The Study Of ZnIn₂S₄ Mediated Photodynamic Therapy Induce Hep G2cells Apoptosis By Bax And Capase-3 Pathway

The incidence and mortality of malignant tumors remain high,which is a serious threat to human health.At present,the commonly used methods for treating tumors include surgery,radiotherapy,chemotherapy,targeted therapy,immunotherapy,etc.Although these methods have certain curative effects on some tumors,the specificity is not obvious,and the side effects are large.For a long time,people have been looking for new therapeutic techniques to effectively solve these problems.Photodynamic therapy is a novel minimally invasive treatment for tumors.It has the advantages of low toxicity,no drug resistance,protection of other organs,and more thorough treatment.Photodynamic reaction refers to a photoreactive reaction involving a biological effect involving aerobic molecules.A photosensitizer is a chemical substance that can absorb the energy of a specific wavelength of light and transmit it to surrounding molecules,thereby producing toxic substances such as reactive oxygen species,which can induce tumor cell death.Objective:This study was to investigate the efficiency of ZnIn₂S₄-PDT-mediated tumor cell apoptosis and the mechanism involved in this process.It is proved that ZnIn₂S₄-PD can induce tumor cell apoptosis and verify the changes of mitochondrial potential level and intracellular reactive oxygen species ROS level with ZnIn₂S₄-PDT.Inducing the correlation of tumor cell apoptosis;confirming the expression of key proteins such as Bax,Caspase-3,Caspase-9 and other tumor cell apoptosis in this process;preliminary clarification that ZnIn₂S₄ can induce tumor cell apoptosis in PDT,and One process is related to the mitochondrial apoptotic pathway.Provide experimental data and theoretical support for the application of ZnIn₂S₄ in PDT.Methods:In this experiment,6 groups were set up:blank control group,ZnIn2S4experimental group,ZnIn₂S₄ control group,ZnIn₂S₄ experimental component group 4,and each group of milliliter culture solution?Hep G2 liver cancer cell density was 1×106cells/ml?.100?g/ml,200?g/ml,300?g/ml,400?g/ml ZnIn₂S₄ particles were added.The ZnIn₂S₄ particles were autoclaved before the experiment.The experimental group and the control group were irradiated with a 300 uv lamp for 60 min,and the irradiation intensity was 1.64?W/cm2.The irradiation distance was adjusted so that the surface temperature to be irradiated was 37±1°C?human body temperature?.To avoid contamination during irradiation.The cells are covered with a piece of quartz glass?through UV light?on the illuminated plate.The following experimental techniques were used to test the results:?1?Apoptosis rate determination:Annexin V-FITC/PI double staining method was used to detect the apoptosis rate of each dose experimental group and control group under the same light conditions;?2?cells Mitochondrial transmembrane potential detection:JC-1 method was used to detect mitochondrial transmembrane potential changes in experimental and control groups under the same light conditions;?3?Bax,Caspases-3,9 enzyme activity detection:cells were treated with different concentrations of ZnIn₂S₄ After the protein concentration was determined,the same amount of supernatant protein was taken,and the activity of Caspase-3,9 was measured according to the reaction system described in the kit;?4?Western Bolt method was used to detect the expression of Caspases-3,9 and Bax protein:cells The expression of each group of proteins was detected according to the operation manual through different concentrations of ZnIn₂S₄;?5?Electron microscopy.Results:?1?The nuclear morphology of treated tumor cells shows typical chromatin condensation,apoptotic bodies,rotating nuclei and cavities,and typical apoptotic changes in nuclear fragmentation.No cell morphology was observed in the blank control group and the ZnIn₂S₄ non-irradiated group?Fig.1?.?2?Flow cytometry results showed that when the concentration of ZnIn₂S₄ increased from 100?g/ml to 400?g/ml,the apoptosis increased significantly?p<0.01??Fig.2?;while the cells alone were 200?g/ml.There was no significant difference in apoptosis between the control group and the control group?p>0.05?,indicating that ZnIn₂S₄ had no significant cytotoxicity against HepG2 liver cancer cells.?3?We examined whether ZnIn₂S₄-PDT-induced apoptosis involves ROS production by using flow cytometry.As shown in Figure 3,ZnIn₂S₄-PDT significantly increased intracellular ROS levels?p<0.01?with increasing concentrations,which was statistically significant compared to the control group.Importantly,ZnIn₂S₄-mediated changes in intracellular ROS levels were positively correlated with changes in ZnIn₂S₄-induced apoptosis rates.All results support that ROS production is directly related to ZnIn₂S₄-PDT-induced apoptosis.?4?In this experiment,we stimulated Hep G2 cells with a photosensitizer ZnIn₂S₄ at concentrations of 100,200,300 and 400?g/ml and recorded by flow cytometry using JC-1 staining?Fig.4?.As the concentration of ZnIn₂S₄increased,the mitochondrial membrane potential decreased significantly.Compared with the control group,the neutrophil membrane potential group showed a significant decrease?p<0.01?.?5?We used ELISA and Western blot to detect changes in the expression levels of apoptosis-related proteins Bax,Caspase-9 and Caspase-3.As shown in Figures 5 and 6,when the Znln2S4 nanoparticles were used to simulate sunlight for 60 minutes,the overall expression of the ELISA assay was positive:Bax\Caspase-3\Caspase-9 was consistently positively correlated;the control group had the lowest expression,and the Znln2S4 group was expressed.The change was significant and the difference was extremely significant?P<0.01?.With the increase of ZnIn₂S₄ dose,the expression level of each index increased,and the difference was significant or extremely significant?P<0.05 or 0.01??Fig.5?.Taken together,these results indicate that induction of tumor cell apoptosis is primarily through activation of the Bax/Caspase-3/Caspase-9 pathway.Conclusion:ZnIn₂S₄ induces apoptosis of Hep G2 tumor cells during ZnIn₂S₄-PDT treatment.ZnIn₂S₄-PDT treatment can increase intracellular ROS levels,and ZnIn₂S₄-PDT treatment can induce Bax,caspase-9caspase-3 activation in a dose-dependent manner.Taken together,our data indicate that ZnIn₂S₄ induces mitochondria-mediated apoptosis in Hep G2 cells by activating the caspase cascade..It provides a basis for deeper foundation and clinical experimental research.