Abstract：
Total triterpenoids from the fruits of Chaenomeles speciosa(TCS) are active components in the prevention and treatment of gastric mucosal damage, which have potential anti-aging effects. However, it is still unclear whether TCS can improve gastric aging, especially its molecular mechanism against gastric aging. On this basis, this study explored the effect and mechanism of TCS on senescent GES-1 cells induced by D-galactose(D-gal) to provide scientific data for the clinical use of TCS to prevent gastric aging. GES-1 cells cultured in vitro and those transfected with overexpression GLS1(GLS1-OE) plasmid of glutaminase 1(GLS1) were induced to aging by D-gal, and then TCS and or GLS1 inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide(BPTES) were given. Cell survival rate, positive rate of β-galactosidase(SA-β-gal) staining, mitochondrial membrane potential(MMP), and apoptosis were investigated. GLS1 activity, levels of glutamine(Gln), glutamate(Glu), α-ketoglutarate(α-KG), urea, and ammonia in supernatant and cells were detected by enzyme-linked immunosorbent assay(ELISA) and colorimetric methods. The mRNA and protein expressions of GLS1 and the related genes of the mitochondrial apoptosis signaling pathway were measured by real-time fluorescence quantitative PCR and Western blot. The results manifested that compared with the D-gal model group and GLS1-OE D-gal model group, TCS significantly decreased the SA-β-gal staining positive cell rate and MMP of D-gal-induced senescent GES-1 cells and GLS1-OE senescent GES-1 cells, inhibited the survival of senescent cells, and promoted their apoptosis(P<0.01). It decreased the activity of GLS1 and the content of Gln, Glu, α-KG, urea, and ammonia in supernatant and cell(P<0.01), reduced the concentration of cytochrome C(Cyto C) in mitochondria and the mRNA and protein expressions of GLS1 and proliferating nuclear antigen in cells(P<0.01). The mRNA expression of Bcl-2 and Bcl-xl, the protein expression of pro-caspase-9 and pro-caspase-3, and the ratio of Bcl-2/Bax and Bcl-xl/Bad in cells were decreased(P<0.01). Cyto C concentration in the cytoplasm, the mRNA expressions of Bax, Bad, apoptosis protease activating factor 1(Apaf-1), and protein expressions of cleaved-caspase-9, cleaved-caspase-3, cleaved-PARP-1 were increased(P<0.01). The aforementioned results indicate that TCS can counteract the senescent GES-1 cells induced by D-gal, and its mechanism may be closely related to suppressing the Gln/GLS1/α-KG metabolic axis, activating the mitochondrial apoptosis pathway, and thereby accelerating the apoptosis of the senescent cells and eliminating senescent cells.
摘要：
木瓜（TCS）果实中的总三萜是预防和治疗胃粘膜损伤的活性成分，具有潜在的抗衰老作用。然而,目前尚不清楚TCS是否能改善胃老化，尤其是其抗胃衰老的分子机制。在此基础上,本研究探讨了TCS对D-半乳糖(D-gal)诱导的衰老GES-1细胞的作用及机制,为临床应用TCS预防胃衰老提供科学依据。用D-gal诱导体外培养的GES-1细胞和用谷氨酰胺酶1（GLS1）的过表达GLS1（GLS1-OE）质粒转染的GES-1细胞衰老，然后给予TCS和/或GLS1抑制剂双-2-(5-苯基乙酰氨基-1,3,4-噻二唑-2-基)乙基硫醚(BPTES)。细胞存活率,β-半乳糖苷酶(SA-β-gal)染色阳性率,线粒体膜电位（MMP），和细胞凋亡进行了研究。GLS1活性，谷氨酰胺（Gln）水平，谷氨酸(Glu),α-酮戊二酸(α-KG)尿素,酶联免疫吸附试验（ELISA）和比色法检测上清液和细胞中的氨。采用实时荧光定量PCR和Westernblot检测GLS1及线粒体凋亡信号通路相关基因的mRNA和蛋白表达。结果表明,与D-gal模型组和GLS1-OED-gal模型组相比,TCS显著降低了D-gal诱导的衰老GES-1细胞和GLS1-OE衰老GES-1细胞的SA-β-gal染色阳性细胞率和MMP,抑制衰老细胞的存活，并促进其凋亡（P＆lt;0.01）。它降低了GLS1的活性和Gln的含量，Glu,α-KG,尿素,上清液和细胞中的氨（P＆lt;0.01），降低线粒体中细胞色素C(CytoC)的浓度和细胞中GLS1和增殖核抗原的mRNA和蛋白表达(P<0.01)。Bcl-2和Bcl-xl的mRNA表达,细胞中pro-caspase-9和pro-caspase-3的蛋白表达以及Bcl-2/Bax和Bcl-xl/Bad的比例降低（P＆lt;0.01）。细胞质中的CytoC浓度，Bax的mRNA表达，糟糕，凋亡蛋白酶激活因子1（Apaf-1），cleaved-caspase-9、cleaved-caspase-3、cleaved-PARP-1蛋白表达增加(P<0.01)。上述结果表明,TCS可以对抗D-gal诱导的GES-1细胞衰老,其机制可能与抑制Gln/GLS1/α-KG代谢轴密切相关,激活线粒体凋亡途径，从而加速衰老细胞的凋亡并消除衰老细胞。
