Transcriptome Analysis of Hepatoprotection of Traditional Medicines Against Acute CCl₄ and Chronic Alcohol-Induced Liver Injury in Mice

Alcohol binge drinking is a risk factor for alcoholism. Alcoholic liver injury is the main alcohol-related disease, manifested from simple steatosis to alcoholic steatohepatitis (inflammation), which can be progressed to liver fibrosis, cirrhosis, and liver cancer with continuous consumption of excessive alcohol [1]. The chronic alcohol-induced liver injury can be reproduced by feeding mice with Lieber-De Carli liquid diet containing 5% alcohol for 28 days or longer, and liver injury was evidenced by histopathology with lipid droplet accumulation, elevated levels of triglyceride in the blood, and aberrant gene expressions, mimicking clinical alcohol steatosis [2,3]. 

Dendrobium nobile Lindl is listed in Pharmacopoeia of China with many beneficial pharmacological effects. At least 82 active ingredients have been isolated from Dendrobium nobile Lindl., including alkaloids (DNLA), glycosides, polysaccharides, phenanthrene, dibenzyl compounds, and others. DNLA can regulate hepatic metabolism gene expression and lipid homeostasis via regulating the bile acids metabolism. In mice feed Lieber-DeCarli liquid diet, DNLA decreased serum triglyceride levels, ameliorated hepatocyte degeneration and prevented steatosis, as evidenced by Oil-red O staining. Alcohol induced 787 differentially expressed genes, while DNLA only had 280 with much less extent. Ingenuity pathway analysis (IPA) showed that DNLA ameliorated alcohol-induced oxidative stress and xenobiotic metabolism disruption. qPCR verified that DNLA alleviated over-activation of Cyp2a4, Cyp2b10 and Abcc4, attenuated oxidative stress (Hmox1, Gstm3, Nupr1), Nrf2 genes (Nqo1, Gclc, Vldlr), and rescued some metabolism genes (Insig1, Xbp1, Socs3, Slc10a2) [3]. 

Rosa roxburghii Tratt. fruit juice (Cili) has been shown to have numerous beneficial effects on health, including antioxidant, anti-ageing, anti-atherosclerotic, antitumor, anti-hyperlipidemia, anti-hyperglycemia effects against diabetes, Cili also had beneficial effects on gut microbiota and hepatoprotection [4]. Cili juice was from Shan-Wang-Guo Co and gavaged to alcohol-fed mice at 4 mL and 8 mL/kg/day for 4 weeks. Cili decreased serum triglyceride levels, improved histopathology and dramatically reduced lipid droplets as compared to model mice. RNA-Seq analyses showed Cili could bring the alcohol-induced aberrant gene pattern towards normal. The qPCR analysis verified that over-activation of CAR and PXR (Cyp2a4, Cyp2b10 and Abcc4) was attenuated by Cili. Cili also alleviated overexpression of oxidative stress responsive genes (Hmox1, Gsta1, Gstm3, Nqo1, Gclc, Vldlr, and Cdkn1a), and rescued alcohol-downregulated metabolism genes (Angptl8, Slc10a2, Ces3b, Serpina12, C6, and Selenbp2). Thus, both DNLA and Cili were effective against chronic alcohol fatty liver, and the mechanisms were associated with decreased oxidative stress, improved lipid metabolism through modulating nuclear receptor CAR-, PXR-and Nrf2-mediated pathways [2,3].

Carbon Tetrachloride (CCl₄) is a widely used hepatotoxicant to study the mechanism of hepatoprotective compounds in traditional Chinese medicines. CCl₄ is activated by cytochrome P450 enzymes, particular CYP2E1 to generate reactive toxic metabolite trichloromethyl radical, which targets critical macromolecules to produce lipid peroxidation in membranes of mitochondria, endoplasmic reticulum, and other critical organelles, leading to inflammation and hepatocellular death, either by apoptosis or by necrosis. 

DNLA has been demonstrated to exhibit protective effects on liver injury induced by CCl₄ in wild-type mice, but not in Nrf2-null mice, and increase the expression of Nrf2 regulated proteins, indicating the activation of the Nrf2/ARE pathway as a mechanism of protection [5]. Further study demonstrated that CCl₄-induced mitochondrial oxidative stress is a critical event leading to liver injury, and DNLA improved mitochondrial oxygen consumption, elevated ATP production, and decreased CCl₄-induced apoptosis in the WT mice, whereas the DNLA-mediated protection on mitochondrial dysfunction was diminished in the Nrf2 null mice, Indicating DNLA effects on CCl₄ –induced mitochondrial dysfunction and mitochondrial oxidative stress were also Nrf2-dependent [6]. 

The molecular mechanisms for DNLA to protect against CCl₄ hepatotoxicity was further studied via RNA-Seq. DNLA brought the CCl₄-induced aberrant gene expression pattern towards normal. GO analysis showed that CCl₄ induced the activation, adhesion and proliferation of immune cells. KEGG analysis showed CCl₄ induced oxidative stress, diseases and compromised adaptive responses. CCl₄ induced differentially expressed genes were attenuated by DNLA. qPCR further verified DNLA attenuated overexpression of S100g, Sprr1, CCL3/7, Saa2/3, IL1rn, Cox7a2 and Rad15. IPA showed that CCl₄-activated mitochondrial oxidative phosphorylation was attenuated by DNLA. Illumina Correlation Engine analysis verified that CCl₄-induced DEGs were highly correlated with the GEO database of CCl₄ hepatotoxicity in rodents, and DNLA dose-dependently attenuated such correlation [7].

The liver is an important organ of detoxification in response to xenobiotics. To “program the liver” is an important concept in that xenobiotics at appropriate doses could evoke adaptive responses to induce a number of molecular events to protect against toxic stimuli, which could be important in hepatoprotection by traditional medicines.

Hua-Feng-Dan is such an example. Hua-Feng-Dan has over 300 years of history in the treatment of stroke and various diseases in China, including liver disorders. Hua-Feng-Dan is composed of 15 herbs, animal-based products, and minerals, and its “Guide drug Yaomu” is subjected to fermentation for 3 months before adding to the recipe. At least 44 volatile and 50 liposoluble components in Hua-Feng-Dan were identified through GC-MS analysis [8]. 

Hua-Feng-Dan and its “Guide Drug Yaomu” at hepatoprotective doses did not produce damage to the liver but produced gene expression changes. RNA-Seq revealed 806 DEGs in Hua-FengDan-treated livers compared to controls, and a low dose of Yaomu produced 235 DEGs in the same direction. GO and KEGG analyses revealed that Hua-Feng-Dan affected metabolism and signaling pathways. IPA upstream analysis pointed toward the activation of MAPK signaling pathways and induced adaptive responses by Hua-Feng-Dan. qPCR verified 10 selected DEGs. Hua-Feng-Dan-induced gene expression changes were highly correlated with the GEO database of chemical-induced adaptive responses in mouse livers [8]. Thus, RNA-Seq analysis revealed Hua-Feng-Dan-induced adaptive responses as mechanisms of hepatoprotection.

Transcriptome analysis is a powerful tool to identify the molecular changes by traditional medicines, mostly acting as adaptive responses to render the host resistant to toxic stimuli, thus protecting against CCl₄-induced acute liver injury, or chronic alcohol-induced fatty liver, and so on, to provide pharmacological basis of their therapeutic effects.

This work was supported by the National Natural Science Foundation of China (grant 82060750), and Guizhou Province Education Department (K-2019(016)).

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