CAJ | 학술논문

为探讨不同浓度黄芩苷对热应激条件下猪近端肾小管细胞(pig kidney proximal tubular cells, LLC-PK1)细胞凋亡率及B细胞淋巴瘤/白血病-2基因(B-cellymphoma-2, Bcl-2)和Bcl-2相关X蛋白基因(Bcl-2 associated X protein, Bax)表达的影响，将培养的LLC-PK1细胞随机分为7个组，Ⅰ组为37℃空白对照组，Ⅱ组为42℃单纯热应激1 h组，Ⅲ、Ⅳ、Ⅴ、Ⅵ和Ⅶ组分别为用不同浓度黄芩苷(0.01、0.1、1、10和100μg/mL)处理组后42℃热应激1 h组，运用实时荧光定量PCR和Western blot分别检测Bcl-2和Bax基因及蛋白的表达，流式细胞仪(Annexin V-FITC/PI双染法)检测细胞凋亡率。结果表明，与Ⅰ组相比，Ⅱ组能显著诱导LLC-PK1细胞Bcl-2 mRNA的表达(P＜0.05)，极显著诱导细胞Bax mRNA和蛋白的表达(P＜0.01)，能极显著降低细胞Bcl-2和Bax mRNA和蛋白的比值(P＜0.01)，极显著增加细胞凋亡率(P＜0.01)，但对细胞Bcl-2蛋白的表达无显著影响(P＞0.05)。黄芩苷处理组与Ⅱ组相比，Ⅳ、Ⅴ和Ⅵ组LLC-PK1细胞Bcl-2 mRNA的表达量均升高，其中Ⅴ组差异极显著(P＜0.01)，Ⅳ及Ⅵ组差异显著(P＜0.05)，Ⅲ及Ⅶ组差异不显著(P＞0.05)，而细胞Bcl-2蛋白的表达量与Ⅱ组相比均差异不显著(P＞0.05)；同样与Ⅱ组相比，黄芩苷处理的各组细胞Bax mRNA及蛋白的表达量均降低，其中Ⅴ及Ⅵ组差异极显著(P＜0.01)，其余各组差异显著(P＜0.05)；除Ⅲ组外，其他各组细胞Bcl-2和Bax mRNA及蛋白的比值与Ⅱ组相比均显著升高(P＜0.05)，其中Ⅴ组细胞Bcl-2和Bax蛋白的比值差异极显著(P＜0.01)；细胞凋亡率仅有Ⅲ组与Ⅱ组相比差异不显著(P＞0.05)，而Ⅴ及Ⅵ组细胞凋亡率极显著低于Ⅱ组(P＜0.01)，其余的Ⅳ和Ⅶ组显著低于Ⅱ组(P＜0.05)。一定浓度范围内的黄芩苷(0.1~100μg/mL)可能通过下调热应激条件下LLC-PK1细胞Bax的表达，从而提高Bcl-2和Bax的比值，降低细胞的凋亡率，对细胞起到保护作用。本研究从分子水平研究黄芩苷缓解热应激对猪LLC-PK1细胞的损害作用，可为明确其解热机制提供理论基础，并为其在临床上的应用提供有价值的参考资料。為探討不同濃度黃芩苷對熱應激條件下豬近耑腎小管細胞(pig kidney proximal tubular cells, LLC-PK1)細胞凋亡率及B細胞淋巴瘤/白血病-2基因(B-cellymphoma-2, Bcl-2)和Bcl-2相關X蛋白基因(Bcl-2 associated X protein, Bax)錶達的影響，將培養的LLC-PK1細胞隨機分為7箇組，Ⅰ組為37℃空白對照組，Ⅱ組為42℃單純熱應激1 h組，Ⅲ、Ⅳ、Ⅴ、Ⅵ和Ⅶ組分彆為用不同濃度黃芩苷(0.01、0.1、1、10和100μg/mL)處理組後42℃熱應激1 h組，運用實時熒光定量PCR和Western blot分彆檢測Bcl-2和Bax基因及蛋白的錶達，流式細胞儀(Annexin V-FITC/PI雙染法)檢測細胞凋亡率。結果錶明，與Ⅰ組相比，Ⅱ組能顯著誘導LLC-PK1細胞Bcl-2 mRNA的錶達(P＜0.05)，極顯著誘導細胞Bax mRNA和蛋白的錶達(P＜0.01)，能極顯著降低細胞Bcl-2和Bax mRNA和蛋白的比值(P＜0.01)，極顯著增加細胞凋亡率(P＜0.01)，但對細胞Bcl-2蛋白的錶達無顯著影響(P＞0.05)。黃芩苷處理組與Ⅱ組相比，Ⅳ、Ⅴ和Ⅵ組LLC-PK1細胞Bcl-2 mRNA的錶達量均升高，其中Ⅴ組差異極顯著(P＜0.01)，Ⅳ及Ⅵ組差異顯著(P＜0.05)，Ⅲ及Ⅶ組差異不顯著(P＞0.05)，而細胞Bcl-2蛋白的錶達量與Ⅱ組相比均差異不顯著(P＞0.05)；同樣與Ⅱ組相比，黃芩苷處理的各組細胞Bax mRNA及蛋白的錶達量均降低，其中Ⅴ及Ⅵ組差異極顯著(P＜0.01)，其餘各組差異顯著(P＜0.05)；除Ⅲ組外，其他各組細胞Bcl-2和Bax mRNA及蛋白的比值與Ⅱ組相比均顯著升高(P＜0.05)，其中Ⅴ組細胞Bcl-2和Bax蛋白的比值差異極顯著(P＜0.01)；細胞凋亡率僅有Ⅲ組與Ⅱ組相比差異不顯著(P＞0.05)，而Ⅴ及Ⅵ組細胞凋亡率極顯著低于Ⅱ組(P＜0.01)，其餘的Ⅳ和Ⅶ組顯著低于Ⅱ組(P＜0.05)。一定濃度範圍內的黃芩苷(0.1~100μg/mL)可能通過下調熱應激條件下LLC-PK1細胞Bax的錶達，從而提高Bcl-2和Bax的比值，降低細胞的凋亡率，對細胞起到保護作用。本研究從分子水平研究黃芩苷緩解熱應激對豬LLC-PK1細胞的損害作用，可為明確其解熱機製提供理論基礎，併為其在臨床上的應用提供有價值的參攷資料。
위탐토불동농도황금감대열응격조건하저근단신소관세포(pig kidney proximal tubular cells, LLC-PK1)세포조망솔급B세포림파류/백혈병-2기인(B-cellymphoma-2, Bcl-2)화Bcl-2상관X단백기인(Bcl-2 associated X protein, Bax)표체적영향，장배양적LLC-PK1세포수궤분위7개조，Ⅰ조위37℃공백대조조，Ⅱ조위42℃단순열응격1 h조，Ⅲ、Ⅳ、Ⅴ、Ⅵ화Ⅶ조분별위용불동농도황금감(0.01、0.1、1、10화100μg/mL)처리조후42℃열응격1 h조，운용실시형광정량PCR화Western blot분별검측Bcl-2화Bax기인급단백적표체，류식세포의(Annexin V-FITC/PI쌍염법)검측세포조망솔。결과표명，여Ⅰ조상비，Ⅱ조능현저유도LLC-PK1세포Bcl-2 mRNA적표체(P＜0.05)，겁현저유도세포Bax mRNA화단백적표체(P＜0.01)，능겁현저강저세포Bcl-2화Bax mRNA화단백적비치(P＜0.01)，겁현저증가세포조망솔(P＜0.01)，단대세포Bcl-2단백적표체무현저영향(P＞0.05)。황금감처리조여Ⅱ조상비，Ⅳ、Ⅴ화Ⅵ조LLC-PK1세포Bcl-2 mRNA적표체량균승고，기중Ⅴ조차이겁현저(P＜0.01)，Ⅳ급Ⅵ조차이현저(P＜0.05)，Ⅲ급Ⅶ조차이불현저(P＞0.05)，이세포Bcl-2단백적표체량여Ⅱ조상비균차이불현저(P＞0.05)；동양여Ⅱ조상비，황금감처리적각조세포Bax mRNA급단백적표체량균강저，기중Ⅴ급Ⅵ조차이겁현저(P＜0.01)，기여각조차이현저(P＜0.05)；제Ⅲ조외，기타각조세포Bcl-2화Bax mRNA급단백적비치여Ⅱ조상비균현저승고(P＜0.05)，기중Ⅴ조세포Bcl-2화Bax단백적비치차이겁현저(P＜0.01)；세포조망솔부유Ⅲ조여Ⅱ조상비차이불현저(P＞0.05)，이Ⅴ급Ⅵ조세포조망솔겁현저저우Ⅱ조(P＜0.01)，기여적Ⅳ화Ⅶ조현저저우Ⅱ조(P＜0.05)。일정농도범위내적황금감(0.1~100μg/mL)가능통과하조열응격조건하LLC-PK1세포Bax적표체，종이제고Bcl-2화Bax적비치，강저세포적조망솔，대세포기도보호작용。본연구종분자수평연구황금감완해열응격대저LLC-PK1세포적손해작용，가위명학기해열궤제제공이론기출，병위기재림상상적응용제공유개치적삼고자료。
The objective of this study is to explore baicalin influence B-cellymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) expression as well as apoptosis rates of heat-stressed pig kidney proximal tubular cells (LLC-PK1). Readily cultured LLC-PK1 cells were randomly divided into seven groups. The cells in groupⅠwere used as blank control cultured at 37℃and cells in groupⅡwere heart-stressed at 42℃for 1 h. Cells in groupsⅢ,Ⅳ,Ⅴ,ⅥandⅦwere cultured additionally with different concentrations (0.01, 0.1, 1, 10 and 100μg/mL) of baicalin at 42℃for 1 h, respectively. Expression of Bcl-2 mRNA and Bax protein were detected with Real-time PCR and Western blot, respectively. Apoptosis rates of LLC-PK1 cells were determined with Annexin V-FITC/PI. Results showed that as compared to groupⅠ, Bcl-2 mRNA in groupⅡincreased significantly (P<0.05), and Bax mRNA and its protein increased extremely significantly (P<0.01), and both ratios between Bcl-2 and Bax mRNA and their proteins decreased significantly (P<0.01), and the apoptosis rates increased extremely significantly(P<0.01). However, there was no evident influence on Bcl-2 protein expression (P>0.05). Compared with groupⅡ, Bcl-2 mRNA expression significantly increased in groupⅣand Ⅵ (P<0.05) and extremely significantly in groupⅤ(P<0.01), but not in groupⅢor Ⅶ (P>0.05), while the Bal-2 protein expression had no difference (P>0.05). Similarly, compared with groupⅡ, both Bax mRNA and its protein expression decreased significantly in groupsⅢ, Ⅳand Ⅶ (P<0.05) and decreased extremely significantly in groupⅤandⅥ(P<0.01). Ratios between Bcl-2 and Bax mRNA and their proteins in all groups except groupⅢhad a significant increase (P<0.05), while the ratio between Bcl-2 and Bax protein in groupⅤincreased extremely significantly (P<0.01). The apoptosis rates decreased extremely significantly in groupⅤ and Ⅵ (P<0.01), and decreased significantly in group Ⅳand Ⅶ(P<0.05). The results indicated that certain concentration (0.1~100 μg/mL) of baicalin protectsed heat-stressed LLC-PK cells by down-regulating Bax expression, increasing the ratio between Bcl-2 and Bax and decreasing apoptosis rates. This experiment reveals some heat-resistant mechanism of baicalin on LLC-PK1 cells exposed to heat stress which will provide valuable reference for its clinical application.