Background: Blocking the Rho A/ROCK Ⅱ/MLC 2(Ras homolog gene family member A/Rho kinase Ⅱ/myosin light chain 2) signaling pathway can initiate neuroprotective mechanisms against neurological diseases such as stroke,...Background: Blocking the Rho A/ROCK Ⅱ/MLC 2(Ras homolog gene family member A/Rho kinase Ⅱ/myosin light chain 2) signaling pathway can initiate neuroprotective mechanisms against neurological diseases such as stroke, cerebral ischemia, and subarachnoid hemorrhage. Nevertheless, it is not clear whether and how disrupting the Rho A/ROCK Ⅱ/MLC 2 signaling pathway changes the pathogenic processes of the blood–brain barrier(BBB) after intracerebral hemorrhage(ICH). The present investigation included the injection of rat caudal vein blood into the basal ganglia area to replicate the pathophysiological conditions caused by ICH. Methods: Scalp acupuncture(SA) therapy was performed on rats with ICH at the acupuncture point “Baihui”-penetrating “Qubin,” and the ROCK selective inhibitor fasudil was used as a positive control to evaluate the inhibitory effect of acupuncture on the Rho A/ROCK Ⅱ/MLC 2 signaling pathway. Post-assessments included neurological deficits, brain edema, Evans blue extravasation, Western blot, quantitative polymerase chain reaction, and transmission electron microscope imaging. Results: We found that ROCK Ⅱ acts as a promoter of the Rho A/ROCK Ⅱ/MLC 2 signaling pathway, and its expression increased at 6 h after ICH, peaked at 3 days, and then decreased at 7 days after ICH, but was still higher than the preintervention level. According to some experimental results, although 3 days is the peak, 7 days is the best time point for acupuncture treatment. Starting from 6 h after ICH, the neurovascular structure and endothelial cell morphology around the hematoma began to change. Based on the changes in the promoter ROCK Ⅱ, a 7-day time point was selected as the breakthrough point for treating ICH model rats in the main experiment. The results of this experiment showed that both SA at “Baihui”-penetrating “Qubin” and treatment with fasudil could improve the expression of endothelial-related proteins by inhibiting the Rho A/ROCK Ⅱ/MLC 2 signaling pathway and reduce neurological dysfunction, brain edema, and BBB permeability in rats. Conclusion: This study found that these experimental data indicated that SA at “Baihui”-penetrating “Qubin” could preserve BBB integrity and neurological function recovery after ICH by inhibiting Rho A/ROCK Ⅱ/MLC 2 signaling pathway activation and by regulating endothelial cell–related proteins.展开更多
RhoA (Ras homolog gene family member A) belongs to the Rho subfamily of GTPases. ROCK (Rho—associated coiled—coil forming protein kinase) is downstream of the active RhoA and affects the generation and secretion of ...RhoA (Ras homolog gene family member A) belongs to the Rho subfamily of GTPases. ROCK (Rho—associated coiled—coil forming protein kinase) is downstream of the active RhoA and affects the generation and secretion of cellular element, which will result in relevant biologic effects. The RhoA/ROCK signaling pathway consists of these serious reactions. Therefore, the activation and inhibition of this pathway are closely related to the occurrence and development of many diseases. The research on the molecular mechanism of these diseases may be instructive and helpful to the clinical treatmen and prognosis of diseases. Recent studies of these typical diseases related to RhoA/ROCK signaling pathway are viewed in this article.展开更多
Disruption of the blood-spinal cord barrier(BSCB)is a critical event in the secondary injury following spinal cord injury(SCI).Mertk has been reported to play an important role in regulating inflammation and cytoskele...Disruption of the blood-spinal cord barrier(BSCB)is a critical event in the secondary injury following spinal cord injury(SCI).Mertk has been reported to play an important role in regulating inflammation and cytoskeletal dynamics.However,the specific involvement of Mertk in BSCB remains elusive.Here,we demonstrated a distinct role of Mertk in the repair of BSCB.Mertk expression is decreased in endothelial cells following SCI.Overexpression of Mertk upregulated tight junction proteins(TJs),reducing BSCB permeability and subsequently inhibiting inflammation and apoptosis.Ultimately,this led to enhanced neural regeneration and functional recovery.Further experiments revealed that the RhoA/Rock1/P-MLC pathway plays a key role in the effects of Mertk.These findings highlight the role of Mertk in promoting SCI recovery through its ability to mitigate BSCB permeability and may provide potential targets for SCI repair.展开更多
Houshiheisan,a classic prescription in traditional Chinese medicine,contains Flos Chrysanthemi,Radix Saposhnikoviae,Ramulus Cinnamomi,Rhizoma Chuanxiong,Radix et Rhizoma Asari,Radix Platycodonis,Rhizoma Atractylodis m...Houshiheisan,a classic prescription in traditional Chinese medicine,contains Flos Chrysanthemi,Radix Saposhnikoviae,Ramulus Cinnamomi,Rhizoma Chuanxiong,Radix et Rhizoma Asari,Radix Platycodonis,Rhizoma Atractylodis macrocephalae,Poria,Rhizoma Zingiberis,Radix Angelicae sinensis,Radix et Rhizoma Ginseng,Radix Scutellariae and Concha Ostreae.According to traditional Chinese medicine theory,Flos Chrysanthemi,Radix Saposhnikoviae,Ramulus Cinnamomi,Rhizoma Chuanxiong,Radix et Rhizoma Asari and Radix Platycodonis are wind-dispelling drugs;Rhizoma Atractylodis macrocephalae,Poria,Rhizoma Zingiberis,Radix Angelicae sinensis and Radix et Rhizoma Ginseng are deficiency-nourishing drugs.A large number of randomized controlled trials have shown that Houshiheisan is effective in treating stroke,but its mechanism of action is unknown.Axonal remodeling is an important mechanism in neural protection and regeneration.Therefore,this study explored the effect and mechanism of action of Houshiheisan on the repair of axons after cerebral ischemia.Rat models of focal cerebral ischemia were established by ligating the right middle cerebral artery.At 6 hours after model establishment,rats were intragastrically administered 10.5 g/kg Houshiheisan or 7.7 g/kg wind-dispelling drug or 2.59 g/kg deficiency-nourishing drug.These medicines were intragastrically administered as above every 24 hours for 7 consecutive days.Houshiheisan,and its wind-dispelling and deficiency-nourishing components reduced the neurological deficit score and ameliorated axon and neuron lesions after cerebral ischemia.Furthermore,Houshiheisan,and its wind-dispelling and deficiency-nourishing components decreased the expression of proteins that inhibit axonal remodeling：amyloid precursor protein,neurite outgrowth inhibitor protein A（Nogo-A）,Rho family small GTPase A（Rho A） and Rho-associated kinase 2（Rock2）,and increased the expression of growth associated protein-43,microtubule-associated protein-2,netrin-1,Ras-related C3 botulinum toxin substrate 1（Rac1） and cell division cycle 42（Cdc42）.The effect of Houshiheisan was stronger than wind-dispelling drugs or deficiency-nourishing drugs alone.In conclusion,Houshiheisan,and wind-dispelling and deficiency-nourishing drugs promote the repair of axons and nerve regeneration after cerebral ischemia through Nogo-A/Rho A/Rock2 and Netrin-1/Rac1/Cdc42 signaling pathways.These effects are strongest with Houshiheisan.展开更多
Myelin-associated glycoprotein（MAG） inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially availabl...Myelin-associated glycoprotein（MAG） inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor（NgR） and paired immunoglobulin-like receptor B（PirB） was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase（ROCK） phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.展开更多
Background:MicroRNAs are closely associated with the progression and outcomes of multiple human diseases,including sepsis.In this study,we examined the role of miR-23a in septic injury.Methods Lipopolysaccharide(LPS)w...Background:MicroRNAs are closely associated with the progression and outcomes of multiple human diseases,including sepsis.In this study,we examined the role of miR-23a in septic injury.Methods Lipopolysaccharide(LPS)was used to induce sepsis in a rat model and H9C2 and HK-2 cells.miR-23a expression was evaluated in rat myocardial and kidney tissues,as well as H9C2 and HK-2 cells.A miR-23a mimic was introduced into cells to identify the role of miR-23a in cell viability,apoptosis,and the secretion of inflammatory cytokines.Furthermore,the effect of Rho-associated kinase 1(ROCK1),a miR-23a target,on cell damage was evaluated,and molecules involved in the underlying mechanism were identified.Results:In the rat model,miR-23a was poorly expressed in myocardial(sham vs.sepsis 1.00±0.06 vs.0.27±0.03,P<0.01)and kidney tissues(sham vs.sepsis 0.27±0.03 vs.1.00±0.06,P<0.01).Artificial overexpression of miR-23a resulted in increased proliferative activity(DNA replication rate:Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:34.13±3.12 vs.12.94±1.21 vs.13.31±1.43 vs.22.94±2.26,P<0.05;HK-2 cells:15.17±1.43 vs.34.52±3.46 vs.35.19±3.12 vs.19.87±1.52,P<0.05),decreased cell apoptosis(Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:11.39±1.04 vs.32.57±2.29 vs.33.08±3.12 vs.21.63±2.35,P<0.05;HK-2 cells:15.17±1.43 vs.34.52±3.46 vs.35.19±3.12 vs.19.87±1.52,P<0.05),and decreased production of inflammatory cytokines,including interleukin-6(Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:59.61±5.14 vs.113.54±12.30 vs.116.51±10.69 vs.87.69±2.97 ng/mL;P<0.05,F=12.67,HK-2 cells:68.12±6.44 vs.139.65±16.62 vs.143.51±13.64 vs.100.82±9.74 ng/mL,P<0.05,F=9.83)and tumor necrosis factor-α(Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:103.20±10.31 vs.169.67±18.84 vs.173.61±15.91 vs.133.36±12.32 ng/mL,P<0.05,F=12.67,HK-2 cells:132.51±13.37 vs.187.47±16.74 vs.143.51±13.64 vs.155.79±15.31 ng/mL,P<0.05,F=9.83)in cells.However,ROCK1 was identified as a miR-23a target,and further up-regulation of ROCK1 mitigated the protective function of miR-23a in LPS-treated H9C2 and HK-2 cells.Moreover,ROCK1 suppressed sirtuin-1(SIRT1)expression to promote the phosphorylation of nuclear factor-kappa B(NF-κB)p65,indicating the possible involvement of this signaling pathway in miR-23a-mediated events.Conclusion:Our results indicate that miR-23a could suppress LPS-induced cell damage and inflammatory cytokine secretion by binding to ROCK1,mediated through the potential participation of the SIRT1/NF-κB signaling pathway.展开更多
Atherosclerosis(AS)is a chronic inflammatory disease,the main causes of which include abnormal lipid metabolism,endothelial injury,physical and chemical injury,hemodynamic injury,genetic factors and so on.These causes...Atherosclerosis(AS)is a chronic inflammatory disease,the main causes of which include abnormal lipid metabolism,endothelial injury,physical and chemical injury,hemodynamic injury,genetic factors and so on.These causes can lead to inflammatory injury of blood vessels and local dysfunction.Bunao-Fuyuan decoction(BNFY)is a traditional Chinese medicine compound that can treat cardiovascular and cerebrovascular diseases,but its effect on AS is still unknown.The aim of this study was to investigate the effect and mechanism of BNFY in proliferation and migration of vascular smooth muscle cells(VSMCs)on AS.At first,the expression ofα-SMA protein in ox-LDL-induced VSMCs,which was detected by immunofluorescence staining and western blot.CCK-8 technique and cloning technique were used to detect the cell proliferation of ox-LDL-induced VSMCs after adding BNFY.Meanwhile,the expression of proliferating protein Ki67 was detected by immunofluorescence staining.Western blot was also used to detect the expression of proliferation-related proteins CDK2,CyclinE1 and P27.Flow cytometry was used to detect the effect of BNFY on cell cycle.The effects of BNFY on proliferation and migration of cells were detected by cell scratch test and Transwell.Western blot was used to detect the expression of adhesion factors ICAM1,VCAM1,muc1,VE-cadherin and RHOA/ROCK-related proteins in cells.We found that the expression of AS markerα-SMA protein increased significantly and cells shriveled and a few floated on the medium after induction of ox-LDL on VSCMs.The proliferation rate of ox-LDL VSMCs decreased significantly after adding different doses of BNFY,and BNFY can inhibit cell cycle.Meanwhile,we also found that cell invasion and migration rate were significantly inhibited and related cell adhesion factors ICAM1,VCAM1,muc1 and VE-cadherin were inhibited too by BNFY.Finally,we found that BNFY inhibited the expression of RHOA,ROCK1,ROCK2,p-MLC proteins in the RHOA/ROCK signaling pathway.Therefore,we can summarize that BNFY may inhibit the proliferation and migration of atherosclerotic vascular smooth muscle cells by inhibiting the activity of RHOA/ROCK signaling pathway.展开更多
基金supported by the National Natural Science Foundation of China(numbers:81774416 and 81473764)。
文摘Background: Blocking the Rho A/ROCK Ⅱ/MLC 2(Ras homolog gene family member A/Rho kinase Ⅱ/myosin light chain 2) signaling pathway can initiate neuroprotective mechanisms against neurological diseases such as stroke, cerebral ischemia, and subarachnoid hemorrhage. Nevertheless, it is not clear whether and how disrupting the Rho A/ROCK Ⅱ/MLC 2 signaling pathway changes the pathogenic processes of the blood–brain barrier(BBB) after intracerebral hemorrhage(ICH). The present investigation included the injection of rat caudal vein blood into the basal ganglia area to replicate the pathophysiological conditions caused by ICH. Methods: Scalp acupuncture(SA) therapy was performed on rats with ICH at the acupuncture point “Baihui”-penetrating “Qubin,” and the ROCK selective inhibitor fasudil was used as a positive control to evaluate the inhibitory effect of acupuncture on the Rho A/ROCK Ⅱ/MLC 2 signaling pathway. Post-assessments included neurological deficits, brain edema, Evans blue extravasation, Western blot, quantitative polymerase chain reaction, and transmission electron microscope imaging. Results: We found that ROCK Ⅱ acts as a promoter of the Rho A/ROCK Ⅱ/MLC 2 signaling pathway, and its expression increased at 6 h after ICH, peaked at 3 days, and then decreased at 7 days after ICH, but was still higher than the preintervention level. According to some experimental results, although 3 days is the peak, 7 days is the best time point for acupuncture treatment. Starting from 6 h after ICH, the neurovascular structure and endothelial cell morphology around the hematoma began to change. Based on the changes in the promoter ROCK Ⅱ, a 7-day time point was selected as the breakthrough point for treating ICH model rats in the main experiment. The results of this experiment showed that both SA at “Baihui”-penetrating “Qubin” and treatment with fasudil could improve the expression of endothelial-related proteins by inhibiting the Rho A/ROCK Ⅱ/MLC 2 signaling pathway and reduce neurological dysfunction, brain edema, and BBB permeability in rats. Conclusion: This study found that these experimental data indicated that SA at “Baihui”-penetrating “Qubin” could preserve BBB integrity and neurological function recovery after ICH by inhibiting Rho A/ROCK Ⅱ/MLC 2 signaling pathway activation and by regulating endothelial cell–related proteins.
文摘RhoA (Ras homolog gene family member A) belongs to the Rho subfamily of GTPases. ROCK (Rho—associated coiled—coil forming protein kinase) is downstream of the active RhoA and affects the generation and secretion of cellular element, which will result in relevant biologic effects. The RhoA/ROCK signaling pathway consists of these serious reactions. Therefore, the activation and inhibition of this pathway are closely related to the occurrence and development of many diseases. The research on the molecular mechanism of these diseases may be instructive and helpful to the clinical treatmen and prognosis of diseases. Recent studies of these typical diseases related to RhoA/ROCK signaling pathway are viewed in this article.
基金Natural Science Foundation of Guangdong Province(2017A030313111)National Natural Science Foundation of China(81974329).
文摘Disruption of the blood-spinal cord barrier(BSCB)is a critical event in the secondary injury following spinal cord injury(SCI).Mertk has been reported to play an important role in regulating inflammation and cytoskeletal dynamics.However,the specific involvement of Mertk in BSCB remains elusive.Here,we demonstrated a distinct role of Mertk in the repair of BSCB.Mertk expression is decreased in endothelial cells following SCI.Overexpression of Mertk upregulated tight junction proteins(TJs),reducing BSCB permeability and subsequently inhibiting inflammation and apoptosis.Ultimately,this led to enhanced neural regeneration and functional recovery.Further experiments revealed that the RhoA/Rock1/P-MLC pathway plays a key role in the effects of Mertk.These findings highlight the role of Mertk in promoting SCI recovery through its ability to mitigate BSCB permeability and may provide potential targets for SCI repair.
基金supported by the National Natural Science Foundation of China,No.81373526
文摘Houshiheisan,a classic prescription in traditional Chinese medicine,contains Flos Chrysanthemi,Radix Saposhnikoviae,Ramulus Cinnamomi,Rhizoma Chuanxiong,Radix et Rhizoma Asari,Radix Platycodonis,Rhizoma Atractylodis macrocephalae,Poria,Rhizoma Zingiberis,Radix Angelicae sinensis,Radix et Rhizoma Ginseng,Radix Scutellariae and Concha Ostreae.According to traditional Chinese medicine theory,Flos Chrysanthemi,Radix Saposhnikoviae,Ramulus Cinnamomi,Rhizoma Chuanxiong,Radix et Rhizoma Asari and Radix Platycodonis are wind-dispelling drugs;Rhizoma Atractylodis macrocephalae,Poria,Rhizoma Zingiberis,Radix Angelicae sinensis and Radix et Rhizoma Ginseng are deficiency-nourishing drugs.A large number of randomized controlled trials have shown that Houshiheisan is effective in treating stroke,but its mechanism of action is unknown.Axonal remodeling is an important mechanism in neural protection and regeneration.Therefore,this study explored the effect and mechanism of action of Houshiheisan on the repair of axons after cerebral ischemia.Rat models of focal cerebral ischemia were established by ligating the right middle cerebral artery.At 6 hours after model establishment,rats were intragastrically administered 10.5 g/kg Houshiheisan or 7.7 g/kg wind-dispelling drug or 2.59 g/kg deficiency-nourishing drug.These medicines were intragastrically administered as above every 24 hours for 7 consecutive days.Houshiheisan,and its wind-dispelling and deficiency-nourishing components reduced the neurological deficit score and ameliorated axon and neuron lesions after cerebral ischemia.Furthermore,Houshiheisan,and its wind-dispelling and deficiency-nourishing components decreased the expression of proteins that inhibit axonal remodeling：amyloid precursor protein,neurite outgrowth inhibitor protein A（Nogo-A）,Rho family small GTPase A（Rho A） and Rho-associated kinase 2（Rock2）,and increased the expression of growth associated protein-43,microtubule-associated protein-2,netrin-1,Ras-related C3 botulinum toxin substrate 1（Rac1） and cell division cycle 42（Cdc42）.The effect of Houshiheisan was stronger than wind-dispelling drugs or deficiency-nourishing drugs alone.In conclusion,Houshiheisan,and wind-dispelling and deficiency-nourishing drugs promote the repair of axons and nerve regeneration after cerebral ischemia through Nogo-A/Rho A/Rock2 and Netrin-1/Rac1/Cdc42 signaling pathways.These effects are strongest with Houshiheisan.
基金supported by the National Natural Science Foundation of China,No.81171178
文摘Myelin-associated glycoprotein（MAG） inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor（NgR） and paired immunoglobulin-like receptor B（PirB） was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase（ROCK） phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.
文摘Background:MicroRNAs are closely associated with the progression and outcomes of multiple human diseases,including sepsis.In this study,we examined the role of miR-23a in septic injury.Methods Lipopolysaccharide(LPS)was used to induce sepsis in a rat model and H9C2 and HK-2 cells.miR-23a expression was evaluated in rat myocardial and kidney tissues,as well as H9C2 and HK-2 cells.A miR-23a mimic was introduced into cells to identify the role of miR-23a in cell viability,apoptosis,and the secretion of inflammatory cytokines.Furthermore,the effect of Rho-associated kinase 1(ROCK1),a miR-23a target,on cell damage was evaluated,and molecules involved in the underlying mechanism were identified.Results:In the rat model,miR-23a was poorly expressed in myocardial(sham vs.sepsis 1.00±0.06 vs.0.27±0.03,P<0.01)and kidney tissues(sham vs.sepsis 0.27±0.03 vs.1.00±0.06,P<0.01).Artificial overexpression of miR-23a resulted in increased proliferative activity(DNA replication rate:Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:34.13±3.12 vs.12.94±1.21 vs.13.31±1.43 vs.22.94±2.26,P<0.05;HK-2 cells:15.17±1.43 vs.34.52±3.46 vs.35.19±3.12 vs.19.87±1.52,P<0.05),decreased cell apoptosis(Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:11.39±1.04 vs.32.57±2.29 vs.33.08±3.12 vs.21.63±2.35,P<0.05;HK-2 cells:15.17±1.43 vs.34.52±3.46 vs.35.19±3.12 vs.19.87±1.52,P<0.05),and decreased production of inflammatory cytokines,including interleukin-6(Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:59.61±5.14 vs.113.54±12.30 vs.116.51±10.69 vs.87.69±2.97 ng/mL;P<0.05,F=12.67,HK-2 cells:68.12±6.44 vs.139.65±16.62 vs.143.51±13.64 vs.100.82±9.74 ng/mL,P<0.05,F=9.83)and tumor necrosis factor-α(Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:103.20±10.31 vs.169.67±18.84 vs.173.61±15.91 vs.133.36±12.32 ng/mL,P<0.05,F=12.67,HK-2 cells:132.51±13.37 vs.187.47±16.74 vs.143.51±13.64 vs.155.79±15.31 ng/mL,P<0.05,F=9.83)in cells.However,ROCK1 was identified as a miR-23a target,and further up-regulation of ROCK1 mitigated the protective function of miR-23a in LPS-treated H9C2 and HK-2 cells.Moreover,ROCK1 suppressed sirtuin-1(SIRT1)expression to promote the phosphorylation of nuclear factor-kappa B(NF-κB)p65,indicating the possible involvement of this signaling pathway in miR-23a-mediated events.Conclusion:Our results indicate that miR-23a could suppress LPS-induced cell damage and inflammatory cytokine secretion by binding to ROCK1,mediated through the potential participation of the SIRT1/NF-κB signaling pathway.
基金supported by the Natural Science Foundation of Zhejiang Province,China(No.LY16H020010)Medicine Health Science and Technology Plan of Zhejiang Province(No.2017194804)Science and Technology Bureau of Wenzhou(No.Y20160021)。
文摘Atherosclerosis(AS)is a chronic inflammatory disease,the main causes of which include abnormal lipid metabolism,endothelial injury,physical and chemical injury,hemodynamic injury,genetic factors and so on.These causes can lead to inflammatory injury of blood vessels and local dysfunction.Bunao-Fuyuan decoction(BNFY)is a traditional Chinese medicine compound that can treat cardiovascular and cerebrovascular diseases,but its effect on AS is still unknown.The aim of this study was to investigate the effect and mechanism of BNFY in proliferation and migration of vascular smooth muscle cells(VSMCs)on AS.At first,the expression ofα-SMA protein in ox-LDL-induced VSMCs,which was detected by immunofluorescence staining and western blot.CCK-8 technique and cloning technique were used to detect the cell proliferation of ox-LDL-induced VSMCs after adding BNFY.Meanwhile,the expression of proliferating protein Ki67 was detected by immunofluorescence staining.Western blot was also used to detect the expression of proliferation-related proteins CDK2,CyclinE1 and P27.Flow cytometry was used to detect the effect of BNFY on cell cycle.The effects of BNFY on proliferation and migration of cells were detected by cell scratch test and Transwell.Western blot was used to detect the expression of adhesion factors ICAM1,VCAM1,muc1,VE-cadherin and RHOA/ROCK-related proteins in cells.We found that the expression of AS markerα-SMA protein increased significantly and cells shriveled and a few floated on the medium after induction of ox-LDL on VSCMs.The proliferation rate of ox-LDL VSMCs decreased significantly after adding different doses of BNFY,and BNFY can inhibit cell cycle.Meanwhile,we also found that cell invasion and migration rate were significantly inhibited and related cell adhesion factors ICAM1,VCAM1,muc1 and VE-cadherin were inhibited too by BNFY.Finally,we found that BNFY inhibited the expression of RHOA,ROCK1,ROCK2,p-MLC proteins in the RHOA/ROCK signaling pathway.Therefore,we can summarize that BNFY may inhibit the proliferation and migration of atherosclerotic vascular smooth muscle cells by inhibiting the activity of RHOA/ROCK signaling pathway.
