Study on the Effect and Mechanism of Gynostemma pentaphyllum Saponins Regulating Long Non coding RNA TUG1/miR-26a Interference with Mitochondrial Apoptosis on Liver Lipid Deposition in ApoE -/- AS Mice
Atherosclerosis (AS) is the main pathological basis in the early stage of cardiovascular and cerebrovascular diseases. The liver is one of the most vulnerable organs in the initiation and development of AS and its related diseases. Its lipid deposition level directly or indirectly reflects the lipid deposition in the artery intima, and can be used as an indicator to predict the level of AS. The majority of the human genome consists of non coding regions, which are transcribed to produce non coding RNAs (ncRNAs). MicroRNAs (miRNAs) and long non coding RNAs (LncRNAs) are two important classifications of ncRNAs. Among them, LncRNA regulates gene expression at different levels through various mechanisms, exerting its biological functions. In recent years, the molecular mechanism of the interaction between miRNA and LncRNA in the occurrence and development of diseases has attracted people’s attention. Taurine upregulates gene 1 (TUG1) is an evolutionarily highly conserved long non coding RNA. Studies have found that TUG1 is related to the occurrence and development of esophageal cancer, gastric cancer, liver cancer, lung cancer and other cancers, but the relationship between TUG1 and liver lipid deposition and atherosclerosis is still rare. Research has found that interfering with the long non coding RNA TUG1 can alleviate LPS induced mitochondrial damage, cell apoptosis, and inflammatory response by upregulating miR-26a.

Gynostemma pentaphyllum (GP) is a perennial herbaceous vine of the genus Gynostemma. Its name first appeared in the Ming Dynasty’s “Saving the Wasteland Materia Medica”. It has the effects of nourishing qi and spleen, resolving phlegm and cough, clearing heat and detoxifying. It was once listed as a “precious Chinese herbal medicine” to be developed under the “Spark Program” of the Ministry of Science and Technology, and was included in the list of health products by the Ministry of Health in 2002. Since the 1970s, people have systematically studied the chemical composition and pharmacological effects of Gynostemma pentaphyllum. Research has found that its main medicinal ingredient is gypenosides (GPs). GPs exert anti AS effects through lipid-lowering, antiplatelet aggregation, and anti thrombotic effects. In the early stage, the research group induced mitochondrial membrane potential damage and decreased enzyme activity of respiratory chain complexes I, II, III, IV, and V in vascular endothelial cells using ox LDL. It was also clarified that the active ingredients of GP, such as GPs, gypenoside XILX, and ginsenoside GRb3, can increase mitochondrial membrane potential; Affects mitochondrial energy metabolism related proteins; Upregulation of ox LDL induces autophagy in endothelial cells, reduces endothelial cell damage, and exerts a protective effect on ox LDL induced endothelial cells. Based on previous research, this article focuses on whether GPs improve liver lipid deposition in ApoE -/- AS mice by interfering with mitochondrial apoptosis through the long non coding RNA TUG1/miR-26a, thereby preventing and treating AS.

With the significant improvement of people’s living standards, the diet structure has also changed. Under the factors of high cholesterol diet and high intensity work pressure, the incidence rate of cardiovascular disease has gradually increased. AS is the most important death factor of cardiovascular disease, which is of great significance for its prevention and treatment. GP series of perennial herbaceous vines belonging to the family Cucurbitaceae and the genus Gynostemma. The whole plant of Gynostemma pentaphyllum is used as medicine, with a cool nature, bitter taste, and slight sweetness. It belongs to the lung, spleen, and kidney meridians, and has the effects of clearing heat and detoxifying, stopping cough, clearing lungs, eliminating phlegm, nourishing the heart and calming the mind, replenishing qi and generating essence. GPs are a group of active ingredients extracted from GP, containing over 80 types of ginsenosides. The glycoside components of all GPs are dammarane type tetracyclic triterpenoids, which have significant effects on the treatment and prevention of cardiovascular diseases such as AS.

Long non coding ribonucleic acid (LncRNA) is a type of functional RNA molecule with a length exceeding 200 nt, which does not have protein coding function and was initially considered as “noise” in the RNA transcription process. However, recent studies have found that LncRNAs are involved in the occurrence and development of various human diseases. It is worth noting that LncRNA also has differential expression in cardiovascular diseases and plays a role in the regulatory network of cardiovascular diseases through different mechanisms, participating in the occurrence and development of cardiovascular diseases. Previous studies have shown that miRNA inhibits mRNA translation or promotes mRNA degradation by binding to complementary sequences on target mRNA, while LncRNA can act as a miRNA sponge to interact with miRNA, affecting mRNA and regulating gene expression. Research has found that under hypoxia/reoxygenation conditions, LncRNA AK088388 regulates autophagy in cardiomyocytes by acting as an endogenous RNA sponge for miR-30a. LncRNA Mexis is an amplifier of ATP binding cassette transporter A1 (ABCA1) gene that relies on LXR transcription, and ABCA1 protein can pump cholesterol out of arterial wall cells, which is crucial for the formation of high-density lipoprotein (HDL) and regulation of cholesterol efflux. It can be seen that the involvement of LncRNA in lipid metabolism and the mechanism of AS occurrence and development cannot be ignored. TUG1 is a highly conserved long non coding RNA in evolution. Current research has shown that TUG1 is not only closely related to the occurrence and development of cancers such as esophageal cancer, gastric cancer, liver cancer, and lung cancer, but also the interaction between LncRNA TUG1 and miR-138-5p may be involved in the pathogenesis and development of chronic heart failure. Furthermore, upregulation of miR-26a can alleviate LPS induced mitochondrial damage and cell apoptosis. Research has found that miRNA-26 plays an important role in cardiovascular disease, with downregulation of its expression in heart disease. miRNA-26 plays a key role in myocardial hypertrophy, with downregulation of miRNA-26a/b expression in both rat models and myocardial cells. The preliminary research of the research group found that the mitochondrial apoptosis pathway plays an important role in the occurrence and development of AS, and GPs have a regulatory effect in this process. Based on the typical pathological characteristics of atherosclerosis in ApoE -/- mice fed with high-fat diet for 12 weeks, this study selected the classic model of atherosclerosis (ApoE -/- C57BL/6J mice) as the main research object, and C57BL/6J mice as the normal control. The focus was on whether GPs improve hepatic lipid deposition in ApoE -/- AS mice by interfering with mitochondrial apoptosis through long non coding RNA TUG1/miR-26a, and thus prevent and treat atherosclerosis.

Research has found that GPs can improve blood lipid levels and liver lipid deposition in ApoE -/- AS mice, consistent with previous studies. On this basis, it was first found that the long non coding RNA TUG1 in ApoE -/- AS mouse liver was significantly upregulated, while miR-26a was significantly downregulated. After intervention with GPs, the above situation showed a reverse result. Meanwhile, indicators related to the mitochondrial apoptosis pathway also underwent changes. The expression trends of Bcl-2, Bax, Cytc, cleaned, caspase-3, cleaned, caspase-9, and cleaned PARP mRNA and protein in the liver of ApoE -/- AS mice were basically consistent. In the model group, the expression of Bcl-2 mRNA and protein was significantly downregulated, while the expression of Bax, Cytc, cleaned caspase-3, cleaned caspase-9, and cleaned PARP mRNA and protein was upregulated. After intervention with GPs, the expression of Bcl-2 mRNA and protein was upregulated, while the expression of Bax, Cytc, cleaned caspase-3, cleaned caspase-9, and cleaned PARP mRNA and protein was downregulated. Mitochondria are the main base for aerobic respiration in cells and an important site for energy supply, playing a crucial role in the occurrence of cell apoptosis. Mitochondria are key regulators of cell apoptosis, serving as the energy factory of cells and participating in oxidative phosphorylation and ATP production. At the same time, mitochondrial dysfunction can also lead to impaired ATP synthesis. The endogenous mitochondrial pathway is one of the main pathways of cell apoptosis, and Cytc is a substance contained in mitochondria that is closely related to cell apoptosis. Under the stimulation of apoptotic signals, the permeability of mitochondrial membrane is in a developmental state, and the release of Cytc and activation of caspase lead to mitochondrial apoptosis. It can be seen that the release of Cytc from mitochondria to the cytoplasm is a key step in triggering the mitochondrial apoptosis pathway. Cytc release is an important event that occurs in the early stages of cell apoptosis. The specific process involves the release of MOMP regulated by mitochondrial MPTP or Bcl-2 family members into the cytoplasm, followed by the further utilization of Cytc released into the cytoplasm. In the presence of ATP/dATP, it can bind with caspase-9 to form an apoptotic complex, which in turn activates caspase-9. Activated caspase-9 can then activate caspase-3, further initiating the caspase cascade reaction and leading to cell apoptosis. Caspase is a type of zymogen that normally exists in an inactive structure. In the caspase dependent mitochondrial pathway, Cytc is released from mitochondria and combines with ATP and Apaf-1 to form a multimer. At the same time, caspase-9 binds to it to form an apoptotic body, which can hydrolyze caspase-9 zymogen and activate caspase-9. Activated caspase-9 can further activate caspase-3, which can cleave the DNA repair enzyme PARP, causing PARP to be cleaved into small fragments and unable to function normally, leading to DNA fragmentation and ultimately causing cell apoptosis. The above results indicate that GPs may improve liver lipid deposition in ApoE -/- AS mice by interfering with mitochondrial apoptosis through long non coding RNA TUG1/miR-26a, thereby preventing and treating AS. The interference mechanism may be related to the expression levels of Bcl2, Bax, Cytc, cleaved caspase-3, cleaved caspase-9, and cleaved PARP that regulate mitochondrial apoptosis. However, its specific targeted regulatory relationship needs to be further validated through subsequent cell experiments. This study is expected to lay the foundation for network target analysis of traditional Chinese medicine in disease prevention and treatment, as well as the analysis of complex mechanisms of multi-component and multi-target traditional Chinese medicine. It will provide stronger experimental evidence for the combination of traditional Chinese and Western medicine in the prevention and treatment of cardiovascular diseases and clinical applications.