OBJECTIVE: Programmed cell death 6 (PDCD6), a Ca ²⁺-binding protein, has been reported to be aberrantly expressed in all kinds of tumors. The aim of this study was to explore the role and mechanism of PDCD6 in hepatocellular carcinomas (HCCs). METHODS: The expression levels of PDCD6 in liver cancer patients and HCC cell lines were analyzed using bioinformatics and Western blotting. Cell viability and metastasis were determined by methylthiazol tetrazolium (MTT) and transwell assays, respectively. And Western blotting was used to test related biomarkers and molecular pathway factors in HCC cell lines. LY294002, a PI3K inhibitor inhibiting AKT, was used to suppress the AKT/GSK3β/β-catenin pathway to help evaluate the role of this pathway in the HCC carcinogenesis associated with PDCD6. RESULTS: The analysis of The Cancer Genome Atlas Database suggested that high PDCD6 expression levels were relevant to liver cancer progression. This was consistent with our finding of higher levels of PDCD6 expression in HCC cell lines than in normal hepatocyte cell lines. The results of MTT, transwell migration, and Western blotting assays revealed that overexpression of PDCD6 positively regulated HCC cell proliferation, migration, and invasion. Conversely, the upregulation of PDCD6 expression in the presence of an AKT inhibitor inhibited HCC cell proliferation, migration, and invasion. In addition, PDCD6 promoted HCC cell migration and invasion by epithelial-mesenchymal transition. The mechanistic investigation proved that PDCD6 acted as a tumor promoter in HCC through the AKT/GSK3β/β-catenin pathway, increasing the expression of transcription factors and cellular proliferation and metastasis. CONCLUSION PDCD6 has a tumor stimulative role in HCC mediated by AKT/GSK3β/β-catenin signaling and might be a potential target for HCC progression.
Humans ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; Proto-Oncogene Proteins c-akt/metabolism* ; beta Catenin/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Glycogen Synthase Kinase 3 beta/metabolism* ; Cell Line ; Cell Proliferation ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Calcium-Binding Proteins/metabolism* ; Apoptosis Regulatory Proteins/genetics*

BACKGROUND: Perturbations in bone marrow mesenchymal stem cell (BMSC) differentiation play an important role in steroid-induced osteonecrosis of the femoral head (SONFH). At present, studies on SONFH concentrate upon the balance within BMSC osteogenic and adipogenic differentiation. However, BMSC apoptosis as well as proliferation are important prerequisites in their differentiation. The hedgehog (HH) signaling pathway regulates bone cell apoptosis. Baicalin (BA), a well-known compound in traditional Chinese medicine, can affect the proliferation and apoptosis of numerous cell types via HH signaling. However, the potential role and mechanisms of BA on BMSCs are unclear. Thus, we aimed to explore the role of BA in dexamethasone (Dex)-induced BMSC apoptosis in this study. METHODS: Primary BMSCs were treated with 10 -6 mol/L Dex alone or with 5.0 μmol/L, 10.0 μmol/L, or 50.0 μmol/L BA for 24 hours followed by co-treatment with 5.0 μmol/L, 10.0 μmol/L, or 50.0 μmol/L BA and 10 -6 mol/L Dex. Cell viability was assayed through the Cell Counting Kit-8 (CCK-8). Cell apoptosis was evaluated using Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining followed by flow cytometry. The imaging and counting, respectively, of Hochest 33342/PI-stained cells were used to assess the morphological characteristics and proportion of apoptotic cells. To quantify the apoptosis-related proteins (e.g., apoptosis regulator BAX [Bax], B-cell lymphoma 2 [Bcl-2], caspase-3, and cleaved caspase-3) and HH signaling pathway proteins, western blotting was used. A HH-signaling pathway inhibitor was used to demonstrate that BA exerts its anti-apoptotic effects via the HH signaling pathway. RESULTS: The results of CCK-8, Hoechst 33342/PI-staining, and flow cytometry showed that BA did not significantly promote cell proliferation (CCK-8: 0 μmol/L, 100%; 2.5 μmol/L, 98.58%; 5.0 μmol/L, 95.18%; 10.0 μmol/L, 98.11%; 50.0 μmol/L, 99.38%, F   =  2.33, P   >  0.05), but it did attenuate the effect of Dex on apoptosis (Hoechst 33342/PI-staining: Dex+ 50.0 μmol/L BA, 12.27% vs. Dex, 39.27%, t  = 20.62; flow cytometry: Dex + 50.0 μmol/L BA, 12.68% vs. Dex, 37.43%, t  = 11.56; Both P  < 0.05). The results of western blotting analysis showed that BA reversed Dex-induced apoptosis by activating the HH signaling pathway, which down-regulated the expression of Bax, cleaved-caspase 3, and suppressor of fused (SUFU) while up-regulating Bcl-2, sonic hedgehog (SHH), and zinc finger protein GLI-1 (GLI-1) expression (Bax/Bcl-2: Dex+ 50.0 μmol/L BA, 1.09 vs. Dex, 2.76, t  = 35.12; cleaved caspase-3/caspase-3: Dex + 50.0 μmol/L BA, 0.38 vs . Dex, 0.73, t  = 10.62; SHH: Dex + 50.0 μmol/L BA, 0.50 vs . Dex, 0.12, t  = 34.01; SUFU: Dex+ 50.0 μmol/L BA, 0.75 vs . Dex, 1.19, t  = 10.78; GLI-1: Dex+ 50.0 μmol/L BA, 0.40 vs . Dex, 0.11, t  = 30.68. All P  < 0.05). CONCLUSIONS BA antagonizes Dex-induced apoptosis of human BMSCs by activating the HH signaling pathway. It is a potential candidate for preventing SONFH.
Humans ; Hedgehog Proteins/metabolism* ; bcl-2-Associated X Protein ; Caspase 3/metabolism* ; Signal Transduction/physiology* ; Apoptosis ; Apoptosis Regulatory Proteins/pharmacology* ; Dexamethasone/pharmacology* ; Mesenchymal Stem Cells/metabolism* ; Bone Marrow Cells

This study investigated the effect of eleutheroside B on apoptosis and autophagy of lung cancer A549 and H460 cells and its molecular mechanism. MTT assay was used to detect the cytotoxicity of eleutheroside B at 5, 10, 15, 20, 25, 30, 35, 40, and 45 mmol·L~(-1) on lung cancer cells. Trypan blue exclusion assay was used to detect the effect of eleutheroside B on the survival rate of lung cancer A549 and H460 cells at different time. Colony formation assay was used to detect the effect of eleutheroside B on the proliferation of lung cancer A549 and H460 cells. AO/EB fluorescence double staining and Hoechst 33342 fluorescence staining were used to detect the effect of eleutheroside B on apoptosis of lung cancer A549 and H460 cells, and Western blot was used to detect apoptosis-related proteins to explore the apoptosis-related molecular mechanism. AO fluorescence staining and Western blot were used to detect the expression of autophagic vesicles and autophagy-related proteins P62 and LC3. The results showed that compared with the control group, eleutheroside B inhibited the growth of lung cancer A549 and H460 cells in a concentration-dependent manner. The optimal effect time of eleutheroside B on lung cancer A549 and H460 cells was 24 h, and the optimal concentrations were 28.64 and 22.16 mmol·L~(-1), respectively. Eleutheroside B could inhibit the colony formation of A549 and H460 cells. Compared with the control group, eleutheroside B could promote the formation of apoptotic bodies and induce cell apoptosis, as well as induce the expression of mitochondrial pathway-related proteins. Under the effect of eleutheroside B, the acidic autophagy vacuole in lung cancer cells increased, LC3Ⅱ expression increased, P62 protein expression decreased, and PI3K, p-Akt, and p-mTOR protein expression decreased in the PI3K/Akt/mTOR pathway. Studies have shown that eleutheroside B can inhibit the growth of lung cancer cells, reduce colony formation, induce apoptosis of lung cancer cells through mitochondrial pathway, and induce autophagy. The mechanism may be related to the PI3K/Akt/mTOR pathway.
Humans ; Lung Neoplasms/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis ; Apoptosis Regulatory Proteins ; Autophagy ; Cell Proliferation ; Cell Line, Tumor ; Glucosides ; Phenylpropionates

RNA modifications affect many biological processes and physiological diseases. The 5-methylcytosine (m⁵C) modification regulates the progression of multiple tumors. However, its characteristics and functions in hepatocellular carcinoma (HCC) remain largely unknown. Here, we found that HCC tissues had a higher m⁵C methylation level than the adjacent normal tissues. Transcriptome analysis revealed that the hypermethylated genes mainly participated in the phosphokinase signaling pathways, such as the Ras and PI3K-Akt pathways. The m⁵C methyltransferase NSUN2 was highly expressed in HCC tissues. Interestingly, the expression of many genes was positively correlated with the expression of NSUN2, including GRB2, RNF115, AATF, ADAM15, RTN3, and HDGF. Real-time PCR assays further revealed that the expression of the mRNAs of GRB2, RNF115, and AATF decreased significantly with the down-regulation of NSUN2 expression in HCC cells. Furthermore, NSUN2 could regulate the cellular sensitivity of HCC cells to sorafenib via modulating the Ras signaling pathway. Moreover, knocking down NSUN2 caused cell cycle arrest. Taken together, our study demonstrates the vital role of NSUN2 in the progression of HCC.
Humans ; ADAM Proteins ; Apoptosis Regulatory Proteins ; Carcinoma, Hepatocellular/metabolism* ; Liver Neoplasms/metabolism* ; Membrane Proteins ; Methyltransferases/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Repressor Proteins ; RNA, Messenger/metabolism* ; Sorafenib ; RNA Methylation/genetics*

OBJECTIVE: To investigate the effect of suppressing high-mobility group box 1 (HMGB1) on neuronal autophagy and apoptosis in rats after intracerebral hemorrhage (ICH) in rats. METHODS: Rat models of ICH induced by intracerebral striatum injection of 0.2 U/mL collagenase Ⅳ were treated with 1 mg/kg anti-HMGB1 mAb or a control anti-IgG mAb injected via the tail immediately and at 6 h after the operation (n=5). The rats in the sham-operated group (with intracranial injection of 2 μL normal saline) and ICH model group (n=5) were treated with PBS in the same manner after the operation. The neurological deficits of the rats were evaluated using modified neurological severity score (mNSS). TUNEL staining was used to detect apoptosis of the striatal neurons, and the expressions of HMGB1, autophagy-related proteins (Beclin-1, LC3-Ⅱ and LC3-Ⅰ) and apoptosis-related proteins (Bcl-2, Bax and cleaved caspase-3) in the brain tissues surrounding the hematoma were detected using Western blotting. The expression of HMGB1 in the striatum was detected by immunohistochemistry, and serum level of HMGB1 was detected with ELISA. RESULTS: The rat models of ICH showed significantly increased mNSS (P < 0.05), which was markedly lowered after treatment with anti- HMGB1 mAb (P < 0.05). ICH caused a significant increase of apoptosis of the striatal neurons (P < 0.05), enhanced the expressions of beclin-1, LC3-Ⅱ, Bax and cleaved caspase-3 (P < 0.05), lowered the expressions of LC3-Ⅰ and Bcl-2 (P < 0.05), and increased the content of HMGB1 (P < 0.05). Treatment with anti-HMGB1 mAb obviously lowered the apoptosis rate of the striatal neurons (P < 0.05), decreased the expressions of Beclin-1, LC3-Ⅱ, Bax and cleaved caspase-3 (P < 0.05), increased the expressions of LC3-Ⅰ and Bcl-2 (P < 0.05), and reduced the content of HMGB1 in ICH rats (P < 0.05). CONCLUSION Down- regulation of HMGB1 by anti-HMGB1 improves neurological functions of rats after ICH possibly by inhibiting autophagy and apoptosis of the neurons.
Animals ; Apoptosis ; Apoptosis Regulatory Proteins/metabolism* ; Autophagy ; Beclin-1 ; Caspase 3/metabolism* ; Cerebral Hemorrhage/therapy* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein/metabolism*

OBJECTIVES: Acute kidney injury (AKI) can be caused by ischemia/reperfusion (I/R), nephrotoxin, and sepsis, with poor prognosis and high mortality. Leptin is a protein molecule that regulates the body's energy metabolism and reproductive activities via binding to its specific receptor. Leptin can inhibit cardiomyocyte apoptosis caused by I/R, but its effect on I/R kidney injury and the underlying mechanisms are still unclear. This study aims to investigate the effect and mechanisms of leptin on renal function, renal histopathology, apoptosis, and autophagy during acute I/R kidney injury. METHODS: Healthy adult male mice were randomly divided into 4 groups: a sham+wild-type mice (ob/+) group, a sham+leptin gene-deficient mice (ob/ob) group, an I/R+ob/+ group, and an I/R+ob/ob group (n=8 per group). For sham operation, a longitudinal incision was made on the back of the mice to expose and separate the bilateral kidneys and renal arteries, and no subsequent treatment was performed. I/R treatment was ischemia for 30 min and reperfusion for 48 h. The levels of BUN and SCr were detected to evaluate renal function; HE staining was used to observe the pathological changes of renal tissue; TUNEL staining was used to observe cell apoptosis, and apoptosis-positive cells were counted; Western blotting was used to detect levels of apoptosis-related proteins (caspase 3, caspase 9), autophagy-related proteins [mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), LC3 I, LC3 II], mTOR-dependent signaling pathway proteins [phosphate and tension homology (PTEN), adenosine monophosphate-activated protein kinase (AMPK), protein kinase B (AKT), extracellular regulated protein kinase (ERK), phosphorylated PTEN (p-PTEN), phosphorylated AMPK (p-AMPK), phosphorylated AKT (p-AKT), phosphorylated ERK (p-ERK)]. RESULTS: There was no significant difference in the levels of BUN and SCr between the sham+ob/+ group and the sham+ob/ob group (both P>0.05). The levels of BUN and SCr in the I/R+ob/+ group were significantly higher than those in the sham+ob/+ group (both P<0.05). Compared with the mice in the sham+ob/ob group or the I/R+ob/+ group, the levels of BUN and SCr in the I/R+ob/ob group were significantly increased (all P<0.05). There was no obvious damage to the renal tubules in the sham+ob/+ group and the sham+ob/ob group. Compared with sham+ob/+ group and sham+ob/ob group, both the I/R+ob/+ group and the I/R+ob/ob group had cell damage such as brush border shedding, vacuolar degeneration, and cast formation. Compared with the I/R+ob/+ group, the renal tubules of the mice in the I/R+ob/ob group were more severely damaged. The pathological score of renal tubular injury showed that the renal tubular injury was the most serious in the I/R+ob/ob group (P<0.05). Compared with the sham+ob/+ group, the protein levels of caspase 3, caspase 9, PTEN, and LC3 II were significantly up-regulated, the ratio of LC3 II to LC3 I was significantly increased, and the protein levels of p-mTOR, p-PTEN, p-AMPK, p-AKT, and p-ERK were significantly down-regulated in the I/R+ob/+ group (all P<0.05). Compared with the sham+ob/ob group, the protein levels of caspase 3, caspase 9, PTEN, and LC3 II were significantly up-regulated, and the ratio of LC3 II to LC3 I was significantly increased, while the protein levels of p-mTOR, p-PTEN, p-AMPK, p-AKT, and p-ERK were significantly down-regulated in the I/R+ob/ob group (all P<0.05). Compared with the I/R+ob/+ group, the levels of p-mTOR, p-PTEN, p-AMPK, p-AKT were more significantly down-regulated, while the levels of caspase 3, caspase 9, PTEN, and LC3 II were more significantly up-regulated, and the ratio of LC3 II to LC3 I was more significantly increase in the I/R+ob/ob group (all P<0.05). CONCLUSIONS Renal function and tubular damage, and elevated levels of apoptosis and autophagy are observed in mice kidneys after acute I/R. Leptin might relieve I/R induced AKI by inhibiting apoptosis and autophagy that through a complex network of interactions between mTOR-dependent signaling pathways.
AMP-Activated Protein Kinases/metabolism* ; Acute Kidney Injury/pathology* ; Animals ; Apoptosis ; Apoptosis Regulatory Proteins/pharmacology* ; Autophagy ; Caspase 3/metabolism* ; Caspase 9/metabolism* ; Female ; Humans ; Ischemia ; Kidney/pathology* ; Leptin/pharmacology* ; Male ; Mammals/metabolism* ; Mice ; Proto-Oncogene Proteins c-akt/metabolism* ; Reperfusion/adverse effects* ; Reperfusion Injury/metabolism* ; TOR Serine-Threonine Kinases/metabolism*

OBJECTIVE: To express matrix remodeling associated 7 (MXRA7) in the human acute myeloid leukemia SHI-1 cell line and to assess the role of MXRA7 in the biological function of SHI-1 cells. METHODS: The full-length cDNA sequence of human MXRA7 was synthesized and subcloned into the lentivirus shuttle vector pRRL-Venus. SHI-1 cells were transfected with the lentivirus which was packaged with 293T cells. The YFP-positive cells were sorted by flow cytometry and the stable cell lines were obtained by expanded culture. The expression and distribution of MXRA7 in SHI-1 cells were verified by real-time qPCR, Western blot and laser confocal techniques. Cell proliferation and cell cycle were measured by flow cytometry, and apoptosis was determined by Annexin V and 7-AAD staining. The expression of apoptosis related proteins were detected by Western blot. RESULTS: The stable SHI-1 cell line overexpressing MXRA7 was established successfully. Laser confocal analysis confirmed that MXRA7 was expressed in the cytoplasm of SHI-1 cells. Compared with the control cell line, the overexpression of MXRA7 showed no effect on the cell proliferation and cell cycle, but reduced the percentage of apoptosis cells induced by methotrexate. Moreover, the expression of BCL-2 protein was increased by overexpression of MXRA7, which can inhibit cell apoptosis. CONCLUSION The SHI-1 stable cell line overexpressing MXRA7 was established successfully, and MXRA7 could inhibit drug-induced apoptosis through increasing the expression of BCL-2 protein.
Apoptosis ; Apoptosis Regulatory Proteins ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Humans ; Membrane Proteins/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism*

: AbstractObjective: To explore the effect and mechanism of curcumin on human T-cell acute lymphoblastic leukemia (T-ALL) cell apoptosis induced by Mcl-1 small molecule inhibitors UMI-77. METHODS: T-ALL cell line Molt-4 was cultured, and the cells were treated with different concentrations of curcumin and Mcl-1 small molecule inhibitor UMI-77 for 24 h. The MTT method was used to detect the cell survival rate after different treatment; According to the results of curcumin and UMI-77, the experimental settings were divided into control group, curcumin group (20 μmol/L curcumin treated cells), UMI-77 group (15 μmol/L Mcl-1 small molecule inhibitor UMI-77 treated cells) and curcumin+ UMI-77 group (20 μmol/L curcumin and 15 μmol/L Mcl-1 small molecule inhibitor UMI-77 treated cells), MTT method was used to detect cell proliferation inhibition rate, Annexin V-FITC/PI double staining method and TUNEL staining were used to detect cell apoptosis, DCFH-DA probe was used to detect cell reactive oxygen species, JC-1 fluorescent probe was used to detect mitochondrial membrane potential, Western blot was used to detect the expression levels of apoptosis-related proteins and Notch1 signaling pathway-related proteins. RESULTS: After the treatment of Molt-4 cells with different concentrations of curcumin and Mcl-1 small molecule inhibitor UMI-77, the cell survival rate was decreased (P<0.05); Compared with the control group, the cell proliferation inhibition rate of the curcumin group and the UMI-77 group were increased, the apoptosis rate of cell was increased, the level of ROS was increased, the protein expression of Bax, Caspase-3 and Caspase-9 in the cells were all increased, and the protein expression of Bcl-2 was reduced (P<0.05); Compared with the curcumin group or UMI-77 group, the cell proliferation inhibition rate and apoptosis rate of the curcumin+UMI-77 group were further increased, and the level of ROS was increased. At the same time, the protein expression of Bax, Caspase-3 and Caspase-9 in the cells were all increased, the protein expression of Bcl-2 was reduced (P<0.05); In addition, the mitochondrial membrane potential of the cells after curcumin treatment was decreased, and the proteins expression of Notch1 and HES1 were reduced (P<0.05). CONCLUSION Curcumin can enhance the apoptosis of T-ALL cells induced by Mcl-1 small molecule inhibitor UMI-77 by reducing the mitochondrial membrane potential, the mechanism may be related to the inhibition of Notch1 signaling pathway.
Apoptosis ; Apoptosis Regulatory Proteins ; Caspase 3/metabolism* ; Caspase 9/pharmacology* ; Cell Line, Tumor ; Curcumin/pharmacology* ; Humans ; Myeloid Cell Leukemia Sequence 1 Protein/metabolism* ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Reactive Oxygen Species/pharmacology* ; Sulfonamides ; Thioglycolates ; bcl-2-Associated X Protein/pharmacology*

Objective: To investigate the effect of arsenic and its main metabolites on the apoptosis of human lung adenocarcinoma cell line A549 and the expression of pro-apoptotic genes Bad and Bik. Methods: In October 2020, A549 cells were recovered and cultured, and the cell viability was detected by the cell counting reagent CCK-8 to determine the concentration and time of sodium arsenite exposure to A549. The study was divided into NaAsO(2) exposure groups and metobol: le expoure groups: the metabolite comparison groups were subdivided into the control group, the monomethylarsinic acid exposure group (60 μmol/L) , and the dimethylarsinic acid exposure group (60 μmol/L) ; sodium arsenite dose groups were subdivided into 4 groups: control group (0) , 20, 40, 60 μmol/L sodium arsenite NaAsO(2). Hoechst 33342/propidium iodide double staining (Ho/PI) was used to observe cell apoptosis and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of Bad and Bik mRNA in cells after exposure. Western blotting was used to detect the protein expressions of Bad, P-Bad-S112, Bik, cleaved Bik and downstream proteins poly ADP-ribose polymerase PARP1 and cytochrome C (Cyt-C) , using spectrophotometry to detect the activity changes of caspase 3, 6, 8, 9. Results: Compared with the control group, the proportion of apoptotic cells in the 20, 40, and 60 μmol/L NaAsO(2) dose groups increased significantly (P<0.01) , and the expression levels of Bad, Bik mRNA, the protein expression levels of Bad, P-Bad-S112, Bik, cleaved Bik, PARP1, Cyt-C were increased (all P<0.05) , and the activities of Caspase 3, 6, 8, and 9 were significantly increased with significantly differences (P<0.05) . Compared with the control group, the expression level of Bad mRNA in the DMA exposure group (1.439±0.173) was increased with a significant difference (P=0.024) , but there was no significant difference in the expression level of Bik mRNA (P=0.788) . There was no significant differences in the expression levels of Bad and Bik mRNA in the poison groups (P=0.085, 0.063) . Compared with the control group, the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to MMA were 0.696±0.023, 0.707±0.014, 0.907±0.031, 1.032±0.016, and there was no significant difference between the two groups (P=0.469, 0.669, 0.859, 0.771) ; the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to DMA were 0.698±0.030, 0.705±0.022, 0.908±0.015, 1.029±0.010, and there was no difference between the two groups (P=0.479, 0.636, 0.803, 0.984) . Conclusion: Sodium arsenite induces the overexpression of Bad and Bik proteins, initiates the negative feedback regulation of phosphorylated Bad and the degradation of Bik, activates the downstream proteins PARP1, Cyt-C and Caspase pathways, and mediates the apoptosis of A549 cells.
A549 Cells ; Adenosine Diphosphate Ribose/pharmacology* ; Apoptosis ; Apoptosis Regulatory Proteins ; Arsenic ; Arsenites ; Cacodylic Acid/pharmacology* ; Caspase 3 ; Caspases/pharmacology* ; Cytochromes c/pharmacology* ; Humans ; Mitochondrial Proteins/pharmacology* ; Poisons ; Propidium/pharmacology* ; RNA, Messenger ; Sincalide/pharmacology* ; Sodium Compounds ; bcl-Associated Death Protein/metabolism*

Objective: To study the underlying mechanism of cadmium-induced apoptosis of mouse spermatocytes (GC-2 spd) . Methods: In March 2021, GC-2 spd cells were exposed to different concentrations of CdCl(2) for 24 hours, namely 5 μmol/L CdCl(2) (low-dose) group and 10 μmol/L CdCl(2) (high-dose) group, and unexposed GC-2 spd cells were used as control group. Mitochondrial morphology was observed in the cells stained with Mito-Track Red CMXRos fluorescent probes by confocal microscopy and the mitochrondrial membrane potential was measured by flow cytometry with JC-1 fluorescent probes. Mitochrondrial proteins, cytosolic proteins and total cellular proteins of GC-2 spd cells were extracted using cell mitochondria isolation kit and RIPA buffer, respectively. The expression of mitochondrial homeostasis regulatory proteins (FIS1 and OPA1), and apoptosis-related proteins (Cytochrome c and cleaved Caspase-3) were examined by Western blot. Results: Compared with the cells in the control group, the relative ratio of JC-1 red/green fluorescence signal in the cells of the low-dose and high-dose CdCl(2) groups decreased significantly (0.740±0.071, 0.570±0.028), with a statistically significant difference (P=0.017, 0.004) ; The morphology of mitochondria changed from long tube to point, and the proportion of cells containing point mitochondria increased significantly (45.1%±3.7% and 25.7%±4.9%), the difference was statistically significant (P=0.005, 0.001) ; The relative expression level of mitochondrial FIS1 in cells of low and high dose CdCl(2) groups was significantly higher (1.271±0.120, 1.693±0.155), the difference was statistically significant (P=0.046, 0.000) ; The relative expression level of OPA1 decreased significantly (0.838±0.050, 0.682±0.040), and the difference was statistically significant (P=0.049, 0.001). Compared with the control group, the relative expression level of cytochrome c protein in the cytoplasm of cells in the low dose group of CdCl(2) was not significantly increased (1.249±0.151), and the difference was not statistically significant (P=0.075). However, the relative expression level in the cytoplasm of cells in the high dose group of CdCl(2) was significantly increased (2.355±0.110), and the difference was statistically significant (P=0.000) ; The relative expression level of Cytochrome c in mitochondria of low and high dose CdCl(2) groups decreased significantly (0.681±0.043, 0.619±0.114), with a statistically significant difference (P=0.004, 0.001) ; Moreover, the level of cleaved Caspase-3 protein in cells gradually increased (5.486±0.544, 11.493±1.739), the difference was statistically significant (P=0.004, 0.000) . Conclusion: Cadmium induced cleaved Caspase-3 mediated apoptosis of GC-2 spd cells via promoting mitochrondrial fission and the release of Cytochrome c from the mitochrondria to the cytosol.
Male ; Mice ; Animals ; Mitochondrial Dynamics ; Caspase 3/metabolism* ; Cadmium/toxicity* ; Cytochromes c/metabolism* ; Fluorescent Dyes ; Apoptosis ; Apoptosis Regulatory Proteins/metabolism* ; Membrane Potential, Mitochondrial