The Molecular Mechanism Of Emodin Treating Acute Kidney Injury Based On Network Pharmacology And Endoplasmic Reticulum Stress Pathway

First PartOBJECTIVE: To dig out targets related to emodin and targets related to acute kidney disease based on network pharmacology.Finding out targets are shared with emodin and acute kidney disease by related software,and using enrichment analysis predicts the related mechanism of emodin in the treatment of acute kidney disease.METHOD: To dig out targets related to emodin with TCMSP database、Pubchem database、Zinc database、Swiss Target Prediction database、STITCH database.To dig out targets related to acute kidney disease with OMIN database、TTD database、GAD database、Di GSe E database.To establish protein-protein network and dig out core targets with cytoscape software and bisogenet and Cyto NCA plug-in.To analyze pathways of core targets with DAVID and Omicshare online instrument.RESULT: We found 37 targets related to emodin by pharmacological database,and we got 2954 related targets after topology analysis.We found 40 targets related to acute kidney disease,and we got 1804 related targets after topology analysis.We got 699 targets shared with emodin and acute kidney disease with Cytoscape analysis.Finally,242 core targets were found by Cyto NCA analysis.Endoplasmic reticulum stress apoptosis pathways was related to emodin and actue kidney disease after enrichment analysisCONCLUSION:(1)There are many targets associated with emodin and actue kidney disease based on network pharmacology.(2)Acute kidney disease treatment with emodin is associated with apoptosis induced by endoplasmic reticulum stress based on enrichment analysis.(3)Network pharmacology provide new methodology to study molecular mechanisms of emodin.Second PartOBJECTIVE:To study effect of emodin on H/R HK-2 cells model for vitality and apoptosis.METHODS: To establish H/R HK-2 cells model and divide into control group(without establishing model),model group(establishing H/R model)and emodin groups with 10μM、30μM、50μM、80μM concentration(establishing H/R model).Different concentration of emodin was added into medium after establishing H/R model.CCK-8 was added at 4h and 12 h after establishing H/R model to detect vitality of HK-2 cells.According to the result of CCK-8,best concentration of emodin was chosen for later experiment.We reestablished H/R HK-2 cells model and we divided into control group,model group and emodin group based on chosen emodin concentration.Apoptosis of HK-2 cells was detected with CFSE/PI at 4h and 12 h after establishing H/R model.RESULT: Decline of vitality of HK-2 cells was observed at 4h and 12 h after establishing H/R HK-2 cells model.Emodin with 10μM、30μM、50μM、80μM concentration improved vitality of HK-2 cells at 4h and 12 h after establishing H/R model.Emodin with 30μM concentration improved vitality of HK-2 cells with most obvious effect.Therefore,emodin with 30μM concentration was chosen for later experiment.The rate of apoptosis of HK-2 cells significantly increased at 4h and 12 h in H/R model.Emodin can inhibit rate of apoptosis of HK-2 cells in H/R model.CONCLUSION:(1)Establishing H/R injury model can cause declined vitality of HK-2 cells and increased apoptosis of HK-2 cells.(2)Emodin can increase the vitality cells and inhibit apoptosis in HK-2 cells induced by H/R.Third PartOBJECTVIE:To study the effect of emodin on endoplasmic reticulum stress related apoptosis pathway of HK-2 cells induced by H/R.METHOD: To establish H/R HK-2 cell model and divide into control group,model group and emodin group.Rt-q PCR and western blot assay were used to detect the GRP78 m RNA and protein,which is biomarker of endoplasmic reticulum stress,and biomarker of downstream of endoplasmic reticulum stress including CHOP、JNK、P53、caspase-12 m RNA and protein,and biomarker of apoptosis including caspase-3、Bcl-2、caspase-9 m RNA and protein.RESULT: At 4h and 12 h after establishing H/R model,the expression of m RNA and protein of GRP78、JNK、P53、caspase-12 and proapoptotic biomarker caspase-3、caspase-9 were increased,however,reduced expression of m RNA and protein of inhibiting apoptotic biomarker bcl-2.Emodin can inhibit expression of JNK、P53、caspase-12、caspase-3、caspase-9 and increase expression of bcl-2.CONCLUSION:(1)H/R model can cause endoplasmic reticulum stress in HK-2 cells and activated downstream of endoplasmic reticulum stress including caspase-12、JNK、P53.(2)H/R model can increase proapoptotic biomarker caspase-3、caspase-9 and reduced expression of inhibiting apoptotic biomarker bcl-2.(3)Emodin can inhibit expression of JNK and P53,which as downstream biomarker of endoplasmic reticulum stress,in HK-2 cells induced by H/R.(4)Emodin can inhibit expression of proapoptotic biomarker caspase-3 、 caspase-9 and increase expression of inhibiting apoptotic biomarker bcl-2.