葛根素对银屑病细胞模型HaCaT细胞增殖和炎症因子影响

Effect of Puerarin on proliferation and inflammatory factors in KGF-induced psoriasis HaCaT cell

目的 探究葛根素(PR)对角质形成细胞生长因子(KGF)诱导的HaCaT细胞银屑病模型的影响及其作用机制。方法 使用40 ng·mL^(-1)的KGF处理细胞0、12、24、48、72 h后使用细胞计数-8(CCK-8)实验检测细胞光密度(OD)值筛选最佳作用时间。然后将细胞分为Control(正常培养)、KGF(KGF)、KGF+PR-L(KGF+3 mg·mL^(-1)PR处理)、KGF+PR-H(KGF+6 mg·mL^(-1)PR处理)、KGF+PR-H+pcDNA-NC(KGF+6 mg·mL^(-1)PR处理,并转染pcDNA-NC)、KGF+PR-H+pcDNA-NLRP3(KGF+6 mg·mL^(-1)PR处理,并转染pcDNA-NLRP3)组。用CCK-8检测细胞增殖能力,用平板克隆实验检测细胞克隆形成能力,用流式细胞术检测细胞周期进程,用蛋白质印迹(Western blot)法检测Nod样受体热蛋白结构域相关蛋白3(NLRP3)、增殖细胞核抗原(PCNA)、细胞周期素D1(Cyclin D1)、周期素依赖性激酶2(CDK2)蛋白表达情况,用酶联免疫吸附(EILSA)实验检测各组细胞白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、转化生长因子-β1 (TGF-β1)、白细胞介素-17(IL-17)炎症因子表达情况。结果 Control、KGF、KGF+PR-L、KGF+PR-H、KGF+PR-H+pcDNA-NC、KGF+PR-H+pcDNA-NLRP3组细胞存活率分别为(100.00±2.79)%,(157.95±5.14)%,(137.90±4.85)%,(114.50±9.30)%,(113.94±9.09)%和(141.91±5.72)%;克隆数目分别为(205.50±14.84),(341.83±21.37),(297.17±20.05),(237.50±11.07),(234.17±16.06)和(307.67±15.16)个;G0/G1期细胞比例分别为(55.25±2.02)%,(51.00±2.04)%,(57.95±2.45)%,(71.50±3.23)%,(70.11±2.77)%和(51.98±2.59)%;IL-17含量分别为(58.80±5.35),(209.85±15.83),(140.34±10.26),(86.69±3.35),(86.65±3.04)和(193.45±5.25)pg·mL^(-1)。以上指标,Control与KGF组比较,KGF+PR-L、KGF+PR-H组与KGF组比较,KGF+PR-L与KGF+PR-H组比较,KGF+PR-H+pcDNA-NC组与KGF+PR-H+pcDNA-NLRP3组比较,差异均有统计学意义(均P<0.05)。结论 葛根素可通过抑制NLRP3的表达,抑制HaCaT细胞增殖能力和炎症反应,并诱导细胞周期阻滞。

Objective To investigate the effect of Puerarin(PR) on keratinocyte growth factor(KGF)-induced psoriasis model of HaCaT cells and its mechanism. Methods Cells were treated with 40 ng· mL^(-1)KGF for 0, 12, 24, 48 and 72 h. Cell counting kit-8 ( CCK-8) was used to detect the optical density( OD) value of cells and screen the optimal treatment time. Then the cells were divided into control( normal culture),KGF( KGF),KGF + PR-L( KGF + 3 mg·mL^(-1)PR treatment),KGF + PR-H( KGF + 6 mg · mL^(-1)PR treatment),KGF + PR-H + pc DNA-NC( KGF + 6 mg · mL^(-1)PR treatment,and transfected with pc DNA-NC),KGF + PR-H + pc DNA-NLRP3( KGF + 6 mg·mL^(-1)PR treatment,and transfected with pc DNA-NLRP3) groups. CCK-8 was used to detect cell proliferation;plate cloning assay was used to detect cell clone formation. Flow cytometry was used to detect cell cycle progression. Western blotting was used to detect the protein expressions of Nod-like receptor thermoprotein dome-associated protein 3( NLRP3),proliferating cell nuclear antigen( PCNA),Cyclin D1 and Cyclin dependent kinase 2( CDK2). Enzyme-liked immunosorbent assay( ELISA) was used to detect interleukin-6( IL-6),interleukin-1β( IL-1β),tumor necrosis factor α( TNF-α) and transforming growth factor-β1( TGF-β1) and interleukin-17( IL-17). Results The cell viabilities of control,KGF,KGF + PR-L,KGF + PR-H,KGF + PR-H + pc DNA-NC,KGF + PR-H +pc DNA-NLRP3 groups were( 100. 00 ± 2. 79) %,( 157. 95 ± 5. 14) %,( 137. 90 ± 4. 85) %,( 114. 50 ± 9. 30) %,( 113. 94 ±9. 09) % and( 141. 91 ± 5. 72) %;the clone numbers were 205. 50 ± 14. 84,341. 83 ± 21. 37,297. 17 ±20. 05,237. 50 ± 11. 07,234. 17 ± 16. 06 and 307. 67 ± 15. 16;the percentages of cells in G0/G1 phase were( 55. 25 ± 2. 02) %,( 51. 00 ±2. 04) %,( 57. 95 ±2. 45) %,( 71. 50 ±3. 23) %,( 70. 11 ±2. 77) % and( 51. 98 ±2. 59) %;IL-17 contents were( 58. 80 ± 5. 35),( 209. 85 ± 15. 83),( 140. 34 ± 10. 26),( 86. 69 ±3. 35),( 86. 65 ± 3. 04) and( 193. 45 ± 5. 25)pg· mL^(-1). Compared control group with KGF group,KGF + PR-L,KGF + PR-H group compared with KGF group,KGF + PR-L group compared with KGF + PR-H group,KGF + PR-H + pc DNA-NC group compared with KGF +PR-H + pc DNA-NLRP3 group,the difference of the above indexes were statistically significant( all P < 0. 05).Conclusion Puerarin can alleviate the development of psoriasis by inhibiting the expression of NLRP3,inhibiting the proliferation of HaCaT cells and inflammatory response,and inducing cell cycle arrest.