The Regulatory Mechanism Of Tongfuyangsui Decoction On Autophagy Of SH-SY5Y Cells Induced By High Copper Based On LKB1-AMPK Pathway

Wilson disease（WD）,WD is a kind of autosomal recessive genetic disease caused by mutations in human ATP7B gene.The disorder of copper metabolism caused by mutant genes causes excessive copper deposition in brain,liver,kidney,cornea and other tissues and organs.Patients may have clinical manifestations such as liver damage,nervous system symptoms,corneal K-F ring and so on.The treatment of WD patients with nervous system symptoms,especially tremor,dystonia,dysphagia and torsion spasm,has always been a difficult problem for clinicians.In previous clinical studies,our team established Tongfu Yangsui decoction（TFSYF）according to the TCM pathogenesis characteristics of early copper cohesion,damp-heat of liver and gallbladder,deficiency of liver and kidney and deficiency of qi and blood in patients with WD.The prescription has achieved a good effect in improving the neurological symptoms of patients with brain type,and has been verified in the experiment of neuroprotective effect of disease animal model TX mice.However,these histological studies can not fully clarify the specific mechanism of TFSYF on nerve cells injured by copper.Autophagy is a process in which damaged organelles and misfolded proteins are presented to lysosomes for digestion and degradation to meet the needs of substance metabolism.this physiological mechanism plays an important role in the pathophysiological process of tumors,neurodegenerative diseases and aging-related diseases.In many autophagy pathways and important regulatory proteins,LKB1-AMPK pathway is an important signal pathway to regulate autophagy.Some studies have shown that copper excessive can eventually lead to cell death by inducing autophagy and apoptosis.further studies have found that copper and dopamine complexes can induce rat dopaminergic neurons to autophagy,resulting in autophagic apoptosis.Neuroimaging in patients with brain-type WD suggests that most patients with brain-type WD have imaging findings of lesions in the basal ganglia,and it has been found that the damage of dopaminergic neurons in the ventral tegmental area caused by excessive copper is an important pathological mechanism of brain injury in patients with WD.Based on this,we propose a hypothesis that TFSYF changes the autophagy effect of dopaminergic neurons induced by excessive copper by regulating LKB1-AMPK pathway.The following research will focus on this hypothesis in order to verify it.This study will provide an important theoretical basis for the protective mechanism of nerve cells in Tongfu Yangsui decoction.Objective:1.To study the protective effect and autophagy effect of TFSYF recipe on dopaminergic neurons induced by excessive copper.2.To verify whether TFSYF has a protective effect on dopaminergic neurons by regulating autophagy based on LKB1-AMPK pathway.Methods:1.MTT method was used to detect the effects of different concentrations of Cu SO₄and different time points on the survival rate of SH-SY5Y cells,and the half inhibition rate of cell proliferation（IC50）was selected to establish the WD nerve cell model.2.Preparation of SD rat serum containing Tongfu Yangsui decoction.3.MTT method was used to screen the best protective concentration of different concentrations of rat serum containing TFYSF on SH-SY5Y cells of WD nerve cell model.4.Construction of AMPK gene silencing and overexpression stable transformants in SH-SY5Y cells by lentivirus transfection technique.5.RT-PCR and Western Blot methods were used to verify the success of the construction of AMPK transgenic cells.6.Grouping:Grouping:（1）the first part is divided into three groups.Normal group:normal cultured SH-SY5Y cells were given 10%blank SD rat serum;model group:cultured SH-SY5Y cells were given 10%SD rat serum at first,and then added the optimal concentration of Cu SO₄;treatment group 1 hour later:the cultured SH-SY5Y cells were given 10%SD rat serum containing TFYSF at first,and then the optimal concentration of Cu SO₄was added 1 hour later.（2）the second part is divided into seven groups.Normal group:normal cultured SH-SY5Y cells were given 10%blank SD rat serum,model group:10%blank SD rat serum was given to cultured SH-SY5Y cells,and then the optimal concentration of Cu SO₄;was added 1 hour later.The cultured SH-SY5Y cells were given 10%TFYSF rat serum at first,and then the optimal concentration of Cu SO₄;was added 1 hour later.AMPK gene silencing model group（AMPK-M）:AMPK-type transgenic SH-SY5Y cells were added with 10%blank SD rat serum,and then the optimal concentration of Cu SO₄;AMPK gene silencing treatment group（AMPK-T）was added 1 hour later:AMPK-type transgenic SH-SY5Y cells were added with 10%TFYSF rat serum,and then the optimal concentration of Cu SO₄;was added 1 hour later.AMPK gene overexpression model group（AMPK+M）:AMPK+type transgenic SH-SY5Y cells plus 10%blank SD rat serum,1 hour later added the optimal concentration of Cu SO₄;AMPK gene overexpression treatment group（AMPK+T）:AMPK+type transgenic SH-SY5Y cells plus 10%TFYSF rat serum,and then added the optimal concentration of Cu SO₄1 hour later.7.The survival rate of cells was detected by CCK8 method.8.The leakage level of lactate dehydrogenase LDH in the cell supernatant was detected by lactate dehydrogenase release assay.9.The survival and death of cells were observed by fluorescence microscope after Calcein-AM/PI staining.10.The relative expression level of reactive oxygen species ROS stained by DCFH-DA fluorescent probe was detected by flow cytometry.11.The level of superoxide dismutase SOD was detected by biochemical-enzyme labeling method.12.Observation of cell ultrastructure by transmission electron microscope.13.Detection of fluorescence intensity of intracellular LC3 protein by immunofluorescence staining.14.The expression levels of LC3-Ⅱprotein,AMPK protein,P-AMPK protein,LKB1protein and m TOR protein were detected by Western Blot.Results:1.The dose detection of Cu SO₄model made by MTT screening showed that Cu SO₄had a certain dose-effect and time-effect relationship on the survival rate of SH-SY5Y cells.When the dose of Cu SO₄increased and the action time prolonged,the cell survival rate decreased gradually（P<0.01）.The cell survival rate after treatment with 527μM for 24hours was 49.89±8.2%,which was close to the semi-inhibition rate of cell proliferation.2.The concentration of serum containing TFYSF screened by MTT showed that 10%of the serum containing TFYSF had the highest survival rate of SH-SY5Y cells induced by Cu SO₄.3.Fluorescence expression of lentivirus transfected cells:there was almost no expression in the normal control group under the fluorescence inverted microscope,while the expression of AMPK in the transgenic experimental group was more obvious under the fluorescence inverted microscope.4.The results of RT-PCR and WB detection showed that the relative expression of AMPK gene and AMPK protein in the AMPK gene silent experimental group was significantly lower than that in the control group（P<0 01）.The relative expression of AMPK gene and AMPK protein in the);AMPK gene overexpression experimental group was significantly higher than that in the control group（P<0 01）.5.The cell survival rate detected by CCK8:compared with the model group,the cell survival rate of the treatment group was significantly higher than that of the model group（P<0.01）,but there was no difference between the AMPK-M group and the model group（P>0.05）,and the cell survival rate of the AMPK+M group was higher than that of the model group（P<0.05）.Compared with the treatment group,the cell survival rate of the AMPK-T group was significantly lower than that of the treatment group（P<0.01）,and the cell survival rate of the AMPK+T group was higher than that of the treatment group（P<0.05）.6.The leakage rate of LDH showed that the level of LDH in the supernatant of the model group was significantly higher than that in the normal group（P<0.01）,the leakage level of LDH in the supernatant of the treatment group was lower than that in the model group（P<0.05）,there was no difference in the AMPK-M group（P>0.05）,and the leakage rate of LDH in the AMPK+M group was lower than that in the model group（P<0.05）.Compared with the treatment group,the leakage rate of LDH in AMPK-T group was significantly lower than that in AMPK+T group（P<0.01）.The leakage rate of LDH in AMPK+T group was significantly lower than that in treatment group（P<0.01）.7.Calcein-AM/PI staining:in the normal group,the morphology of cells was regular,and the green fluorescence area was significantly more than the red area,indicating that the number of living cells was significantly more than that of dead cells;in the model group,the number of red fluorescence area was slightly more than that of green fluorescence area,indicating that the number of dead cells was slightly more than that of living cells in the treatment group,which indicated that the number of living cells in the treatment group was more than that of dead cells.There was no significant difference between the red fluorescence region and the green fluorescence region in the AMPK-M group,indicating that there was no difference in the number of dead cells and living cells;in the AMPK-T group,the number of living cells was slightly more than that in the red fluorescence region,indicating that the number of living cells in the AMPK+M group was more than that in the red area,indicating that the number of living cells in the experimental group was more than that of dead cells.In the AMPK+T group,the green fluorescence area was significantly more than the red area,indicating that the number of living cells in the visual field area was significantly more than that of dead cells in this group.8.The level of ROS was detected by flow cytometry:compared with the normal group,the level of ROS in the model group was significantly higher than that in the model group（P<0.01）,the level of ROS in the treatment group was significantly lower than that in the model group（P<0.01）,the level of ROS in the AMPK-M group was higher than that in the AMPK-M group（P<0.05）,and the ROS level in the AMPK+M group was significantly lower than that in the model group（P<0.01）.Compared with the level of ROS in the treatment group,the level of ROS in the AMPK-T group was significantly higher than that in the treatment group（P<0.01）,but there was no significant difference in the ROS level in the AMPK+T group（P>0.05）.9.Determination of SOD level:compared with the normal group,the level of SOD in the model group was significantly lower than that in the normal group（P<0.01）,the level of SOD in the treatment group was significantly higher than that in the model group（P<0.01）,there was no difference in the level of SOD in the AMPK-M group（P>0.05）,and the level of SOD in the AMPK+M group was significantly higher than that in the normal group（P<0.01）.The level of cellular SOD in AMPK-T group was significantly lower than that in treatment group（P<0.01）,but there was no significant difference in cellular SOD level between AMPK+M group and treatment group（P>0.05）.10.The ultrastructure of the cells was observed by transmission electron microscope:in the normal group,the morphology and structure of nucleus and mitochondria were normal,the nuclear membrane was clear,and autophagosomes could not be seen in the cells.In the model group,a large number of vacuoles could be seen,the nuclear morphology was irregular,the nuclear membrane was not clear,the number of mitochondria decreased,the structure of mitochondria was irregular and vacuolated,the contents could be seen in some vacuoles,and some mitochondria were wrapped into the vesicles.More autophagosomes can be seen in the cells.In the treatment group,the nuclear morphology and structure was more regular,the nuclear membrane was not clear,the organelles were abundant,the mitochondria were slightly swollen and the number of autophagosomes increased significantly.In AMPK-M group,the shape of nucleus was irregular,the nuclear membrane was clear,the structure of mitochondria was damaged seriously and the number was small,some mitochondria were vacuolated and a small amount of autophagosomes could be seen.In AMPK-T group,the shape of nucleus was irregular,the nuclear membrane was clear,the number of mitochondria was less and the structure was incomplete,and the number of autophagosomes was more.In the AMPK+M group,the nuclear morphology was irregular and the nuclear membrane was clear,with a large number of slightly swollen mitochondria,some mitochondria vacuolated and a large number of autophagosomes.In AMPK+T group,the nuclear morphology was regular and the nuclear membrane was clear,with a large number of slightly swollen mitochondria,no vacuoles and a large number of autophagosomes.11.LC3 immunofluorescence expression:compared with the normal group,the LC3immunofluorescence intensity of the model group was significantly higher than that of the model group（P<0.01）,the LC3 immunofluorescence intensity of the treatment group was significantly higher than that of the model group（P<0.01）,the LC3immunofluorescence intensity of the AMPK-M group was significantly lower than that of the model group（P<0.01）,and the LC3 immunofluorescence intensity of the AMPK+M group was higher than that of the model group（P<0.05）.Compared with the treatment group,the LC3 immunofluorescence intensity of AMPK-T group was significantly lower than that of the treatment group（P<0.01）,but there was no significant difference in LC3 immunofluorescence intensity of AMPK+T group（P>0.05）.12.The expression of autophagy effector protein LC3 was detected by WB:compared with the normal group,the expression level of LC3-Ⅱprotein in the model group was significantly higher than that in the model group（P<0.01）,the expression level of LC3-Ⅱprotein in the treatment group was significantly higher than that in the model group（P<0.01）,and the expression level of LC3-Ⅱprotein in the AMPK-M group was significantly higher than that in the model group（P<0.01）.The expression of LC3-Ⅱprotein in AMPK+M group was significantly higher than that in control group（P<0.01）.Compared with the treatment group,the expression of LC3-Ⅱprotein in AMPK-T group was significantly decreased（P<0.01）,but there was no significant difference in LC3-Ⅱprotein expression in AMPK+T group（P>0.05）.13.The expression levels of AMPK protein,P-AMPK protein,LKB1 protein and m TOR protein were detected by WB:compared with the normal group,the expression levels of AMPK,P-AMPK,m TOR and LKB1 in the model group were significantly higher than those in the normal group（P<0.01）,and the expression levels of AMPK,P-AMPK,m TOR and LKB1 in the treatment group were significantly higher than those in the normal group（P<0.01）.Compared with the model group,the protein expression levels of AMPK and P-AMPK in the treatment group were significantly higher than those in the model group（P<0.01）;The expression levels of AMPK,P-AMPK and m TOR in the AMPK-M group were significantly lower than those in the model group（P<0.01）,but there was no difference in the expression of LKB1 protein between the two groups（P>0.05）;In AMPK+M group,there was no significant difference in the expression of LKB1 protein between the AMPK+M group and the model group（P>0.05）,AMPK protein increased（P<0.05）,and the expression level of P-AMPK protein increased significantly（P<0.01）,while m TOR protein decreased significantly（P<0.01）.Compared with the treatment group,the expression level of AMPK,P-AMPK and m TOR protein in AMPK-T group was significantly lower than that in the treatment group（P<0.01）,but there was no difference in the expression level of LKB1 protein（P>0.05）.The protein expression levels of AMPK,P-AMPK and m TOR in AMPK+T group were significantly lower than those in treatment group（P<0.01）,but there was no significant difference in LKB1 protein expression between the two groups（P>0.05）.Conclusion:1.High copper environment can enhance the level of oxidative stress and reduce the effect of antioxidation in SH-SY5Y cells.Tongfu Yangsui decoction can significantly change the phenomenon of excessive intracellular oxidation in high copper environment.2.High copper environment can induce autophagy in SH-SY5Y cells,but this autophagy effect is not enough to resist the damage caused by oxidative stress.Tongfu Yangsui decoction can improve the autophagy effect of SH-SY5Y cells to reduce the level of intracellular oxidation and achieve the protective effect on cells.3.The cell experiment of AMPK stable transgenic strain showed that Tongfu Yangsui decoction further activated autophagy of SH-SY5Y cells by regulating LKB1-AMPK signal pathway,and then played a role in protecting SH-SY5Y cells.