Citrus grandis ‘Tomentosa’ is a commonly used Chinese medicine for resolving phlegm and cough with a long history of application. Modern pharmacological research show that the main active site are flavonoids ingredients, with antioxidant anti-inflammation antipyretic analgesic effect. ROF is one of the main flavonoids. In previous reports indicated, the protective effects of ROF against alcohol-induced liver injury had not been studied in depth. Therefore, this study is mainly to clarify the protective effect of ROF and its mechanism.
In this study, the hepatoprotective effect of ROF from Citrus grandis ‘Tomentosa’ on the ethanol-induced liver injury model in vivo and in vitro were investigated. Results suggested that ROF reduced the liver injury induced by ethanol through its anti-inflammatory and antioxidant effects, and the underlying mechanism may be associated with the reduced release of inflammatory cytokines and apoptosis-related genes by inhibiting the TLR4 / NF-kB signaling pathway.
There were many pathogenic factors in the development of ALD. Among them, long term excessive consumption of alcohol was a key factor in ALD, which lead to chronic liver damage. Mice chronic alcoholic liver model was used to simulate the long-term drinking of human beings. Liver index refers to the percentage of liver and body weight, which is one of the indicators to judge whether the liver is healthy. When alcoholic liver injury occurs, the liver pathology may show hepatomegaly, unclear boundary, watery degeneration and inflammatory cell infiltration, which eventually leads to weight increase and liver index increase. ALT and AST play important roles in the synthesis and decomposition of amino acids in the body, and are important indicators reflecting liver function in clinic. When the liver is gradually damaged, ALT and AST will continue to release into the blood, so that the level of them in blood can reflect the degree of liver damage. The expression of AST and ALT in the serum of alcohol induced mice increased significantly, while the expression of them decreased significantly after administration of ROF, total FLA and BIF.
Metabolized alcohol will produce a large number of oxidative free radicals and consume a large number of enzymes. SOD and GSH are important antioxidant enzymes in the body. The activity level of these enzymes directly reflects the body’s antioxidant capacity and plays an important role in eliminating excessive free radicals. Compared with the control group, GSH and SOD in the ethanol group were significantly decreased, while the expression levels of them were increased after administration. The destruction of the body’s antioxidant system leads to lipid peroxidation of the liver cell membrane and the increase of MDA level. When the antioxidant enzymes in the human body decrease, it will inhibit the metabolism of acetaldehyde, resulting in the output of triglyceride disorder, and then a large amount of deposition in the liver. The content of TG and MDA in the therapeutic group was decreased compare with the ethanol group. Hepatic CYP2E1 thought to be a major source of ROS production and could be induced by alcohol. The results showed the hepatic CYP2E1 protein expression were reduced after ROF treatment, suggesting that ROF might exert a beneficial effect on CYP2E1-mediated oxidative stress.
The protective effects of ROF against ALD was studied using ethanol-induced LO2 cells in vitro. MTT experiments found that 100 µmol·L− 1 ROF could significantly enhance cell viability. Therefore, the concentration of ROF was selected as 25, 50 and 100 µ mol·L− 1 in the follow-up experimental study. When the concentration of ethanol was selected by MTT, the cell survival rate could reach 36% when the concentration of ethanol was 200 mmol·L− 1. Annexin V could bind phosphatidylserine (PS) specifically, which indicated that cells were apoptotic. Annexin V-FITC/PI double staining and flow cytometry were used to evaluate the inhibitory effect of ROF on ethanol induced apoptosis of LO2 cells. The results showed that the early and late apoptosis rates in the ethanol group were significantly increased, 50 µmol·L− 1 and 100 µmol·L− 1 ROF treatment were significantly decreased.
Bax and Bcl-2 are two important regulatory factors of apoptosis. They have opposite effects on cell apoptosis. The increase of Bax expression level leads to cell apoptosis. On the contrary, when the level of Bcl-2 is higher, it inhibits cell apoptosis. Caspase-3 as the executive factor of apoptosis, its significantly increased expression level can promote cell apoptosis. Studies have shown that excessive alcohol concentration in LO2 cells will promote the overexpression of Caspase-3 factor and the imbalance of Bax and Bcl-2 factor expression, thus promoting a large number of cell apoptosis.
Compared with the blank group, the expression levels of Bax and caspase-3 mRNA were increased and the expression levels of Bcl-2 mRNA were decreased in LO2 cells treated with ethanol alone for 24 h. Compared with the ethanol group, the expression level of Bax mRNA decreased, while the expression level of Bcl-2 mRNA increased. In order to further study its apoptotic effect, we detected the expression of Caspase-3 and Cleaved Caspase-3 by Western Blot. The protein expression level of ethanol treated cells was increased. However, ROF significantly down regulated the expression of Caspase-3 and Cleaved Caspase-3 in LO2 cells. It is suggested that ROF can promote the proliferation of LO2 cells induced by ethanol and inhibit their apoptosis.
Inflammatory response injury plays a key role in the occurrence of ALD. Endotoxin activates Kupffer cells that release some inflammatory factors, including TNF-α, IL-1β, and IL-6, which can injure the normal cells of the human body and then induce liver injury. In our experiment, ELISA was used to detect the mice serum and PCR was used to detect the expression of inflammatory factors in the supernatant of LO2 cells. The results show that ROF can effectively reduce the production of TNF-α, IL-6 and IL-1β, thus reducing the inflammatory reaction in vivo and in vitro.
TLR4 is closely related to inflammation of liver injury induced by alcohol. It can activate NF-kB pathway, regulate the expression of inflammatory factors, and promote the production of inflammatory cytokines, such as TNF-α and IL-6. Our study shows that ROF can significantly reduce the expression of TLR4 and NF-kB, thereby inhibiting the production of TNF-α and IL-6. In addition, TLR4 / NF-kB can also target the NLRP3 to induce liver inflammation. Activation of NLRP3 can transform IL-1β secretion. The results clarified that ethanol could increase the inflammatory level of NLRP3 and promote the secretion of IL-1β. ROF treatment significantly reduced the production of these inflammatory factors, suggesting that ROF treatment may play an anti-inflammatory role by inhibiting the nuclear translocation of NF-kB and affecting the activation of NLRP3 by reducing the expression of TLR4.
In conclusion, ROF had protected effects on ethanol-induced mouse ALD model and ethanol-induced LO2 cells. This study suggests that ROF may be used as a potential therapeutic for ALD, and provided research ideas for the development and utilization of Citrus grandis ‘Tomentosa’ resources. We suggest that ROF be considered as candidate potential anti-ALD option and it is worth further exploring clinical trials in future research.