The Mechanism Of Liver Drug Metabolizing Enzyme CYP3A4 Under Hypoxic Regulation

The liver is one of the important metabolic organs of the human body.The CYP450 family of drug metabolizing enzymes contained in the liver is crucial in the process of drug conversion in the body.CYP3A4 accounts for a large proportion in the CYP450 family,which has a wide range of substrates including clinical drugs.It is a key metabolic enzyme for drug conversion in vivo.There are many factors that could affect the normal function of drug metabolizing enzymes,including hypoxia,which could also fluctuate other physiological processes of the body to a certain extent.Besides,China is the country with the largest plateau area in the world.In recent years,the number of people entering the plateau from the plain has been gradually increased.As a major feature of the plateau,hypoxia would also affect the physiological processes of the human body,including the metabolism of drugs.However,the current plateau medication is still used according to the plain area,which would influence the rationality,safety and effectiveness of the drug among plateau population.From the perspective of nuclear receptors and inflammatory factors,our team has initially explored the mechanism of regulating CYP3A1 in rat liver under hypoxic environment in the rapid plateau,while the regulation mechanism of CYP3A4 in human liver under hypoxic environment has been unclear.Considering that there may exist discrepancy in regulation between different species,this study selected CYP3A4 in the human liver cells as the research's aim and explored its regulatory mechanism under hypoxic environment in order to provide more useful data and theory for rational plateau medication in the future.This research was mainly conducted through cell experiments.According to requirements,the human normal liver cells L-02 were placed in a 1% hypoxic environment for different treatments,and experiments were performed after common culture in normal control groups.The main objectives of this project are as follows.The first one is to investigate the effects of hypoxia on human normal liver cells L-02.Compare the biochemical indicators of L-02 cells between the normal control group and the hypoxic group at different time.The second one is to investigate the expression changes of drug metabolizing enzyme CYP3A4 and possible regulatory factors including nuclear receptors PXR,RXR?,HNF4?,and related cofactors PGC-1?,SRC-1 respectively in the human normal liver cells L-02 under hypoxic environment,and the expression of HIF-1? protein was used as a detection index to determine the success of hypoxia modeling,and then Real-time PCR and Western Blot were used to determine changes in mRNA and protein expression levels of these factors.The last one is to examine the regulatory role of HIF-1? on the expression of CYP3A4 and possible nuclear receptors in human normal liver cells L-02 during hypoxia.The effects of PXR,RXR?,HNF4? and related cofactors PGC-1?,SRC-1 expression levels were measured using Real-time PCR and Western Blot.The correlation between HIF-1? and nuclear receptor PXR and CYP3A4 and their possible regulatory pathways were analyzed by the late investigations.The first part of the results showed that after different periods of hypoxia treatment,ALT,a marker of liver function evaluation in human normal liver cells L-02,significantly increased after hypoxia,similar to the increase of MDA and LDH,while T-SOD significantly decreased after hypoxia.Under hypoxic conditions,the liver tissue of the body may be damaged to a certain degree.As the main organ of drug metabolism,the processes mediated by drug metabolizing enzymes and the normal physiological activities of the liver would be affected,which may disturb the internal environment of the body.The second part of the results showed that the hypoxia model of human normal liver cell L-02 was successful.After hypoxia treatment,the mRNA and protein expression levels of the drug metabolizing enzyme CYP3A4 in the cells could change and show the opposite trend,which is also consistent with the mRNA and protein expression of PXR,PGC-1? and SRC-1,while the mRNA and protein expression of RXR? and HNF4? were all decreased.Combined with existing researches,the decline of the final expression of drug metabolizing enzymes would delay the rate of drug metabolism and may increase drug toxicity.We speculated that the nuclear receptor PXR and related regulatory factors in L-02 cells under hypoxic environment may be an upstream regulatory link of the drug metabolizing enzyme CYP3A4.Besides,the reason of the inconsistent trend of mRNA and protein expression may possibly be due to the interference of certain factors during the post-transcriptional translation process.The last part of the results showed that HIF-1? is able to inhibit the expression of the drug metabolizing enzyme CYP3A4 and other possible related regulatory factors in human normal liver cells L-02 except for the cofactor SRC-1 that is positively correlated with HIF-1? after designing a single factor experiment.Combining literature investigation,we can speculate that HIF-1? may indirectly regulate CYP3A4 expression through PXR-related circuits under hypoxia.On the other hand,mRNA stability experiments proved that HIF-1? would destabilize the mRNA of CYP3A4,PXR and PGC-1?.In summary,the main conclusion of this study is that the normal physiological activity of human normal liver cells L-02 could be affected after 1% hypoxia treatment at different time.The decreased CYP3A4 expression could block the normal drug metabolism in vivo.In addition,HIF-1? may indirectly regulate CYP3A4 expression through nuclear receptor PXR and related factors and reducing the mRNA stability of some of the factors.