| Objective: Different doses of monobutyl phthalate(MBP),mono(2-ethyl)hexyl phthalate(MEHP)and their combined exposure in vitro to intervene in mouse testicular stromal cells(TM-3),And then explore the effects of MBP and MEHP on TM-3 autophagy,the phosphatase deleted on chromos ome 10 and the tensin homologous protein / threonine kinase(PTEN / AKT)pathway,and explore the interaction of joint infection And the possible relationship between autophagy and apoptosis.Methods: Method: 1,pre-experiment CCK-8 experiment setting MBP,MEHP concentration 0(control group),50,100,200,400,800μmol / L,acting on TM-3 cells for 24 h,refer to the half inhibition calculated by modified Kou’s method Dose to determine the subsequent exposure dose;set MBP,MEHP concentration 0,200,400,800 μmol / L and MBP + MEHP concentration 400 μmol / L,use the CCK-8 method to detect the infection time set to 24,36,48 h MBP,The effect of MEHP on the activity of TM-3 cells;2.Use inverted phase contrast microscope to observe the effect of each expo sure group on the normal morphology of TM-3 cells;3.The MDC fluorescence method was used to detect the different concentrations of MBP,MEHP,and MBP + MEHP on TM-3.Induction of cell autophagy;4.Observe the changes of cell ultrastructure after transmission electron microscope;5.Use Annexin V-FITC / PI method to detect the changes of TM-3 cell apoptosis in each exposure group;6,Western blot was used to detect TM-3 cell autophagy-related protein microtubule-associated protein 1 light chain 3(LC3),ubiquitin-binding protein p62(p62),PTEN,AKT and apoptosis-related protein Bcl-2 related X protein(Bax),B lymphoma-2(Bcl-2)protein relative content changes.Results: 1.The results of CCK-8 showed that compared with the control group,the cell survival rate decreased with the increase of the MBP and MEHP exposure dose(P<0.05).The results of MDC method showed that compared with the control group,the signal intensity of 200 μmol / L MBP and MEHP group had no statistically significant difference(P>0.05);the signal intensity of 400,800 μmol/L MBP and MEHP group was not statistically significant.As the concentration increases,it increases(P<0.01).Western blot analysis showed that compared with the control group,the relative content of autophagy-related proteins LC3 II / LC3 I and p62 protein in the 200 μmol / L MBP and MEHP groups was not statistically significant(P>0.05);400,800 μmol / L MBP The relative content of LC3 II / LC3 I and PTEN protein in MEHP group increased(P <0.05);the relative content of p62 protein in 200 μmol / L MBP group was not statistically different from that of control group(P>0.05),400,800 μmol / L The relative content of p62 protein in MBP group decreased;the relative content of p62 protein in 200,400,and 800 μmol / L MEHP group decreased;the relative content of p-AKT / AKT protein in MBP and MEHP group decreased(P<0.01).2.Compared with the control group,the results of CCK-8 showed that the survival rate of TM-3 cells infected with MBP and/or MEHP decreased;the results of electron microscope observation of cell structure changes were as follows: control group: mitochondria were visible in the cytoplasm The endoplasmic reticulum structure is relatively complete,and vacuoles are occasionally seen;MBP exposure group: autophagosomes with a complete double membrane structure are seen,the number of mitochondria is reduced,t he endoplasmic reticulum cavity is expanded,and the memb rane-like structure in the cytoplasm is increased.Increased lysosomes;MEHP-infected group: the phenomenon of cellular vacuolization was further intensified,the number of organelles in the cytoplasm was severely reduced,and autophagolysosomes were visib le;MBP + MEHP combined-infected group: intact mitochondria were still visible in the cytoplasm,There is vacuolation,multivesicular structure,and autophagosomes can be seen.The MDC results showed that the fluorescence signal intensity of each exposure group increased.Western blot analysis showed that the relative content of LC3 II / LC3 I protein increased(P<0.01),the relative content of p62 protein decreased(P<0.01),and the relative content of PTEN protein in MBP,MEHP and MBP + MEHP exposure grou ps compared with the control group.Increase(P<0.05),the relative content of p-AKT / AKT protein decreased(P <0.05).Analysis of the interaction between MBP and MEHP combined infection by 2 × 2 factorial design variance showed that the activity of MBP and MEHP combined infection on TM-3 cells,the fluorescence signal intensity of autophagic vesicles,LC3 II / LC3 I and p62 protein The expression levels had interactions,and the positive reaction rate o f the combined MBP + MEHP exposure group <MBP exposure group positive reaction rate + MEHP exposure group positive reaction rate,and the interactions all showed antagonism(P <0.01).3.The results of CCK-8 showed that compared with the control group,the cell survival rate increased after 24 h of 50 μmol / L MBP + MEHP exposure;and the cell survival rate decreased with the increase of MBP + MEHP dose(P <0.05).The cell survival rate of 3-MA at the concentration of 1 mmol / L was not statistically different from that of the control group(P> 0.05);the cell survival rate decreased with the increase of the concentration of 3-MA in the intervention agent(P<0.05).MDC results showed that the fluorescence signal intensity of the control group was low,the fluorescence signal intensity of the MBP + MEHP exposure group increased,and the fluorescence signal intensity of the 3-MA + MBP + MEHP intervention group was lower than that of the MBP + MEHP group,but higher than the control group;Annexin V-FITC / PI method was used to detect the change of apoptosis in each treatment group.The results showed that: compared with the early apoptosis rate of the control group,the early apoptosis rate of cells in the MBP + MEHP group inc reased(P<0.05);compared with MBP + MEHP Compared with the group,the early apoptosis rate of cells in the 3-MA + MBP + MEHP intervention group increased(P<0.05).Western blot analysis showed that compared with the control group,the relative content o f autophagy marker protein LC3 II / LC3 I protein increased,the relative content of p62 protein decreased(P<0.01),and the relative content of apoptosis-related protein Bax increased in the MBP + MEHP exposure group.The relative content of Bcl-2 decreased(P<0.01);compared with the MBP + MEH P group,the expression of autophagy-related protein in the 3-MA + MBP + MEHP group was: the content of LC3 II / LC3 I decreased(P<0.05),and the content of p62 increased(P<0.01),the apoptosis-related protein Bax increased(P<0.05),and Bcl-2 decreased(P<0.01).Conclusion: 1.MBP,MEHP,MBP + MEHP can destroy the basic morphology of TM-3 cells,destroy the endoplasmic reticulum,mitochondria an d other ultrastructures,and induce the production of autophagosomes in the cytoplasm;2.MBP,MEHP and MBP + MEH P are infected Increased autophagy levels in TM-3 cells,and PTEN positively regulates cell autophagy by inhibiting AKT phosphorylation;3.The interaction of MBP + MEHP combined infection appears to be antagonistic;4.Caused by MBP + MEHP infection TM-3 autophagy and apoptosis increased,and autophagy may play a protective role by inhibiting apoptosis. |
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