疏肝祛瘀解毒方介导p53通路诱导铁死亡抑制裸鼠肝癌生长的研究

Decoction for soothing liver and removing stasis and toxicity inhibits he⁃patocellular carcinoma proliferation in nude mice by inducing ferropto⁃sis via p53 pathway

目的:基于肿瘤蛋白53(tumor protein 53,p53)/溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11/xCT)/谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)通路探讨疏肝祛瘀解毒方(SGQYJDF)诱导铁死亡抑制裸鼠肝癌增殖。方法:通过将人肝癌细胞株SK-Hep-1注射于裸鼠右腋皮下,建立异种皮下移植瘤模型,成模后分为模型对照组、SGQYJDF低、中、高剂量组和SGQYJDF中剂量联合Sorafenib组,按分组连续灌胃14 d,期间观察裸鼠肿瘤体积;灌胃14 d后,测量肿瘤质量并计算生长抑制率,采用HE染色观察各组肿瘤组织病理学变化;比色法检测各组肿瘤组织内丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)和亚铁离子(ferrous ions,Fe^(2+))水平;免疫组织化学法(immunohistochemistry,IHC)检测各组肿瘤组织内增殖标志物(Kiel 67 antigen,Ki-67)及GPX4表达;免疫荧光法(immunofluorescence,IF)检测肿瘤组织内p53和xCT的表达;Western blot法检测p53、xCT和GPX4的表达水平。结果:(1)在肿瘤增殖方面,通过观察裸鼠肿瘤体积及质量并计算生长抑制率,与模型对照组比较,SGQYJDF能够呈剂量相关性的降低肿瘤体积(P<0.01),抑制肿瘤质量增长(P<0.01),各组肿瘤组织均未见明显浸润,同时通过IHC观察计算肿瘤组织内Ki-67阳性细胞率可知,与模型对照组比较,SGQYJDF能够呈剂量相关性降低肿瘤组织Ki-67阳性细胞率(P<0.01),抑制其增殖能力;(2)在铁死亡生化指标方面,通过检测计算相对含量,与模型对照组比较,SGQYJDF能够呈剂量依赖性提高肿瘤组织内Fe^(2+)含量和MDA含量,同时降低GSH的含量(P<0.01);(3)在蛋白表达方面,通过IHC、IF与Western blot实验,与模型对照组比较,SGQYJDF能够呈剂量相关性地上调p53(P<0.05)的表达,同时抑制xCT(P<0.05)和GPX4(P<0.01)的表达。此外,在实验结果中,我们发现人体等效量的SGQYJDF与Sorafenib联用,其效应高于两倍人体等效量的SGQYJDF。结论:提示SGQYJDF可能通过上调裸鼠皮下移植瘤p53的表达、抑制xCT和GPX4的表达从而诱导其发生铁死亡,抑制其增殖。

AIM:To investigate the inhibitory effect of the“decoction for soothing liver and removing stasis and toxicity(SGQYJDF)”on hepatocellular carcinoma(HCC)proliferation in nude mice by inducing ferroptosis via the tumor protein 53(p53)/solute carrier family 7 member 11(SLC7A11/xCT)/glutathione peroxidase 4(GPX4)pathway.METHODS:An ectopic subcutaneous tumor model was established by injecting SK-Hep-1 cells subcutaneously into the right axilla of nude mice.Upon formation of tumor,the mice were randomly divided into five groups(i.e.,control group,low-,medium-and high-dose SGQYJDF groups and medium-dose SGQYJDF plus Sorafenib group).Each group of mice was orally administered with the corresponding therapy for 14 consecutive days,during which the tumor size was observed regularly.At the end of treatment,the tumor growth inhibition rate was calculated based on tumor mass,and histopathological changes were observed by HE staining.Then,the levels of malondialdehyde(MDA),glutathione(GSH)and ferrous ions(Fe^(2+))were detected by colorimetric assays.The expression of the proliferation markers Ki-67 and GPX4 was detected by immunohistochemistry(IHC).The expression of p53 and xCT was detected by Immunofluorescence(IF).And the expression of p53,xCT and GPX4 was determined by Western blot.RESULTS:(1)SGQYJDF was found to dose-dependently decrease tumor volume(P<0.01)and inhibit tumor mass growth(P<0.01),and meanwhile,reduce the percentage of Ki-67-positive cells(P<0.01)and their proliferation ability in tumor tissues,as compared to the control group.(2)In terms of Ferroptosis-related indicators,SGQYJDF was found to dose-dependently increase the levels of Fe^(2+)and MDA but decrease the level of GSH in tumor tissues(P<0.01),as compared to the control group.(3)In terms of protein expression,SGQYJDF was found to dose-dependently upregulate the expression of p53(P<0.05)but inhibit the expression of xCT(P<0.05)and GPX4(P<0.01).Notably,medium-dose SGQYJDF plus sorafenib showed a stronger effect than high-dose SGQYJDF.CONCLUSION:Our findings suggest that SGQYJDF can induce Ferroptosis to inhibit the proliferation of subcutaneously transplanted tumor tissues in nude mice by upregulating the expression of p53,and inhibiting the expression of xCT and GPX4.