The Study Of The Inhibitory Effect Of Sanguinarine On Mitophagy And Its Mechanism

Sanguinarine(SAN)is a plant-derived isoquinoline alkaloid,which has been reported to have a wide range of biological activities.This study found that sanguinarine has the activity of inhibiting mitochondrial autophagy,and mitochondrial autophagy is an important process for cells to control the quality of mitochondria and eliminate damaged mitochondria.Whether it is too high or too low,the level of mitochondrial autophagy will seriously affect cells.Normal physiological functions cause a series of diseases.In addition,mitochondrial dysfunction will lead to abnormal function and development of high-energy-demanding organs.The heart and brain are the two most energy-consuming organs in the organism.Therefore,this article also focuses on the cardiotoxicity and neurotoxicity caused by sanguinarine.In addition to research,we have also conducted a systematic study on the developmental toxicity of sanguinarine.In order to study the inhibitory effect of SAN on mitochondrial autophagy and its molecular mechanism,as well as its developmental toxicity to the nervous system,heart development and other organs,we conducted the following studies:Methods and results:1.The inhibitory effect of SAN on mitochondrial autophagyWe use SAN and a traditional mitochondrial autophagy model agent carbonyl cyanide metachlorophenylhydrazone(CCCP)to treat the Hela-PINK1 and Hela-Parkin cell lines.After the treatment is completed,the cells were observed and recorded under the microscope to study the influence of PINK1 and Parkin on the distribution of two mitochondrial autophagy-related proteins.In addition,we conducted experiments with concentration dimension and time dimension variables to determine the concentration-and time-dependence of SAN treatment on mitochondrial autophagy.Western Blot was used to study the effect of SAN on the expression levels of autophagy marker proteins,such as LC3 B,P62 and mitochondrial outer membrane receptor protein TOM20.We furtherly clarified the underlying molecular mechanism of SAN in the inhibition of mitochondrial autophagy.Experimental results show that CCCP can cause mitochondrial autophagy,lead to mitochondrial co-localization of PINK1 and Parkin in the early stage of mitochondrial autophagy,up-regulate the expression of autophagy marker proteins,such as LC3 B,P62 and mitochondrial outer membrane protein TOM20.While SAN treatment can significantly inhibit the mitochondrial autophagy and offset all the changes induced by CCCP including the co-localization of PINK1 and Parkin in mitochondria and the up-regulated expression of autophagy marker proteins.2.SAN induces Parkinson’s disease and neurotoxicity by inhibiting mitochondrial autophagyWe used 24-144 hpf zebrafish to evaluate the neurotoxicity of SAN,and selected1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP,a traditional nerve injury modeling agent)as a positive control.Zebrafish were divided into two groups treated with SAN alone,SAN and N-Acetyl-L-cysteine(N-Acetyl-L-cysteine,NAC,a kind of ROS antioxidant),relatively.After treatment,the development of neuron and brain vascular were tested.Apoptotic staining in brain and behavioral test were performed.The expression level of genes related with nerve damage and mitochondrial autophagy were detected by q PCR.In addition,in order to furtherly study the underlying mechanism of SAN toxicity,PC12 nerve cells were used for the experiments in vitro.Following treatment of PC12 cells with SAN and NAC,apoptosis detection with Hoechst33342-PI double staining was performed,Ed U Proliferation level and ROS reactive oxygen levels were also detected.In addition,the protein level changes of α-syn,TH,caspase3,PARP,BAX,Bcl2,PINK1 and Parkin in PC12 cells caused by SAN and NAC treatment were examined by Western Blot.The results showed: Compared with the blank control group,the development of zebrafish neurons in the MPTP positive control group were delayed and lacked,accompanied with increased brain cell apoptosis,loss of blood vessels,and Parkinson-like behaviors,mitochondrial autophagy and nerve damage.The gene expression profiles in PC12 cells are altered compared with the MPTP treatment group.SAN also caused a similar phenomenon,which appear concentration-dependent.But after NAC was added together with SAN,the level of apoptosis decreased significantly,indicating that ROS is involved in the process of SAN-induced apoptosis.The results of in vitro toxicity studies show that SAN can induce PC12 cell apoptosis and increase the level of ROS reactive oxygen species,and decrease the proliferation ability.After adding NAC,the level of ROS and the level of apoptosis in PC12 cells decrease significantly,which indicates that ROS mediates lead to the occurrence of apoptosis.3.SAN causes cardiotoxicity through mitochondrial autophagyWe used 48-96 hpf zebrafish juveniles to evaluate the cardiotoxicity of SAN in the body.Zebrafish at 48 hpf were treated with SAN and NAC until to 96 hpf,and then embryonic development,structure and function development of heart and inferior intestinal veins(SIVs)development were examined.Meanwhile,heart histopathological section staining,blood cell staining,apoptotic cells staining in the heart,and detection for the expression levels of genes envolved in heart development,angiogenesis,and apoptosis were performed.In addition,in order to further study the toxic mechanism of SAN,we selected the cardiomyocyte HL-1 cell line to study the toxicity of SAN in vitro.We treated HL-1 cells with SAN and NAC,and then performed Hoechst33342-PI double staining.apoptosis detection,Ed U proliferation level detection and ROS level detection.In addition,the protein levels of PUMA,caspase3,PARP,BAX,Bcl2,caspase9,p-P38,p-P38,p-Erk and p-JNK in HL-1 cells caused by SAN and NAC treatment were detected by Western Blot.The results show that SAN can cause structural and functional abnormalities during heart development,including decreased heart rate,decreased red blood cell count,hemodynamic changes,cardiomyocyte apoptosis,increased SV-BA distance,and intestinal venous congestion.By TUNEL staining and AO staining,cell apoptosis in the heart region of zebrafish was observed.In addition,after SAN treatment,the expression level of genes which play a key role in heart development and function were altered,such as: sox9 b,myl7,nkx2.5 and bmp10.And apoptosis-related genes,such as caspase3,caspase9,bax and bcl2,were also altered by SAN.The results of studies in vitro on SAN cardiotoxicity indicate that SAN can induce apoptosis of HL-1 cells and a significant increase in ROS levels.In addition,the MAPK pathway(JNK and P38)is significantly enhanced and participates in SAN-induced cardiotoxicity.Our findings will be helpful for better understanding the toxic effects of SAN on the heart.4.Developmental toxicity in zebrafish caused by SANIn order to have a more comprehensive understanding of the toxic effects of SAN,a systematic developmental toxicity study was carried out using 4hpf zebrafish for SAN treatment until to 96 hpf.The embryo developmental toxicity was evaluated according to the mortality,deformity rate,and hatching rate of embryos in each group.The developmental toxicity of individual organs such as cardiac,liver and neuron were examined.Zebrafish behavioral test was also perfomed.For further mechanism research,antioxidant system,oxidation level and apoptotic cells in zebrafish body were detected,relatively.The expression levels of genes involved in Nrf2 pathway,Wnt pathway,apoptosis and mitochondrial autophagy were detected by q PCR..The results show that compared with the control group,SAN treatment can cause a significant increase in deformity rate,pericardial edema and spinal curvature.The hatch ability and body length of zebrafish juveniles in the SAN-treated group were significantly reduced.SAN also affects the development of the heart,liver and nervous system.Further studies showed that the level of ROS reactive oxygen species increased significantly,the activity of total superoxide dismutase decreased significantly,and the concentration of malondialdehyde increased significantly.Compared with the control group,more apoptotic cells were detected in the SANtreated group.In addition,the gene expression results showed that in the SAN-treated group,oxidative stress and apoptosis pathways were induced,while Nrf2 and Wnt pathways were inhibited.In summary,these results will help to understand the toxicity caused by SAN and its potential molecular mechanism.