| Part Ⅰ MDM4 involved in the mechanism of methamphetamine-activated microgliaBackground:Methamphetamine(METH)is a kind of neuromuscular synthetic drug,one of the amphetamine-type stimulants.Its molecular structure is similar to the neurotransmitter catecholamine.It is easily soluble in water,alcohol and other solvents.Because it looks like ice crystal,it looks like ice crystal.Also known as "ice poison",its widespread abuse in the world,its tendency to endanger public health and social security is becoming more and more serious.It is one of the most serious drugs for public health and social safety worldwide.METH was first used as a drug to control appetite,and later turned into a psychotropic drug,which can bring a short-lived state of mind and body to the smoker,and at the same time also endanger serious side effects.After taking methamphetamine,it mainly causes damage to the central nervous system,cardiovascular system,liver and kidney.At present,it has been shown in the literature that the target organ of the toxicity of methamphetamine is the central nervous system of the human body,which can cause apoptosis of autophagy in the central nervous system,inflammatory reaction of autophagy and autophagy and can cause small The activation of glial cells and the like,the dysfunction of cells in the central nervous system described above will further lead to the appearance of neurodegenerative symptoms such as Huntington,Parkinson,etc.,ultimately affecting the normal function of the nervous system.Our group has been working on a series of methamphetamine neurotoxicity studies.In this regard,our group has found that methamphetamine can cause neuronal apoptosis and inflammatory response of star glue.In addition,there are a large number of clinical and post-mortem pathological anatomical data showing that in addition to neuronal apoptosis and astrocyte activation in the brain of long-term use of ice,there is also a neuroinflammatory reaction.The neuroinflammatory response plays an important role in the central nervous system and plays an important role.When it comes to the inflammatory response of the nervous system,it is necessary to mention the specialized monocyte-microglia in the nervous system.The microglia account for about 10-20%of the number of cells in the brain,and the number of neurons is the same.Quite,it is the front line of the brain to deal with harmful external stimuli.Microglia,a specialized immune cell in the central nervous system,plays an important role in maintaining normal brain function,and it can continuously eliminate apoptotic bodies produced by apoptosis in other cells of the central nervous system.Foreign substances,local infections,etc.,microglia play an important role in maintaining the central nervous system homeostasis,especially in the development of central nervous system diseases such as Parkinson’s,multiple sclerosis and Alzheimer’s disease.The research group has not yet conducted in-depth research on the effects of METH on microglia.In the preliminary pre-experiment,it was found that METH can activate microglia,cause microglia to secrete related inflammatory factors,and pass low-throughput screening.By MDM4,METH4 increased significantly after METH treatment of microglia,and it was reasonably hypothesized that MDM4 had an effect on METH-induced microglia activation.This study aims to demonstrate that MDM4 participates in the regulation of METH-induced microglia activation by in vitro experiments,and further explores the potential pathway and possible mechanism of METH-induced microglia activation.Purpose:This study aims to explore the mechanism of action of MDM4 in METH-induced microglia activation by establishing an in vitro model of METH neurotoxicity using immunological and molecular biology techniques,and to regulate MDM4 expression by molecular biological means,and to detect MDM4.The regulation of microglia activation and the possible molecular pathways involved in the study of possible molecular mechanisms of METH-induced microglia activation,laying the foundation for further clarifying the regulatory mechanism of MDM4 in METH-activated microglia activityMethod:1.METH induced microglia autophagy and establishment of MDM4 elevated model(1)Inoculate BV2 cells into 6-well plates,culture the cells at 5%C02 and 37℃,and use METH in DMEM high glucose medium dissolved in 2%FBS until 60-80%confluence.The concentration was 0 mmol/L,0.4 mmol/L,0.8 mmol/L,1 mmol/L,2 mmol/L,and 3 mmol/L for 24 hours.The total protein was extracted,and the expression level of MDM4 and the expression level of autophagy-related molecules were detected by Western Blot.The appropriate METH concentration was determined according to the trend of MDM4 after METH concentration gradient treatment.(2)According to the trend of MDM4 and autophagy in the results of the above experiments,select the most suitable METH treatment concentration,and treat the concentration at 0h,6h,12h,24h,36h and 48h to establish a time gradient treatment experiment.After the corresponding time,the total protein was extracted,and the expression level of MDM4 and the expression level of autophagy were detected by Western Blot.The appropriate METH administration time was determined according to the trend of MDM4 after METH time gradient treatment.2.The role of MDM4 in autophagy and activation of microglia(1)According to the mRNA sequence of MDM4,the siRNA interference fragment of MDM4 was designed,and the siRNA was transfected by the lipo-3000 liposome transfection instructions.The BV2 was divided into 8 groups,which were blank control,control + small interference 1.Control + small interference 2,control + small interference 3,administration group,administration + small interference 1,administration + small interference 2,administration + small interference 3,24 hours after treatment,Western Blot detected the expression level of MDM4 cells Changes in autophagy and inflammatory response,depending on the results,select effective specific siRNA.(2)Select MDM4-specific chemical inhibitor GSC-207895,and set 5 concentration gradients according to the concentration recommended by the existing literature.The range concentrations are 0μM/L,1μM/L,2μM/L,3μM/L,4μM/L,pre-treated for 24 h,then treated with METH at a final concentration of 2 mM for 24 hours to extract total cellular protein,and Western Blot was used to detect the changes of autophagy and inflammatory response in the expression level of MDM4.3.MDM4 mediates METH-induced microglia activation via autophagy pathway(1)According to the mRNA sequence of ATG5,the siRNA interference fragment of ATG5 was designed and transfected into siRNA using lipo-3000 liposome transfection instructions.BV2 was divided into 4 groups,which were blank control group,control+ small interference,After administration for 24 hours,Western Blot was used to detect the expression level of autophagy-related molecules in MDM4 expression level,and the level of inflammatory factors was detected by qRT-PCR.(2)Selecting the autophagy-specific chemical inhibitor 3-MA,according to the treatment concentration and time recommended by the existing literature,ie 2 mM,after treatment for 24 h,the final concentration of 2 mM METH was treated for 24 hours,divided into 4 groups,respectively METH(-)3MA(-)group,METH(-)3MA(+)group,METH(+)3MA(-)group and METH(+)3MA(+)group,Western Blot detected MDM4 expression level and autophagy The expression level of the relevant molecule was measured and the level of inflammatory factor change was detected by qRT-PCR.result:1.METH induced microglia autophagy and establishment of MDM4 elevated model(1)BV2 cells were treated with 0 mmol/L,0.4 mmol/L,0.8 mmol/L,1 mmol/L,2 mmol/L and 3 mmol/L final concentrations of METH.The results of Western Blot showed that MDM4 was treated with METH.The increase of MDM4 was significantly higher than that of the control group of 0 mmol/L at 2 mmol/L,and the expression of autophagy was similar to that of MDM4.(2)BV2 cells were treated with 2 mmol/L METH final concentration for 0h,6h,12h,24h,36h,48h.The results of Western Blot showed that MDM4 increased with METH,and MDM4 protein level also increased with MDM4.Protein expression began to rise after 6 hours of METH treatment,peaked at 24 hours,remained relatively high at subsequent levels,and began to decline at 48 hours,2.The role of MDM4 in autophagy and activation of microglia(1)After treatment with MDM4 siRNA Oligo fragment,MDM4 protein expression showed a downward trend compared with METH group.Using METH+siRNA3,the expression of MDM4 was significantly lower than that of the METH group.With METH+siRNA1 or METH+siRNA2,the expression level of MDM4 was not significantly decreased compared with the METH group.At the same time,the autophagy and inflammation Maker genes were detected.The results showed that compared with the control group,the levels of autophagy and inflammatory protein were increased in the METH group,while the expression of related proteins was decreased in the METH+small interference group compared with the METH group.(2)Select MDM4-specific chemical inhibitor GSC-207895,set 5 concentration gradients,the concentration range is 0μM/L,1μM/L,2μM/L,3μM/L,4μM/L,pretreatment for 24h,then give After treatment with METH at a final concentration of 2 mM for 24 hours,the expression level of MDM4 increased with the MDM4-specific chemical inhibitor GSC-207895,which showed a trend of decreasing first,and significantly inhibited the expression of MDM4 from 2 mM,3 mM.reach the peak.At the same time,autophagy and inflammation-related marker genes were detected.The results were as follows,compared with the METH group;while METH+GSC-207895 showed a decrease in autophagy and inflammation-related protein expression compared with the METH group.3.MDM4 mediates METH-induced microglia activation via autophagy pathway(1)After treatment with ATG5 siRNA Oligo,it was found that the expression of ATG5 was down-regulated as compared with METH.At the same time,the expression levels of autophagy and inflammation-related molecules were detected.The results were as follows.Compared with the Control group,the expression of autophagy and related inflammatory molecules was up-regulated in METH group.However,METH+ small interference was not changed in MDM4 compared with METH group,only inflammation-related molecules.The expression is reduced.(2)Select autophagy-specific chemical inhibition of 3-MA,pre-treated at a final concentration of 2 mM for 24 h,and then treated with METH at a final concentration of 2 mM for 24 hours,and simultaneously detected the expression levels of autophagy and inflammation-related molecules.Compared with the METH group,MMT4 in the METH＋3MA group did not change,the autophagy-related molecules decreased significantly,and the inflammatory molecules decreased significantly.in conclusion:1.METH treatment of BV2 cells increased MDM4 and autophagy pathway molecules.After METH treatment of BV2 cells,knockdown of MDM4 and treatment with MDM4 inhibitor decreased MDM4,and autophagy and inflammatory pathway molecules decreased,suggesting that MDM4 regulates autophagy and inflammation.After METH treatment of BV2 cells,MTG4 was not significantly altered after knockdown of ATG5 and treatment with autophagy inhibitor.The autophagy and inflammatory pathway molecules decreased,suggesting that autophagy is located downstream of MDM4 and upstream of the inflammatory pathway.Part Ⅱ The mechanism of luteolin in relieving the hepatotoxicity of methamphetamineBackground:While studying the central nervous system damage of methamphetamine,our research group also targeted the screening of natural drugs that antagonize the central nervous system toxicity of methamphetaline.Luteolin is a natural compound of flavonoids,mainly found in fruits,vegetables and herbs.A large number of reports have reported that luteolin has many biological activities such as anti-inflammatory,anti.tumor and anti-oxidation,especially in The role of the central nervous system:Fu,H.Sun,Y et al reported that luteolin can reduce the central nervous system inflammation,oxidative stress and apoptosis caused by spinal cord ischemia-reperfusion injury;Y.Kwon reports Luteolin may be a potential therapeutic drug for Alzheimer’s disease;SF Nabavi et al reported that luteolin can alleviate cognitive impairment caused by α βpeptide in rats;M.Zuiki et al confirmed that luteolin can be relieved by Astrocyte proliferation induced by IL-6 secreted by differentiated neurons of pluripotent stem cells.Based on the above literature reports that luteolin relieves central nervous system damage,we tried to hypothesize that luteolin can alleviate the neurotoxicity caused by METH.Because luteolin mainly relies on the active ingredients that are metabolized in the body to exert its biological effects.Therefore,it is verified that the model of luteolin to alleviate the central nervous system toxicity caused by METH is carried out in animals.Unfortunately,many previous pre-experiments did not find that luteolin can alleviate the neurotoxicity caused by METH.Unexpectedly,during the systemic pathological examination of pre-experimented rats,the liver caused by METH to the liver was found.Toxicity can be alleviated by luteolin.After repeated animal experiments,pathology showed that luteolin can effectively alleviate the hepatotoxicity caused by METH,and there are also reports that luteolin can alleviate the poisoning drugs such as carbon tetrachloride,mercuric chloride and alcohol.The resulting liver damage proves the liver protection effect of luteolin.However,the specific mechanism by which luteolin relieves the hepatotoxicity caused by methamphetamine is unknown.Qualcomm’s second-generation sequencing RNA-seq technology provides the possibility to explore possible regulatory molecules and pathways.Therefore,this study used RNA-seq to study the molecular mechanism and regulatory network of luteolin to slow down the hepatotoxicity induced by METH.Purpose:This study intends to establish a model of hepatotoxicity in METH animals,on the basis of which it can be used to detect the hepatotoxicity of luteolin against METH,and use transcriptome sequencing technology to detect the transcription of genes that may regulate the hepatotoxicity of METH.Expression,potential pathways and possible molecular mechanisms to further study the molecular mechanism and regulatory network of luteolin on the hepatotoxicity induced by METH.Method:1.Luteolin reduces the establishment of animal model of hepatotoxicity induced by METH200g-220g SD male rats were selected,control group,METH group,luteolin plus METH group,no control in the control group,METH group,administered at 15mg/kg,once every 12 hours,a total of 8 needles In the luteolin + METH group,the first 3 days of luteolin was intragastrically administered 100 mg/kg each time for three consecutive days,followed by METH treatment,liver tissue fixation,HE staining,serum and AST in serum.ALT concentration.2.RNA-seq results analysisThe liver tissues of the rats in the model were established and stored at-80℃.Each group consisted of 3 samples.The three groups were the control group,the METH group,the luteolin plus the METH group,and the total RNA was extracted.The machine was sequenced,and the preliminary results were analyzed after sequencing.3.RNA-seq results verificationThe differentially expressed genes in the results were selected,and the gene expression was verified by qRT-PCR to verify the reliability and reproducibility of the sequencing results.Result:1.Luteolin reduces METH-induced hepatotoxicity(1)Histopathology showed that the liver tissue of the blank control group was intact,and the diffuse balloon-like changes were observed in the liver tissue of the METH group.The degree of the luteolin + METH group was between the blank control group and the METH group,which was lighter.The cell edema-like changes.(2)Detection of biochemical indicators,AST and ALT in serum,AST and ALT in blank control group were normal,AST and ALT levels in METH group were significantly increased,and AST and ALT in serum of luteolin + METH group were in blank control group.There was a significant decrease between the METH group and the METH group.2.RNA-seq results analysis1859 DGEs were identified in the METH treated group,including 873 up-regulated and 786 down-regulated DGEs compared to the control group.At the same time,899 up-regulated and 978 down-regulated DGEs were filtered in the group pretreated with luteolin compared to the METH-treated group.Finally,it was found that 497 DGEs including 314 up-regulations and 183 down-regulated DGEs may have protective effects.Using 497 differentially expressed genes for GO analysis and KEGG analysis,eight pathways were enriched by KEGG pathway analysis,oxidative phosphorylation,nonalcoholic fatty liver disease(NAFLD),PPAR signaling pathway and AMPK signaling pathway were mainly Enrichment pathway.3.RNA-seq results verificationTo confirm the accuracy of the mRNA sequencing results,6 genes were selected to verify the sequencing results using quantitative real-time PCR.Leap2,Fasn,Fabp5 and Pnpla3 were up-regulated DGE in the METH group compared to the control group,and Mbp and Calm3 were genes that did not change in the 3 groups.The results of qRT-PCR were basically consistent with the results of 1RNA sequencing.in conclusion:1.Luteolin can effectively alleviate hepatotoxicity caused by METH2.497 differentially expressed genes may be involved in the hepatotoxicity of luteolin,and the possible pathways and potential mechanisms are analyzed based on this.2.The results of qRT-PCR are basically consistent with the results of mRNA sequencing,suggesting that the RNA-Seq results have high accuracy and reproducibility. |
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