Tanulmány Bekapcsol A hatás Acteosid (an hatóanyag összetevő Cistanche Deserticola) On NAFLD By Inducing Autofágia

2 Találatok

2.1 Összehasonlítás LC3-Ⅱ fehérje expresszió szintek ben HepG2 sejtek kezelt ACT és ECH a relatív expresszió a LC3-Ⅱ fehérje in HepG2 sejtek kezelt BafA1 (0..-ben/ BafA1 ({}}.67±0.0}4) volt szignifikánsan magasabb mint hogy a in a hepG2 sejtek in a a a hepG2 sejtek in a a a a hepG2 sejtek in a in hepG2 sejtek in a the rapamicin+BafA1 group (1.24±% 3b{{30}}}......06) volt magasabb mint hogy a (0.28±{{{40}}}.02). a a kifejezés LC{3-Ⅱ fehérje in hepG2 sejtek in a a ECH+BafA1 csoport [0.43±0.02 és 1.36±0.11, illet] volt siffinkányian magasabb mint azok in a ECH csoport és a a ACT csoport [(0,18±0.01 és 0,28±0.02, illet]], és a a különbségek statisztikailag szignifikáns (P<0.05).

TCM GYÓGYNÖVÉNY CISZTANCHE MÁJ VÉDELEM

2.2 Összehasonlítás LC3-Ⅱ fehérje expresszió szintek ben HepG2 sejtek kezelt eltérő koncentrációkkal a ACT

The LC3-Ⅱ protein expression levels of HepG2 cells in the CON group (0.79±0.07) were compared with those in the STA group (0.50±0.04), with no significant difference (P>{{{{10}}}}}.05); the LC{3-Ⅱ fehérje expresszió szint a HepG2 sejtek in a BafA1 kombinált 25, 50, és 1{0}{}μmol/L ACT groups (1.17±0.30, 1.83±% 7b{31}}.53, és 0.97±0.25, illetve) szignifikánsan magasabb mint azok a a 25, 50, és 100 μmol/L ACT csoportok (0.38±0.09, 0.35±0.18, és 0.30±0.15, illet), szignifikáns különbségekkel (P<0.05). The LC3-Ⅱ protein expression level of HepG2 cells in the 50 μmol/L ACT group was the highest. See Figure 1.

Megjegyzés: A. Fehérje immunblotting; B. Félkvantitatív analízis eredmények; összehasonlítva a a a ACT csoport nélkül BafA1,aP<0.05.
Ábra 1 Összehasonlítás LC{3-Ⅱ fehérje expresszió szintek ben HepG2 sejtek kezelt különböző koncentrációkkal a ACT

2.3 Összehasonlítás életképesség a HepG2 sejtek kezelt különböző koncentrációkkal ACT 24 és 48 h

A OD értékek a HepG2 sejtek beavatkozás után {{{1}, 2{{}}, 4{{1{0}}, 6{{{{, 80, és 10{{}}}}μmol/L ACT for 24 h were {{{24}.98±0.17, 0.99% 26plusmn;{{30}}}.., 15, 0.99±{0.19, 1.00±{0.12, {0.94±0.15, és {0.94±0.20, illet; és a a OD értékek a HepG2 sejtek beavatkozás után for 48 h volt 3.06±0.27% 2c .96±0.27, 2.88±0.27, 3.08±0.15, 3.01±0.13, és .09±0.22, illetőleg. volt nem szignifikáns különbség a a a OD értékek után 24 és 48 h of beavatkozás különböző koncentrációkkal a ACT (F=0}.213, P{=0.96, 0.36). Lásd ábra 2.

Megjegyzés: A. HepG2 sejtek kezelték különböző koncentrációkkal a ACT for 24 h; B. HepG2 sejtek kezelték különböző koncentrációkkal of ACT for 48 h.

Ábra 2 OD értékek at 570 nm után 24 és 48 h kezelés eltérő koncentrációkkal a ACT

2.4 Összehasonlítás TG szintek és Olaj Piros O festés be a a be vitro cella modell NAFLD után ACT beavatkozás

A TG szint a HepG2 sejtek a a a modell csoport (34,15±1,90) volt szignifikánsan magasabb mint hogy a a üres kontroll csoport (14,17±2,74) és a a ACT beavatkozás csoport (19,11±1,68), és a a különbségek statisztikailag szignifikáns (P<0.05). The number of stained lipid droplets in HepG2 cells in the ACT intervention group was significantly reduced (see Figure 3).

Note: A. TG level; B. Oil red O staining (20×); compared with the model group, aP<0.05.
Ábra 3 Összehasonlítás a TG szint és Olaj piros O festés NAFLD in vitro sejt modell után ACT kezelés

2.5 Összehasonlítás autofagoszómák és lipid cseppek után ACT kezelés összehasonlítva a a üres kontroll csoport, a lipid cseppek a HepG2 sejtek a a a rapamicin kezelés csoport és a a ACT beavatkozás csoport szignifikánsan, és a LC3 autofagoszómák a HepG2 sejtek a a ACT beavatkozás csoport sist szignifikánsan, és kolokalizált lipid cseppekkel a térben. Lásd ábra 4.

3 Vita

NAFLD is the most common chronic liver disease in the world, accounting for about 1/4 of the world's total population, and has become a global public health problem. Overnutrition is the main factor driving NAFLD. Excessive intake of nutrients by liver cells causes excessive fat deposition in the cells, leading to lipotoxic reactions such as cell stress (oxidative stress and endoplasmic reticulum stress), inflammasome activation, and cell apoptosis, and subsequently causing a series of changes in liver tissue phenotypes such as inflammatory stimulation, tissue regeneration, and fibrosis [14-15]. In recent years, studies have shown that autophagy plays an important role in preventing the development of human NAFLD. The results of autophagy-related index detection in the livers of patients with NAFLD and non-alcoholic fatty liver disease and the livers of obese and high-fat diet mice showed that the autophagy process in hepatocytes was blocked, which led to the massive accumulation of intracellular TG and other lipid droplets [16]. Other studies have shown that after the key gene 7 of hepatocyte autophagy was specifically knocked out in mice fed a high-fat diet, the level of TG in mouse hepatocytes increased significantly [17]. However, after overexpression of the Atg7 gene or intervention with autophagy inducers such as rapamycin and carbamazepine, fatty degeneration in the mouse liver was significantly reduced [18-20].
This suggests that liver fatty degeneration can be significantly reduced by improving liver autophagy activity, providing an effective strategy for the treatment of NAFLD. Natural products, as a major source of new drug development, are widely used in the prevention and treatment of various liver diseases. As an excellent product commonly used in traditional Chinese medicine, Cistanche deserticola has attracted widespread attention for its pharmacological effects such as neuroprotection, immunomodulation, antioxidant, anticancer, and liver protection. So far, more than 120 compounds have been isolated from Cistanche deserticola, including phenylethanoid glycosides, terpenoids and their glycosides, lignans and their glycosides, oligosaccharides, polyols, and polysaccharides [21]. Among them, phenylethanoid glycosides are the most studied class of compounds in the active ingredients of Cistanche deserticola. ECH and ACT are representative compounds of Cistanche deserticola phenylethanoid glycosides, which have positive effects in antioxidants, protecting the liver, myocardium, nerve cells and enhancing memory [22-23]. However, there is no relevant literature report on whether Cistanche deserticola phenylethanoid glycosides are involved in the autophagy process in protecting the liver.

Autophagy is a self-degradation process that is ubiquitous in the body. It is a catabolic process in which autophagosomes degrade cellular metabolic waste. The autophagy process generally includes three processes: autophagy initiation, autophagic membrane extension and autophagic lysosome formation, which are mainly regulated by rapamycin target protein and phosphatidylinositol-3 kinase signaling pathways. Autophagosomes are markers of cellular autophagy. In the process of autophagosome formation, a series of autophagy-related proteins including Atg5~12 and LC3 play an important role. Therefore, this study constructed an autophagy evaluation system, detected the expression level of autophagy marker protein LC3 at the molecular level, and evaluated the autophagy induction ability of two phenylethanoid glycosides of Cistanche deserticola in HepG2 cells. The results showed that after the addition of BafA1 treatment on the basis of the intervention of the autophagy inducer rapamycin, the intracellular LC3-Ⅱ of HepG2 cells increased significantly, indicating that the autophagy evaluation system can accurately evaluate the autophagy induction activity of drugs; after ECH and ACT were treated with BafA1, the expression of LC3-Ⅱ protein in HepG2 cells in the ECH+BafA1 group and the ACT+BafA1 group was significantly higher than that in the ECH group and the ACT group, and the differences were statistically significant (P<0.05). However, since the autophagy process is very complex, it is easily changed by external environmental stimuli. Therefore, this study used BafA1 to block the autophagic flow, thereby more accurately evaluating the autophagy inducing ability of ECH and ACT. The results showed that after using BafA1 to block the autophagic flow, the intracellular autophagosomes could not be degraded in the autophagolysosomes, resulting in a significant increase in the overall autophagy level of the cells. After the intervention of rapamycin, Ech, and ACT in HepG2 cells, the LC3-Ⅱ protein expression levels of HepG2 cells in the rapamycin+BafA1 group and the ACT+BafA1 group were higher than those in the BafA1 group alone. Although the expression levels of HepG2 cells in the ECH+BafA1 group were significantly higher than those in the ECH group alone, the differences were statistically significant (P<0.05), the expression levels of LC3-Ⅱ protein in HepG2 cells were lower than those in the BafA1 group alone, indicating that ACT has a significant ability to induce autophagy in HepG2 cells. Therefore, this study took ACT as the research object and further studied its pharmacological activity in inducing autophagy and Erőkifejtés máj védelem and other biological functions. Secondly, safety is a primary consideration in drug development. In order to evaluate the safety of ACT, this study further tested the effect of ACT on the viability of HepG2 cells. The results showed that the viability of HepG2 cells was almost unaffected by intervention with different concentrations of ACT, indicating that ACT can be used as a candidate drug to further study its pharmacological effects and molecular mechanisms.

Autofágia a nagyon konzervált biológiai folyamat sejtekben, és enyhe változások a gyógyszer koncentrációk fog befolyásolja a szint a sejtes autofagoszómák. További meghatározása a optimális koncentráció a ACT indukálni a autofagoszómák in HepG2 sejtek, ez tanulmány használt különböző koncentrációk a ACT kombinált BafA1 kezelni HepG2 sejtek és a a expresszió szint LC3-Ⅱ fehérje sejtekben sejtekben. A eredmények megmutatta hogy 50 μmol/L ACT szignifikánsan növelhetné a a autofagoszómák a HepG2 sejtekben. Ezért, ezt tanulmány használt 50 μmol/L ACT to beavatkozni a a NAFLD sejt modell konstruált in vitro, és előzetesen feltárt a kapcsolat között az máj védelem funkció és autofágia.
Related studies on NAFLD cell models have shown that after in vitro intervention with free fatty acids, the TG level of HepG2 cells increased significantly, and the aggregation of oily lipid droplets could be clearly observed in the cells [24-25]. In this study, 100 μmol/L PA was used to intervene in HepG2 cells in vitro for 24 h. The TG level of HepG2 cells in the model group was significantly higher than that in the blank control group. At the same time, the aggregation of lipid droplets could be clearly observed in the cells, indicating that the NAFLD cell model was successfully constructed. In order to further observe the effect of ACT on the NAFLD cell model, 50 μmol/L ACT was used to intervene in the NAFLD in vitro cell model, and the effect of ACT on NAFLD was evaluated by TG level detection and Oil Red O staining. The results showed that after ACT intervention, the TG level and the number of oily lipid droplets in HepG2 cells were significantly lower than those in the model group, indicating that ACT has the activity of reducing TG in the PA-induced NAFLD cell model. In order to further study the relationship between ACT's lipid-lowering activity and cellular autophagy, this study used LC3-Ⅱ specific protein antibodies and BODIPY fluorescent probes to co-stain autophagosomes and lipid droplets. It was observed that the number of autophagosomes in HepG2 cells after ACT intervention was significantly increased compared with the control group, which was consistent with the results of protein immunoblotting in this study, further confirming that ACT has autophagy-inducing activity in HepG2 cells. In addition, after drug intervention, intracellular autophagosomes and lipid droplets co-localized, indicating that ACT's effect on improving PA-induced NAFLD fatty degeneration may involve the involvement of cellular autophagy. However, in the NAFLD cell model, whether ACT exerts its pharmacological activity of reducing intracellular TG levels through the lipophagic pathway still needs to be further explained by blocking the intracellular autophagy process.
In összegzés, között a kettő feniletanol glikozid vegyületek a Cistanche sivatagicola, ACT van a szignifikáns képesség indukál autofágia in HepG2 sejtek. At a a PA-indukált máj steatózis sejt modell, CT szignifikáns csökkent a TG szint in HepG2 sejtek. Után ACT beavatkozás, autofagoszómák és lipid cseppek in HepG2 sejtek had a bizonyos társlokalizáció kapcsolat, jelzése hogy autofágia lehet lehet a új út érintett a farmakológiai folyamat a ACT in terms of máj védelem. Lásd 5. In későbbi vizsgálatok, a a hatásmechanizmus a ACT in lowering lipidek lesz tovább tanulmányozott részletesen autofágiával blokkolókkal to lay a foundation for for new drug development.