谷超 趙璐璐 李剛 高原 王勝男 李曉佳 元曉榮 王琪雯 包勒朝魯 韓瑞蘭

中圖分類(lèi)號(hào) R692.5;R589.5;R965 文獻(xiàn)標(biāo)志碼 A 文章編號(hào) 1001-0408（2021）20-2458-09

DOI 10.6039/j.issn.1001-0408.2021.20.05

摘 要 目的：探討氯化鑭對(duì)高磷致血管平滑肌細(xì)胞（VSMCs）鈣化的抑制作用及其機(jī)制。方法：在MTT法篩選氯化鑭作用濃度及時(shí)間的基礎(chǔ)上，將人VSMCs分為對(duì)照組（1 mmol/L磷溶液）、氯化鑭高濃度對(duì)照組（1 mmol/L磷溶液+60 μmol/L氯化鑭）、模型組（3 mmol/L磷溶液）、氯化鈉組（3 mmol/L磷溶液+180 μmol/L氯化鈉）、核因子κB（NF-κB）信號(hào)通路激動(dòng)劑+氯化鑭組（3 mmol/L磷溶液+1 μg/mL脂多糖+60 μmol/L氯化鑭）、陽(yáng)性對(duì)照組（3 mmol/L磷溶液+100 μmol/L焦磷酸鈉）和氯化鑭低、中、高濃度組（3 mmol/L磷溶液+15、30、60 μmol/L氯化鑭），采用茜素紅S染色法和Von Kossa染色法檢測(cè)經(jīng)磷溶液作用6 d、相應(yīng)藥物作用2 d后各組細(xì)胞的鈣化情況，采用Western blot法檢測(cè)細(xì)胞中腫瘤壞死因子受體相關(guān)蛋白6（TRAF6）、核因子κB抑制蛋白α（IκBα）、NF-κB p65、骨形態(tài)發(fā)生蛋白2（BMP-2）、平滑肌22α（SM22α）、Runt相關(guān)轉(zhuǎn)錄因子2（Runx2）蛋白的表達(dá)水平，采用實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)法檢測(cè)細(xì)胞中TRAF6、IκBα、BMP-2、SM22α、Runx2 mRNA的表達(dá)水平。結(jié)果：與對(duì)照組比較，氯化鑭高濃度對(duì)照組細(xì)胞沒(méi)有出現(xiàn)鈣化，模型組和氯化鈉組細(xì)胞均出現(xiàn)明顯鈣化且光密度（OD）值均顯著升高（P<0.01），細(xì)胞質(zhì)中TRAF6、BMP-2蛋白及其mRNA的表達(dá)水平和Runx2 mRNA的表達(dá)水平以及細(xì)胞核中NF-κB p65、Runx2蛋白的表達(dá)水平均顯著升高（P<0.01），細(xì)胞質(zhì)中IκBα、SM22α蛋白及其mRNA的表達(dá)水平和NF-κB p65蛋白的表達(dá)水平均顯著降低（P<0.01）。與模型組比較，氯化鑭各濃度組和陽(yáng)性對(duì)照組細(xì)胞的鈣化均明顯改善，OD值均顯著降低，上述蛋白及mRNA的表達(dá)水平均不同程度逆轉(zhuǎn)（P<0.05或P<0.01）。與氯化鑭高濃度組比較，NF-κB信號(hào)通路激動(dòng)劑+氯化鑭組細(xì)胞出現(xiàn)明顯鈣化，OD值顯著升高，細(xì)胞質(zhì)以及細(xì)胞核中上述指標(biāo)均顯著逆轉(zhuǎn)（P<0.05或P<0.01）。結(jié)論：氯化鑭可抑制高磷誘導(dǎo)的VSMCs鈣化，其機(jī)制可能與抑制NF-κB信號(hào)通路活化有關(guān)。

關(guān)鍵詞 氯化鑭;高磷;血管鈣化;核因子κB信號(hào)通路;人血管平滑肌細(xì)胞

Discussion on the Inhibitory Mechanism of Lanthanum Chloride on Vascular Smooth Muscle Cell Calcification Induced by High Phosphorus Based on NF-κB Signaling Pathway

GU Chao1，ZHAO Lulu1，LI Gang1，GAO Yuan1，WANG Shengnan1，LI Xiaojia1，YUAN Xiaorong1，WANG Qiwen1，Baolechaolu2，HAN Ruilan1（1. School of Pharmacy， Inner Mongolia Medical University， Hohhot 010110， China; 2. Collaborative Innovation Center of Mongolian Medicine，Inner Mongolia Medical University， Hohhot 010110， China）

ABSTRACT? ?OBJECTIVE： To discuss the inhibitory effect of lanthanum chloride on the calcification of vascular smooth muscle cells （VSMCs） induced by high phosphorus and its mechanism. METHODS： On the basis of screening the action concentration and time of lanthanum chloride by MTT method， human VSMCs were divided into control group （1 mmol/L phosphorus solution）， lanthanum chloride high concentration control group （1 mmol/L phosphorus solution+60 μmol/L lanthanum chloride）， model group （3 mmol/L phosphorus solution）， sodium chloride group （3 mmol/L phosphorus solution+180 μmol/L sodium chloride）， nuclear factor κB （NF-κB） signaling pathway agonist+lanthanum chloride group （3 mmol/L phosphorus solution+1 μg/mL lipopolysaccharide+60 μmol/L lanthanum chloride）， positive control group （3 mmol/L phosphorus solution+100 μmol/L sodium pyrophosphate）， and lanthanum chloride low， medium， and high concentration groups （3 mmol/L phosphorus solution+15， 30， 60 μmol/L lanthanum chloride）. Alizarin red S staining and Von Kossa staining were used to detect cell calcification in each group after treated with phosphorus solution for 6 d and relevant medicine for 2 d. Western blot assay was used to detect the protein expression of TNF-α receptor associated protein 6 （TRAF6）， nuclear factor κB inhibitor protein α （IκBα）， NF-κB p65， bone morphogenetic protein 2 （BMP-2）， smooth muscle 22 α （SM22α） and Runt related transcription factor 2 （Runx2）. Real-time fluorescence quantitative polymerase chain reaction was used to detect mRNA expression of TRAF6， IκBα， BMP-2， SM22α and Runx2. RESULTS： Compared with control group， no cell calcification was observed in the lanthanum chloride high concentration control group， while obvious cell calcification and significant increase of OD value were observed in model group and sodium chloride group （P<0.01）; protein and mRNA expression of TRAF6 and BMP-2 in cytoplasm as well as mRNA expression of Runx2， protein expression of NF-κB p65 and Runx2 in nucleus were significantly increased （P<0.01）; protein and mRNA expression of IκBα and SM22α as well as protein expression of NF-κB p65 in cytoplasm were significantly decreased （P<0.01）. Compared with model group， cell calcification was significantly improved in lanthanum chloride groups and positive control group， while OD values were significantly reduced; the expression levels of the above-mentioned protein and mRNA were reversed to varying degrees （P<0.05 or P<0.01）. Compared with lanthanum chloride high concentration group， obvious cell calcification was observed in NF-κB signaling pathway agonist+lanthanum chloride group， and OD value was significantly increased; the above indexes were significantly reversed in cytoplasm and nucleus （P<0.05 or P<0.01）. CONCLUSIONS： Lanthanum chloride can inhibit the calcification of VSMCs induced by high phosphorus， and its mechanism may be related to the inhibition of NF-κB signaling pathway activation.