内质网应激（endoplasmic reticulum stress, ERS）介导的未折叠蛋白反应（unfolded protein response, UPR）在多种疾病进程中扮演着举足轻重的角色，这些疾病包括但不限于代谢失调、恶性肿瘤（癌症）、神经退行性疾病、心血管病变以及生殖系统的功能异常等。活化转录因子6（activating transcription factor 6, ATF6）作为内质网应激的关键分子被广泛研究。因此，在深入探讨这些疾病的发生发展机制，并寻求有效治疗策略的过程中，ATF6的调控作用显得尤为关键和重要。

ATF6的下游基因RAS相关蛋白Rab-7a（ras-related protein Rab-7a, RAB7A）作为RAS癌基因超家族的一员，在细胞内囊泡转运的过程中扮演了举足轻重的角色。在本课题组的前期研究中，RAB7A在乙型肝炎病毒（hepatitis B virus, HBV）的生命周期中发挥着显著作用，进而影响了HBV在宿主体内的复制效率。此外，RAB7A对肝细胞癌（hepatocellular carcinoma, HCC）具有促进作用。RAB7A在肝脏疾病的代谢过程中通过多种机制发挥作用，包括调控脂肪降解、影响信号转导以及参与自噬过程等。众所周知，缺氧微环境是肝脏疾病中常见的一种病理状态，尤其是在慢性肝病、肝硬化以及肝脏肿瘤等疾病中。在这种环境下，肝细胞和其它细胞面临着氧气供应不足的挑战，导致细胞代谢和功能发生一系列的变化。因此，RAB7A与肝脏疾病缺氧微环境下的物质代谢之间存在着密切的关联。

为了探究RAB7A在缺氧环境下如何调控HepG2细胞糖代谢的机制，首先使用氯化钴化学诱导法构建缺氧细胞模型，通过检测乳酸和ATP的含量以及PAS染色实验，检测并观察到在缺氧的条件下，RAB7A增加了HepG2细胞的乳酸生成、ATP生成和糖原含量。通过RT-qPCR和Western Blot实验，发现RAB7A能够上调参与葡萄糖代谢的关键酶，尤其是醛缩酶A（aldolase A, ALDOA）的表达水平。接下来，通过Co-IP实验确定了RAB7A与缺氧诱导因子1-α（hypoxia inducible factor 1 subunit alpha, HIF1A）之间的相互作用。然而，尽管RAB7A与HIF1A在功能上有所关联，但通过RT-qPCR和Western Blot实验发现它们的表达水平并不相互影响。在过表达HIF1A的条件下，通过双荧光素酶报告基因实验，发现过表达/敲降RAB7A后，ALDOA的启动子活性无显著性变化，这进一步说明了HIF1A起着更为主要的调控作用。此外，通过双荧光素酶报告基因实验、凝胶电泳迁移率实验（EMSA）以及染色质免疫沉淀实验（ChIP），发现HIF1A能够与ALDOA的启动子结合，更进一步的ChIP实验发现在缺氧条件下RAB7A能够与HIF1A蛋白结合，进而促进HIF1A的转录因子活性。这种活性的提升可能使得HIF1A更加有效地与ALDOA的启动子结合，从而上调ALDOA的表达。ALDOA作为糖酵解和糖生成过程中的关键酶，其表达和功能的提升进一步推动了糖代谢的进行。

男性不育是临床上常见的复杂疾病，其成因多种多样。雄性生殖器官如果发育不良，或者生殖细胞发生障碍，都可能使其失去原有的正常生殖功能，进而导致不育现象的发生。生殖细胞易受到胞内或胞外刺激并诱发ERS和UPR反应，这会对睾丸和精子造成损害，导致不育。

为了针对ERS过程的关键分子ATF6，探索ATF6调控生殖相关基因热休克蛋白A1L（heat shock protein family A member 1 like, HSPA1L）的分子机制，首先基于雄性ATF6敲除小鼠睾丸组织转录物组测序信息，筛选ATF6下游5个生殖相关基因；在HEK-293T细胞中转染ATF6过表达质粒，通过RT-qPCR和Western Blot验证过表达效率；通过双荧光素酶报告基因实验选择启动子活性较高的下游基因并检测过表达ATF6对其启动子活性的影响；通过Gene-Regulation预测ATF6和下游基因启动子可能的结合位点；RT-qPCR和Western Blot检测在HEK-293T细胞中过表达ATF6对于下游基因表达的影响；利用EMSA确定ATF6与下游基因启动子是否结合。实验结果表明，转染后HEK-293T细胞中ATF6的mRNA和蛋白表达显著增高。转录物组测序及双荧光素酶报告基因实验筛选出ATF6下游的生殖相关基因HSPA1L。HSPA1L也称附睾分泌性精子结合蛋白，属于分子伴侣家族中的一员，在雄性生殖细胞发育和精子成熟过程中发挥着重要作用，参与细胞内蛋白质折叠和修复的过程。本研究表明，ATF6能够促进HSPA1L的截短启动子活性。过表达ATF6后，HSPA1L的表达量明显升高。ATF6蛋白可能与HSPA1L的启动子DNA序列aagtcgtcac相结合。

综上所述，在HepG2细胞中初步揭示了ATF6下游基因RAB7A在缺氧条件下增强葡萄糖代谢的分子机制，这一发现不仅有助于深入理解细胞在缺氧环境下的代谢适应机制，还为未来针对RAB7A在基于缺氧条件下葡萄糖代谢的肝病中的相关研究提供了一个潜在的新方向。另外，内质网应激的关键分子ATF6可能通过与生殖相关的基因HSPA1L的启动子结合，进而调控该基因的表达水平。这一发现对于未来关于内质网应激（ERS）相关男性不育症的预防和治疗研究具有深远的潜在影响，为这一领域的深入探索奠定了坚实的基础。

The unfolded protein response (UPR) mediated by endoplasmic reticulum stress (ERS) plays an important role in the process of a variety of diseases, including but not limited to metabolic disorders, malignant tumors (cancer), neurodegenerative diseases, cardiovascular diseases and reproductive system dysfunction. ATF6 as a key molecule of endoplasmic reticulum stress has been widely studied. Therefore, the regulatory role of ATF6 is particularly critical and important in the process of exploring the occurrence and development mechanism of these diseases and seeking effective treatment strategies.

As a member of RAS oncogene superfamily, ras-related protein Rab-7a (RAB7A) , a downstream gene of ATF6, plays an important role in the process of vesicle transport in cells. In the previous study of our research group, RAB7A plays a significant role in the life cycle of hepatitis B virus (HBV), and then affects the replication efficiency of HBV in the host. In addition, RAB7A has a promoting effect on hepatocellular carcinoma (HCC). RAB7A plays a role in the metabolic process of liver diseases through a variety of mechanisms, including regulating fat degradation, affecting signal transduction, and participating in autophagy. As we all know, hypoxic microenvironment is a common pathological state in liver diseases, especially in chronic liver diseases, liver cirrhosis and liver tumors. In this environment, hepatocytes and other cells are facing the challenge of insufficient oxygen supply, leading to a series of changes in cell metabolism and function. Therefore, there is a close relationship between RAB7A and material metabolism in the hypoxic microenvironment of liver diseases.

In order to explore the mechanism of how RAB7A regulates glucose metabolism in HepG2 cells under hypoxic environment, the hypoxic cell model was constructed by chemical induction with cobalt chloride. By detecting the content of lactic acid and ATP and PAS staining, it was observed that RAB7A increased the production of lactic acid, ATP and glycogen in HepG2 cells under hypoxic conditions. Through RT-qPCR and Western Blot, it was found that RAB7A could up regulate the expression of key enzymes involved in glucose metabolism, especially aldolase A (ALDOA). Next, RAB7A and hypoxia inducible factor 1-alpha (HIF1A) were determined by Co-IP experiment. However, although RAB7A and HIF1A are functionally related, RT-qPCR and Western Blot showed that their expression levels do not affect with each other. Under the condition of overexpression of HIF1A, through the dual luciferase reporter gene assay, it was found that the promoter activity of ALDOA had no significant change after overexpression/knockdown of RAB7A, which further indicated that HIF1A played a more important regulatory role. In addition, through the dual luciferase reporter gene assay, EMSA and ChIP experiments, it was found that HIF1A could bind to the promoter of ALDOA. Further ChIP experiments found that RAB7A could bind to HIF1A protein under hypoxia conditions, thereby promoting the transcription factor activity of HIF1A. This increase in activity enables HIF1A to more effectively bind to the promoter of ALDOA, thereby up regulating the expression of ALDOA. ALDOA is a key enzyme in the process of glycolysis and glycogenesis. The improvement of its expression and function further promotes the progress of glucose metabolism.

Male infertility is a common and complex disease in clinic, and its causes are various. If the male reproductive organs are stunted or the germ cells are blocked, they may lose their original normal reproductive function, and then lead to infertility. Germ cells are sensitive to intracellular or extracellular stimulation and induce ERS and UPR which can damage testis and sperm and lead to infertility.

In order to explore the molecular mechanism of ATF6, which is the key molecule of ERS, regulating the reproductive related gene heat shock protein family A member 1 like (HSPA1L). Firstly, using the transcriptome sequencing information of testis tissue of male ATF6 knockout mice, five reproductive related genes downstream of ATF6 were screened. The ATF6 overexpression plasmid was transfected into HEK-293T cells, and the overexpression efficiency was verified by RT-qPCR and Western Blot. The downstream genes with high promoter activity were selected by dual luciferase reporter gene assay and the effect of overexpression of ATF6 on its promoter activity was detected. The possible binding sites of ATF6 and downstream gene promoters were predicted by Gene-Regulation. RT-qPCR and Western Blot were used to detect the effect of overexpression of ATF6 on downstream gene expression in HEK-293T cells. EMSA was used to determine whether ATF6 binds to downstream gene promoters. The results showed that the levels of ATF6 gene expression, both at the mRNA and protein levels, were significantly enhanced in HEK-293T cells post-transfection. HSPA1L, a reproductive related gene downstream of ATF6, was screened by transcriptome sequencing and dual luciferase reporter gene assay. HSPA1L, also known as epididymal secretory sperm binding protein, is a member of the molecular chaperone family. It plays an important role in the development of male germ cells and sperm maturation, and participates in the process of protein folding and repair in cells. This study shows that ATF6 could promote the truncated promoter activity of HSPA1L . After overexpression of ATF6, the expression of HSPA1L was significantly increased . ATF6 protein can bind to the promoter DNA sequence aagtcgtcac of HSPA1L.

In conclusion, the molecular mechanism by which RAB7A, a downstream gene of ATF6, enhances glucose metabolism under hypoxia was initially revealed in HepG2 cells. This finding not only contributes to the in-depth understanding of the metabolic adaptation mechanism of cells in hypoxic environment, but also provides a potential direction for the future research on RAB7A in liver diseases based on glucose metabolism under hypoxic conditions. Additionally, ATF6, the key molecule of endoplasmic reticulum stress, regulates the expression level of HSPA1L gene by binding to the promoter of the reproduction related gene. This finding has far-reaching potential impact on the prevention and treatment of endoplasmic reticulum stress (ERS) - related male infertility in the future, and has laid a solid foundation for further exploration in this field.