体外法研究壳聚糖对无乳链球菌诱导的奶牛乳腺上皮细胞炎性损伤的保护作用

In Vitro Study on Protective Effect of Chitosan against Inflammation Injury of Bovine Mammary Epithelial Cells Induced by Streptococcus agalactiae

本试验采用体外法研究壳聚糖(CTS)对无乳链球菌(S.agalactiae)诱导的奶牛乳腺上皮细胞(BMECs)炎性损伤的保护作用。首先,采用不同浓度(0、15.625、31.25、62.5、125、250、500和1000 mg/mL)的CTS处理BMECs 12和24 h后,以四甲基偶氮唑蓝(MTT)法检测BMECs活性,根据BMECs活性筛选出CTS的适宜作用时间(24 h)和作用浓度(31.25、125和250 mg/mL),用于后续试验。然后,采用双因素试验设计,以不进行CTS处理和S.agalactiae诱导的BMECs作为对照组,以S.agalactiae诱导6 h的BMECs作为S.agalactiae组,以不同浓度(31.25、125和250 mg/mL)CTS处理24 h的BMECs作为CTS组,以不同浓度(31.25、125和250 mg/mL)CTS处理24 h后再经S.agalactiae诱导6 h的BMECs作为CTS(-)+sgc组,每组4个重复。使用荧光定量PCR的方法检测各组BMECs的白细胞介素(IL)-6、IL-1β、肿瘤坏死因子-α(TNF-α)、IL-8、Toll样受体2(TLR2)、髓样分化因子88(MyD88)、白细胞介素-1受体相关激酶4(IRAK 4)、肿瘤坏死因子受体相关因子6(TRAF6)和转化生长因子β活化激酶1(TAK1)的mRNA表达量,使用Western blot的方法检测各组BMECs的核因子-κB(NF-κB)抑制蛋白-α(IκB-α)、磷酸化NF-κB-p65(p-NF-κB-p65)、磷酸化p38(p-p38)、磷酸化细胞外信号调节激酶1/2(p-ERK1/2)和磷酸化c-Jun氨基末端激酶(p-JNK)的蛋白表达量。结果显示:1)31.25和125 mg/mL的CTS显著或极显著降低了BMECs的IL-6、IL-8、TLR2、MyD88、IRAK4和TRAF6 mRNA表达量(P<0.05或P<0.01);31.25、125和250 mg/mL的CTS极显著降低了S.agalactiae诱导的BMECs的IL-6、IL-1β、TNF-α、IL-8、TLR2、MyD88、IRAK4、TRAF6和TAK1 mRNA表达量(P<0.01)。2)31.25、125和250 mg/mL的CTS显著或极显著降低了BMECs和S.agalactiae诱导的BMECs的p-NF-κB-p65、p-p38、p-ERK1/2和p-JNK蛋白表达量(P<0.05或P<0.01)。由上述结果可知,CTS可以通过抑制NF-κB和丝裂原活化蛋白激酶(MAPK)信号通路转导来降低S.agalactiae诱导BMECs产生炎症反应,从而有效保护细胞,降低炎性损伤作用。

The aim of this study was to investigate the protective effect of chitosan(CTS)against Streptococcus agalactiae(S.agalactiae)-induced inflammatory injury of bovine mammary epithelial cells(BMECs)using an in vitro method.Firstly,the BMECs were treated with different concentrations(0,15.625,31.25,62.5,125,250,500 and 1000 mg/mL)of CTS for 12 and 24 hours,respectively.The BMECs viability was detected by methyl thiazoyl terazolium(MTT)assay,and the appropriate action time and action concentrations(31.25,125 and 250 mg/mL)were screened out according to BMECs viability for follow-up experiment.Then,a two-factor experiment design was adopted and divided into four groups with 4 replicates in each group.BMECs in control group were not treated with CTS and not stimulated by S.agalactiae,BMECs in S.agalactiae group were stimulated by S.agalactiae for 6 hours,BMECs in CTS groups were treated with CTS at different concentrations(31.25,125 and 250 mg/mL)for 24 hours,and BMECs in CTS(-)+sgc group were treated with CTS at different concentrations(31.25,125 and 250 mg/mL)for 24 hours,and then stimulated by S.agalactiae for 6 hours.The mRNA expression levels of interleukin(IL)-6,IL-1β,tumor necrosis factor-α(TNF-α),IL-8,Toll-like receptor 2(TLR2),myeloid differentiation factor 88(MyD88)、IL-1 receptor associated kinase 4(IRAK4),tumor necrosis factor receptor-associated factor 6(TRAF6)and transforming growth factor-β activated kinase 1(TAK1)in BMECs were detected by quantitative real-time PCR method,and the protein expression levels of nuclear transcription factor-κB(NF-κB)inhibitor-α(IκB-α),phosphorylated NF-κB-p65(p-NF-κB-p65),phosphorylated p38(p-p38),phosphorylated extracellular signal-regulated kinase 1/2(p-ERK1/2)and phosphorylated c-Jun N-terminal kinase(p-JNK)in BMECs were detected by Western blot method.The results showed as follows:1)CTS at 31.25 and 125 mg/mL significantly or extremely significantly decreased the mRNA expression levels of IL-6,IL-8,TLR2,MyD88,IRAK4 and TRAF6 in BMECs(P<0.05 or P<0.01);CTS at 31.25,125 and 250 mg/mL extremely significantly decreased the mRNA expression levels of IL-6,IL-1β,TNF-α,IL-8,TLR2,MyD88,IRAK4,TRAF6 and TAK1 in BMECs induced by S.agalactiae(P<0.01).2)CTS at 31.25,125 and 250 mg/mL significantly or extremely significantly reduced the protein levels of p-NF-κB-p65,p-p38,p-ERK1/2 and p-JNK in BMECs and BMECs induced by S.agalactiae(P<0.05 or P<0.01).It can be concluded that CTS can effectively protect cells and reduce inflammatory injury by inhibiting the NF-κB and mitogen activated protein kinase(MAPK)signaling pathways of BMECs and inhibiting S.agalactiae-induced inflammatory response.