Oridonin Confers Liver Protection Against ROS Through Activation Of The Nrf2-mediated Defensive Response

BackgroundHerbal medicine, Donglingcao (Rabdosia rubescens), has been traditionally used in China, which is the major source of Donglingcao. Oridonin, a tetracyclic diterpenoid compond isolated from Rabdosia rubescens, has many kinds of pharmacological andphysiological effects, such as anti-inflammation, anti-tumor. In addition, Donglingcao tea has also been used in folk as a nature health care product to prevent many kinds of disease. However, the mechanisms of action are not well-documented. Nrf2-Keapl pathway regulates a cytoprotective response and is tightly related with antioxidant response, anti-inflammation and anti-tumor. In this study, we investigated how oridonin affect this signal pathway and confer the protection of liver cell against reactive oxygen specices, ROS..Liver is a major disintoxicating organ in human body. Many deleterious substances, for example, ethyl alcohol, are metabolized through liver. In the metabolizing process, ROS are produced, which is the major oxidize damage source.Nrf2 is a critical transcriptional factor that regulates a cytoprotective response. Many of its downstream target genes are important in maintaining the cellular antioxidant response and xenobiotic metabolization. Under basal conditions, Nrf2 is sequestered in the cytoplasm by Keap1, Keap1 acts both as a repressor of the Nrf2 transactivation and as a sensor of phase 2 inducers. Electrophiles and oxidants disrupt the Keapl-Nrf2 complex, resulting in nuclear accumulation of Nrf2, where it enhances the transcription of phase 2 genes via a common upstream regulatory element, the antioxidant response element,ARE, which is a duplication regulation factor that lies upstream section of some oxidation resistance protein and some II phase disintoxicating substrat gene. After Nrf2 and its downstream target genes activating, cells remove toxic and carcinogenic chemicals. In this way, Nrf2 can reduce tumor susceptivity, protect nerve tissue, reduce atherosclerosis, prolong life.Oridonin also known as rubesecensin A, is a diterpenoid purified from Rabdosia rubescens. Now,we found that oridonin can active Nrf2 and increase the expression of its downstream genes. In this study, we investigated how oridonin affect this signal pathway and confer the protection of liver cell against reactive oxygen specices, ROS..ObjectiveTo confirm that oridonin can protect the liver cells at the dose dependent way. To prove that oridonin confers liver protection against ROS through activation of the Nrf2-mediated defensive response through a dose dependent way.MethodsA pGL4.22-ARE-TATA-luciferrase plasmid is constructed, and transfect chang's liver cells. After oridonin added, results of fluorescence show that it can promote the ARE fluorescein enzyme report gene to duplicate. Oridonin can improve the level of expression of Nrf2 and its downstream genes,and this is detected by Western Blotting and Real Time PCR; the level of oxygen free radical is examinated by FACS. After those results are established, the Nrf2 gene expression level is decreased by RNA silence technique, and detect if the effect of anti- oxidation damage still exit. First the plasmid is construted, Nrf2-Knock-down, Chang's Liver cells are packed infected by Retroviral, and Nrf2-Knock-down Chang's Liver cell line is selected. The Nrf2 gene is silenced in what we have slected by Western Blotting. After oridonin and ethyl alcohol added, the levels of active oxygen are detected and AST is chose to determine if oridonin protect live cells by Nrf2 pathway.ResultsOridonin can strengthen the Nrf2 expression in the liver cells, promote its downstream gene duplication, reduce the active oxygen, and protect livers cells from the damage by ethyl alcohol. After the Nrf2 silence, anti-oxidation damage function vanishs. Those results show that oridonin protect liver cells from oxidation damages, and this function is achieved by Nrf2 pathway.ConclusionWe have identified oridonin as a novel class of Nrf2 activators. Oridonin can active Nrf2 and its downstream genes expression, which reduce the oxidative damage of ROS and the deleterious substance. and illustrated the mechanistic action of oridonin in Nrf2 activation. Furthermore, we have demonstrated the feasibility of using natural compounds targeting Nrf2as a therapeutic approach to protect humans from various environmental insults that we have to face every day.