c-Fos),Nuclear factor of activated T-cells cytoplasmic 1(NFATc1)expressions,and Extracellular regulated protein kinases(ERK),c-Jun N-terminal kinase(JNK),NF-kappa B-p65(p65)phosphorylation in osteoclasts.In conclusion,CEE significantly inhibits OVX-induced osteoporosis and RANKL-induced osteoclastogenesis,potentially through modulating the Estrogen Receptor(ER)/RANK/NFATc1 signaling pathway.展开更多
文摘Objective:To investigate the regulatory effect of miR-146a overexpression on corneal inflammatory response in a mouse model of dry eye,and to analyze its relationship with the IRAK1/TRAF6/NF-кB signaling pathway.Methods:A total of 50 SPF-grade BALB/c mice were randomly divided into five groups,with10 mice in each group.Except for the control group,the other four groups were treated with 0.2%benzalkonium chloride(BAC)solution in both eyes to construct a dry eye model.After successful modeling,the control group and model group received NC agomir;the miR antagonist group received miR-146a antagomir;the miR agonist group received miR-146a agomir;and the pathway agonist group received miR-146a agomir+NF-κB activator 2.After four weeks of treatment,the expressions levels of miR-146a,inflammatory factors,and IRAK1/TRAF6/NF-κB signaling pathway proteins were observed and compared among the five groups.Results:After four weeks of treatment,there was a statistically significant difference in the relative expression of miR-146a in the five groups(F=61.058,P<0.001),which was significantly higher in the miR agonist group than in the other four groups.After4 weeks of treatment,there were statistically significant differences in the expression levels of IL-1β,IL-6,IL-8 and TNF-αin the five groups(F=84.757,103.658,55.477,46.762;P<0.001).After four weeks of treatment,there were statistically significant differences in the protein expression levels of IRAK1,TRAF6,NF-κB and IκBαin the five groups(F=62.975,77.173,67.108,29.381;P<0.001),except for the control group,the expression levels of IRAK1,TRAF6 and NF-κB proteins in the miR agonist group were significantly lower than those in the other three groups,and the expression levels of IκBαprotein were significantly higher than those in the other three groups.Conclusion:Overexpression of miR-146a can negatively regulate corneal inflammatory response in dry eye mice through the IRAK1/TRAF6/NF-κB signaling pathway,which can provide new insights for the clinical treatment of dry eye disease.
基金Natural Science Foundation of Xinjiang Uygur Autonomous Region(No.2022D01C604)。
文摘Objective:The objective of this study was to investigate the alterations and potential implications of the Osteoprotegerin(OPG)/Receptor Activator of Nuclear Factor-kappa B Ligand(RANKL)/Receptor Activator of Nuclear Factor-kappa B(RANK)signaling pathway factors in a murine model of sepsis-associated acute kidney injury(SA-AKI).This research aimed to offer novel insights into the mechanistic exploration of SA-AKI.Methods:The SA-AKI model group(CLP group)was established through cecal ligation and puncture surgery(CLP),while the control group consisted of sham-operated animals(Sham group)subjected only to laparotomy without cecal ligation and puncture.Blood samples were collected 24 h post-surgery,and murine kidney tissues were harvested upon euthanasia.Serum levels of Serum Creatinine(Scr)and Blood Urea Nitrogen(BUN)were quantified using assay kits.Furthermore,serum levels of interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α),and interleukin-1 beta(IL-1β)were assessed through enzyme-linked immunosorbent assay(ELISA).Renal tissue pathological alterations were examined employing hematoxylin-eosin staining(HE),and the mRNA and protein levels of OPG,RANKL,and RANK in murine kidney tissues were determined via reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and Western blotting.Results:Comparative analysis revealed that,in comparison to the Sham group,the CLP group demonstrated a significant elevation in the levels of Scr,BUN,IL-6,TNF-α,and IL-1β,with statistically significant disparities(all P<0.05).Histopathological examination of the CLP group's kidneys unveiled glomerular congestion,edema,partial ischemic wrinkling,enlargement of interstitial spaces,the presence of necrotic epithelial cells in select renal tubules,tubular luminal dilation,varying degrees of interstitial edema,and infiltration by a limited number of inflammatory cells.In parallel,relative to the Sham group,the CLP group exhibited substantial upregulation in mRNA expression of OPG and RANK in renal tissues,while RANKL mRNA expression experienced marked downregulation,with statistically significant distinctions(all P<0.05).Moreover,in comparison with the Sham group,the CLP group demonstrated an elevation in protein expression of OPG and RANK in kidney tissues,whereas RANKL protein expression displayed significant downregulation,with statistically significant differences(all P<0.05).Conclusion:In a murine sepsis model,augmented expression of OPG and RANK,coupled with diminished RANKL expression,suggests the potential involvement of the OPG/RANKL/RANK signaling pathway in the pathophysiological progression of SA-AKI.
基金Hainan provincial health industry research project(No.20A200001)General project of natural science foundation of Hainan province(No.817306)Science research project of colleges and universities(No.Hnky2019-40)。
文摘Objective:To investigate the association of Micro-rna(miR)-146a-5p expression with preeclampsia,and further explore the potential mechanism involved.Methods:Compared with the blank control group,the expressions of miR-146a-5p and TRAF6 were detected in lipopolysaccharide(LPS)-induced JEG-3 cells.Chorionic carcinoma cell JEG-3 in vitro culture are divided into control,miR-146a-5p mimic+lipopolysaccharide(lps),miR-146a-5p mimic and miR-146a-5p inhibitor groups.qRT-PCR analysis were used to detect the mRNA of miR-146a-5p,IL-1β,IL-6,IL-8 and TNF-α.Western blot assays were carried out to determine the protein expression of TRAF6/NF-кB pathway related proteins.Results:1.miR-146a expression in miR-146a mimic group were significantly higher than the other three groups(P<0.05).2.Compared with the control group,the expression level of miR-146a-5p in JEG-3 cells induced by LPS was significantly increased,and the expression level of TRAF6 was significantly reduced(P<0.05).3.Compared with the control group,the mRNA expression levels of IL-1β,IL-6,IL-8,and TNF-αdecreased significantly after using miR-146a mimic(P<0.05).After adding miR-146a inhibitor,the mRNA expression levels of IL-1β,IL-6,IL-8,and TNF-αwere significantly increased(P<0.05).However,compared with the mimic+LPS group,the difference was not statistically significant(all P>0.05).The results of Western Blot showed that the expression of TRAF6 and NF-κB protein in JEG-3 cells decreased significantly after adding miR-146a mimic and increased after adding miR-146a inhibitor.Conclusion:MiR-146-5p can affect the inflammation response of Maternal-fetal interface by inhibiting TRAF6/NF-кB signaling pathway in preeclampsia.
基金supported by grants from the National Natural Science Foundation (31872979, 31572366)the National Key Research and Development Program of China (2017YFD0502002)the National Basic Research Programs of China (2015CB943102)。
文摘Background: Intramuscular fat(IMF) content is a vital parameter for assessing pork quality. Increasing evidence has shown that microRNAs(miRNAs) play an important role in regulating porcine IMF deposition. Here, a novel miRNA implicated in porcine IMF adipogenesis was found, and its effect and regulatory mechanism were further explored with respect to intramuscular preadipocyte proliferation and differentiation.Results: By porcine adipose tissue miRNA sequencing analysis, we found that miR-146a-5p is a potential regulator of porcine IMF adipogenesis. Further studies showed that miR-146a-5p mimics inhibited porcine intramuscular preadipocyte proliferation and differentiation, while the miR-146a-5p inhibitor promoted cell proliferation and adipogenic differentiation. Mechanistically, miR-146a-5p suppressed cell proliferation by directly targeting SMAD family member 4(SMAD4) to attenuate TGF-β signaling. Moreover, miR-146a-5p inhibited the differentiation of intramuscular preadipocytes by targeting TNF receptor-associated factor 6(TRAF6) to weaken the AKT/mTORC1 signaling downstream of the TRAF6 pathway.Conclusions: MiR-146a-5p targets SMAD4 and TRAF6 to inhibit porcine intramuscular adipogenesis by attenuating TGF-β and AKT/mTORC1 signaling, respectively. These findings provide a novel miRNA biomarker for regulating intramuscular adipogenesis to promote pork quality.
基金supported by the National Natural Science Foundation of China(Grant Nos.31770811,31471318 and 31271453)the Fundamental Research Funds for the Central Universities(Grant No.20720190082,China)+1 种基金the Regional Demonstration of Marine Economy Innovative Development Project(Grant No.16PYY007SF17,China)the Fujian Provincial Science&Technology Department(Grant No.2017YZ0002-1,China)
文摘Although different types of drugs are available for postmenopausal osteoporosis,the limitations of the current therapies including drug resistances and adverse effects require identification of novel anti-osteoporosis agents.Here,we defined that norlichexanthone(NOR),a natural product,is a ligand of estrogen receptor-alpha(ERα)and revealed its therapeutic potential for postmenopausal osteoporosis.We used mammalian-one hybrid assay to screen for ERαmodulators from crude extracts of several plant endophytes.As a result,NOR purified from the extract of endophyte ARL-13 was identified as a selective ERαmodulator.NOR directly bound to ERαwith an affinity in nanomolar range,revealing that it is a natural ligand of ERα.NOR induced osteoblast formation in MC3T3-E1 precursor cells.Conversely,NOR inhibited receptor activator of nuclear factor-kappa B ligand(RANKL)-induced osteoclast formation in both RAW264.7 macrophages and mouse primary monocytes.Mechanistically,NOR inhibited RANKL-induced association of ERαand TRAF6 to prevent ERα-mediated TRAF6 activation via Lys63-linked ubiquitination.Importantly,NOR exhibited potent anti-osteoporosis efficacy in an ovariectomized mouse model.Comparing to estrogen,NOR was of much less capability in stimulating endometrial hyperplasia and promoting mammalian cancer cell proliferation.Taken together,our study identified NOR as a natural and high affinity ligand of ERαwith substantial anti-osteoporosis but less estrogenic activity.
基金supported by the National Natural Science Foundation of China(Nos.81273816,81774379,and 81370974)the Natural Science Foundation of Hunan Province,China(Nos.2017JJ2338 and 2020JJ4860)+1 种基金the Fundamental Research Funds for the Central Universities of Central South University(No.502211706)the Fund for the Key Laboratory of Hunan Province,China(No.2017TP1004).
文摘Cyathulae Radix,a traditional Chinese medicine and a common vegetable,boasts a history spanning millennia.It enhances bone density,boosts metabolism,and effectively alleviates osteoporosis-induced pain.Despite its historical use,the molecular mechanisms behind Cyathulae Radix’s impact on osteoporosis remain unexplored.In this study,we investigated the effects and mechanisms of Cyathulae Radix ethanol extract(CEE)in inhibiting osteoporosis and osteoclastogenesis.Eight-week-old female mice underwent ovariectomy and were treated with CEE for eight weeks.Micro-computed tomography(micro-CT)assessed histomorphometric parameters,bone tissue staining observed distal femur histomorphology,and three-point bending tests evaluated tibia mechanical properties.Enzyme-linked immunosorbent assay(ELISA)measured serum estradiol(E2),receptor activator for nuclear factor B ligand(RANKL),and osteoprotegerin(OPG)levels.Osteoclastogenesis-related markers were analyzed via Western blotting(WB)and quantitative real-time polymerase chain reaction(qRT-PCR).Additionally,CEE effects on RANKL-induced osteoclast formation and bone resorption were investigated in vitro using tartrate-resistant acid phosphatase(TRAP)staining,qRT-PCR,and WB assay.Compared with the ovariectomy(OVX)group,CEE treatment enhanced trabecular bone density,maximal load-bearing capacity,and various histomorphometric parameters.Serum E2 and OPG levels significantly increased,while Receptor activator of nuclear factor-κB(RANK)decreased in the CEE group.CEE downregulated matrix metallopeptidase 9(MMP-9),Cathepsin K(CTSK),and TRAP gene and protein expression.In bone marrow macrophages(BMMs),CEE reduced mature osteoclasts,bone resorption pit areas,and MMP-9,CTSK,and TRAP expression during osteoclast differentiation.Compared with DMSO treatment,CEE markedly inhibited RANK,TNF receptor associated factor 6(TRAF6),Proto-oncogene c-Fos(c-Fos),Nuclear factor of activated T-cells cytoplasmic 1(NFATc1)expressions,and Extracellular regulated protein kinases(ERK),c-Jun N-terminal kinase(JNK),NF-kappa B-p65(p65)phosphorylation in osteoclasts.In conclusion,CEE significantly inhibits OVX-induced osteoporosis and RANKL-induced osteoclastogenesis,potentially through modulating the Estrogen Receptor(ER)/RANK/NFATc1 signaling pathway.
