淫羊藿苷对脂多糖诱导成骨细胞骨架F-actin损伤的保护作用
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摘要
目的考察淫羊藿苷对成骨细胞骨架F-actin损伤的保护作用及其对相关信号通路的调控。方法采用脂多糖诱导大鼠成骨细胞损伤模型。实验分为空白对照组、模型组和0.1,1,10μg·mL~(-1)淫羊藿苷组,MTT法观察淫羊藿苷对成骨细胞活性的影响,TRITC-Phalloidin荧光染色观察淫羊藿苷对细胞骨架的影响,ELISA法测定各组成骨细胞中F-actin的含量,RT-PCR法测定细胞骨架RhoA和Cofilin的mRNA表达量。结果与空白对照组比较,100μg·mL~(-1)脂多糖能显著降低成骨细胞存活率(P<0.01);与模型组比较,1～10μg·mL~(-1)淫羊藿苷预处理后能显著提高细胞存活率(P<0.05或P<0.01);而0.1μg·mL~(-1)淫羊藿苷则无显著影响。与模型组比较,1～10μg·mL~(-1)淫羊藿苷能显著抑制成骨细胞F-actin解聚(P<0.05),抑制Rho A的mRNA表达(P<0.05或P<0.01),0.1～10μg·mL~(-1)淫羊藿苷能抑制Cofilin的mRNA表达(P<0.01)。结论淫羊藿苷对脂多糖诱导的细胞骨架F-actin损伤具有保护作用,其机制与抑制细胞骨架相关因子RhoA和Cofilin的mRNA表达有关。
OBJECTIVE To investigate the protective effect of icariin on F-actin damage of osteoblast cytoskeleton and the regulation of related signaling pathways. METHODS Lipopolysaccharide(LPS) was used to induce osteoblasts to establish an injury model in vitro. Experimental group: control group, model group and 0.1, 1, 10 μg·mL~(-1) icariin groups. MTT method was used to observe the effect of icariin on the activity of osteocytes. TRITC-Phalloidin fluorescence staining was used to observe the effect of icariin on cytoskeleton. ELISA method was used to determine the content of F-actin in osteocytes. RT-PCR method was used to determine the expression of cytoskeleton-related factors Rho A and Cofilin mRNA. RESULTS Compared with the control group, 100 μg·mL~(-1) LPS could significantly reduce the survival rate of osteoblasts(P<0.01). Compared with the model group, the survival rate could significantly improve after pretreatment with 1~(-1)0 μg·mL~(-1) icariin(P<0.05 or P<0.01), while0.1 μg·mL~(-1) icariin had no significant effect. Compared with the model group, 1~(-1)0 μg·mL~(-1) icariin could significantly inhibit F-actin depolymerization in osteoblasts(P<0.05), inhibit the mRNA expression of Rho A(P<0.05 or 0.01), and icariin at concentration of 0.1~(-1)0 μg·mL~(-1) could inhibit the mRNA expression of Cofilin(P<0.01). CONCLUSION Icariin has protective effect on LPS-induced cytoskeleton F-actin injury model, and its mechanism may be related to inhibiting the expression of cytoskeleton-related factors Rho A and Cofilin mRNA.
OBJECTIVE To investigate the protective effect of icariin on F-actin damage of osteoblast cytoskeleton and the regulation of related signaling pathways. METHODS Lipopolysaccharide(LPS) was used to induce osteoblasts to establish an injury model in vitro. Experimental group: control group, model group and 0.1, 1, 10 μg·mL~(-1) icariin groups. MTT method was used to observe the effect of icariin on the activity of osteocytes. TRITC-Phalloidin fluorescence staining was used to observe the effect of icariin on cytoskeleton. ELISA method was used to determine the content of F-actin in osteocytes. RT-PCR method was used to determine the expression of cytoskeleton-related factors Rho A and Cofilin mRNA. RESULTS Compared with the control group, 100 μg·mL~(-1) LPS could significantly reduce the survival rate of osteoblasts(P<0.01). Compared with the model group, the survival rate could significantly improve after pretreatment with 1~(-1)0 μg·mL~(-1) icariin(P<0.05 or P<0.01), while0.1 μg·mL~(-1) icariin had no significant effect. Compared with the model group, 1~(-1)0 μg·mL~(-1) icariin could significantly inhibit F-actin depolymerization in osteoblasts(P<0.05), inhibit the mRNA expression of Rho A(P<0.05 or 0.01), and icariin at concentration of 0.1~(-1)0 μg·mL~(-1) could inhibit the mRNA expression of Cofilin(P<0.01). CONCLUSION Icariin has protective effect on LPS-induced cytoskeleton F-actin injury model, and its mechanism may be related to inhibiting the expression of cytoskeleton-related factors Rho A and Cofilin mRNA.
引文
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[3]ETIENNEMANNEVILLE S,HALL A.Rho GTPases in cell biology[J].Nature,2002,420(6916):629-635.
[4]TKACH V,BOCK E,BEREZIN V.The role of RhoA in the regulation of cell morphology and motility[J].Cytoskeleton,2010,61(1):21-33.
[5]BERNSTEIN B W,BAMBURG J R.ADF/Cofilin:a functional node in cell biology[J].Trends Cell Biol,2010,20(4):187-195.
[6]ESWARAPPA S M,PAREEK V,CHAKRAVORTTY D.Role of actin cytoskeleton in LPS-induced NF-kappaBactivation and nitric oxide production in murine macrophages[J].Innate Immun,2008,14(5):309-318.
[7]ALHADIDI Q,SHAH Z A.Cofilin mediates LPS-induced microglial cell activation and associated neurotoxicity through activation of NF-κB and JAK-STAT pathway[J].Mol Neurobiol,2018,55(2):1676-1691.
[8]ZHANG Y,SHEN L,MAO Z,et al.Icariin enhances bone repair in rabbits with bone infection during post-infection treatment and prevents inhibition of osteoblasts by vancomycin[J].Front Pharmacol,2017(8):784.Doi:10.3389/fphar.2017.00784.
[9]SHOU D,ZHANG Y,SHEN L,et al.Flavonoids of herba epimedii enhances bone repair in a rabbit model of chronic osteomyelitis during post-infection treatment and stimulates osteoblast proliferation in vitro[J].Phytother Res,2017,31(2):330-339.
[10]HUANG X W,WANG N N,WANG X P,et al.Comparison of absorption and utilization of four flavonoids Epimedii folium in osteoblasts[J].Chin J Clin Pharm(中国临床药学杂志),2016,25(4):222-226.
[11]WU J F,DONG J C,XU C Q.Effects of icariin on inflammation model stimulated by lipopolysaccharide in vitro and in vivo[J].Chin J Integrat Tradit West Med,2009,29(29):330-334.
[12]KONG L,LIU J,WANG J,et al.Icariin inhibits TNF-α/IFN-γinduced inflammatory response via inhibition of the substance P and p38-MAPK signaling pathway in human keratinocytes[J].Int Immunopharmacol,2015,29(2):401-407.
[13]KUSTERMANS G,PIETTE J,LEGRAND-POELS S.Actintargeting natural compounds as tools to study the role of actin cytoskeleton in signal transduction[J].Biochem Pharmacol,2008,76(11):1310-1322.
[14]BANAN A,KESHAVARZIAN A,ZHANG L,et al.NF-kappaB activation as a key mechanism in ethanol-induced disruption of the F-actin cytoskeleton and monolayer barrier integrity in intestinal epithelium[J].Alcohol,2007,41(6):447-460.
[15]XU H,FENG W,ZHANG H,et al.Actin cytoskeleton mediates BMP2-Smad signaling via calponin 1 in preosteoblast under simulated microgravity[J].Biochimie,2017(138):184-193.
[16]SCHUMANN R R,LEONG S R,FLAGGS G W,et al.Structure and function of lipopolysaccharide binding protein[J].Science,1990,249(4975):1429-1431.
[17]GUO C,YUAN L,WANG J,et al.Lipopolysaccharide(LPS)induces the apoptosis and inhibits osteoblast differentiation through JNK pathway in MC3T3-E1 cells[J].Inflammation,2014,37(2):621-631.
[18]NAKAO J,FUJII Y,KUSUYAMA J,et al.Low-intensity pulsed ultrasound(LIPUS)inhibits LPS-induced inflammatory responses of osteoblasts through TLR4-MyD88 dissociation[J].Bone,2014(58):17-25.
[19]XU M X,SUN X X,LI W,et al.LPS at low concentration promotes the fracture healing through regulating the autophagy of osteoblasts via NF-κB signal pathway[J].Eur Rev Med Pharmacol Sci,2018,22(6):1569-1579.
[20]XU C Q,LIU B J,WU J F,et al.Icariin attenuates LPS-induced acute inflammatory responses:Involvement of PI3K/Akt and NF-κB signaling pathway[J].Eur J Pharmacol,2010,642(1):146-153.