Cadmium induces apoptosis in primary rat osteoblasts through caspase and mitogen-activated protein kinase pathways

Exposure to cadmium (Cd) induces apoptosis in osteoblasts (OBs); however, little information is available regarding the specific mechanisms of Cd-induced primary rat OB apoptosis. In this study, Cd reduced cell viability, damaged cell membranes and induced apoptosis in OBs. We observed decreased mitochondrial transmembrane potentials, ultrastructure collapse, enhanced caspase-3 activity, and increased concentrations of cleaved PARP, cleaved caspase-9 and cleaved caspase-3 following Cd treatment. Cd also increased the phosphorylation of p38-mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK)1/2 and c-jun N-terminal kinase (JNK) in OBs. Pretreatment with the caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, ERK1/2 inhibitor (U0126), p38 inhibitor (SB203580) and JNK inhibitor (SP600125) abrogated Cd-induced cell apoptosis. Furthermore, Cd-treated OBs exhibited signs of oxidative stress protection, including increased antioxidant enzymes superoxide dismutase and glutathione reductase levels and decreased formation of reactive oxygen species. Taken together, the results of our study clarified that Cd has direct cytotoxic effects on OBs, which are mediated by caspase- and MAPK pathways in Cd-induced apoptosis of OBs.     

Regulation by Jun N-terminal kinase/stress activated protein kinase of cytokine expression in Mycobacterium avium subsp paratuberculosis-infected bovine monocytes

OBJECTIVE: To evaluate activation of Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway in bovine monocytes after incubation with Mycobacterium avium subsp paratuberculosis (Mptb) organisms. SAMPLE POPULATION: Bovine monocytes obtained from 4 healthy adult Holstein dairy cows. PROCEDURES: Bovine monocytes were incubated with Mptb organisms with or without a specific inhibitor of the JNK/SAPK pathway (SP600125) for 2, 6, 24, or 72 hours. Expression of interleukin (IL)-1beta, IL-10, IL-12, IL-18; transforming growth factor-beta (TGF-beta); and tumor necrosis factor-alpha (TNF-alpha) and the capacity of Mptb-infected monocytes to acidify phagosomes and kill Mptb organisms were evaluated. Phosphorylation status of JNK/SAPK was evaluated at 10, 30, and 60 minutes after Mptb incubation. RESULTS: Compared with uninfected control monocytes, Mptb-infected monocytes had increased expression of IL-10 at 2 and 6 hours after incubation and had increased expression of TNF-alpha, IL-1beta, IL-18, and TGF-beta at 2, 4, and 6 hours. Additionally, Mptb-infected monocytes had increased expression of IL-12 at 6 and 24 hours. Addition of SP600125 (specific chemical inhibitor of JNK/SAPK) resulted in a decrease in TNF-alpha expression at 2, 6, and 24 hours, compared with untreated Mptb-infected cells. Addition of SP600125 resulted in a decrease in TGF-beta expression at 24 hours and an increase in IL-18 expression at 6 hours. Addition of SP600125 failed to alter phagosome acidification but did enhance the capacity of monocytes to kill Mptb organisms. CONCLUSIONS AND CLINICAL RELEVANCE: Activation of JNK/SAPK may be an important mechanism used by Mptb to regulate cytokine expression in bovine monocytes for survival and to alter inflammatory and immune responses.     

Butyrate-induced apoptosis and cell cycle arrest in bovine kidney epithelial cells: involvement of caspase and proteasome pathways

Beyond their nutritional effect, short-chain fatty acids, especially butyrate, modulate cell differentiation, proliferation, motility, and in particular, they induce cell cycle arrest and apoptosis. A bovine kidney epithelial cell line (Madin-Darby bovine kidney; MDBK) was used to investigate the cell cycle regulatory and apoptotic effects of butyrate. Butyrate not only induced apoptosis but also induced cell cycle arrest at the G1/S boundary and M/G2 in MDBK cells (P < 0.01). The cell responses were concentration-dependent (r(2) = 0.9482, P <0.001). In examining possible mechanisms for the apoptosis and cell cycle arrest induced by butyrate, the results showed that butyrate treatment activates caspase-3 activities and induces accumulation of acetylated histone. At least two proteins, cdc6 and cdk1, become targeted for destruction on butyrate treatment. These two proteins are downregulated (P < 0.01 and P < 0.05, respectively) by proteolytic pathways. Moreover, the proteasome inhibitor MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal) reverses the cell cycle arrest induced by butyrate, indicating a multiprotein crosstalk wherein the ubiquitination/ proteasome pathway interacted with the caspase-signaling pathway. Because the proteasome inhibitor MG-132 blocked activation of caspase-3, these results functionally locate the proteasome pathway upstream of the caspase pathway. All these results indicate that butyrate functions as both a nutrient and signaling molecule regulating cell growth and proliferation.     

Caspase activation in equine influenza virus induced apoptotic cell death.

Equine influenza virus (EIV) is the leading cause of acute respiratory infection in horses worldwide. In recent years, the precise mechanism by which influenza infection kills host cells is being re-evaluated. In this report, we examined whether caspases, a group of intracellular proteases, are activated following EIV infection and contribute to EIV-mediated cell death. Western blotting analysis indicated that a nuclear target of caspase-3, poly(ADP-ribose) polymerase (PARP) was proteolytically cleaved in EIV-infected MDCK cells, but not in mock-infected cells. In comparison with caspase-3 specific inhibitor Ac-DEVD-CHO, a general caspase inhibitor Boc-D-FMK provided much stronger inhibition of EIV-induced cytopathic effect and apoptosis. Our results suggest that EIV may activate more than one caspase. Caspase activation and cleavage of its cellular targets may play a critical role in EIV-mediated cytotoxicity.     

Susceptibility of Bovine Umbilical Cord Endothelial Cells to Bovine Herpesviruses and Pseudocowpox Virus

The purpose of the study was to determine the susceptibility of bovine umbilical cord endothelial (BUE) cells to bovine herpesvirus (BHV) 1, BHV2, BHV4 and BHV5, and to pseudocowpox virus. The detection limits and growth curves of these viruses in BUE cells were compared with those in Vero, Madin-Darby bovine kidney (MDBK), or bovine fetal diploid lung (BFDL) cells. Detection limits were determined by inoculating cell cultures with serial 10-fold dilutions of these viruses, and growth curves by titration of virus, harvested at various times after infecting cells at a multiplicity of infection of 0.1. The detection limits of BHV2 and BHV4 were lower in BUE cells than in Vero or MDBK cells, and cytopathic effects were observed earlier in BUE cells. In addition, BHV2 and BHV4 grew to higher titres in BUE cells than in Vero or MDBK cells. BUE cells appeared to be equally susceptible to BHV5, but less susceptible to BHV1.1 and BHV1.2 than MDBK cells. The study showed that BUE cells are highly susceptible to BHV2 and BHV4, and that the use of BUE cells can improve the laboratory diagnosis of these viruses. The use of BUE cells could also improve the isolation and growth of pseudocowpox virus.     

High sensitivity of fibroblast cell lines derived from LEC rats to heat treatment

A fibroblast cell line derived from LEC rat was approximately twofold more sensitive to heat treatment at 45 degrees C than were that from WKAH rat in terms of heating time required to attain 50% loss of survival in a colony forming assay. The present study was carried out for understanding the mechanism underlying the higher sensitivity of LEC rat cells to heat treatment. Although apoptosis was not found in WKAH rat cells, the percentages of apoptotic cells in LEC rat cells significantly increased after heat treatment. LEC rat cells showed significantly lower sensitivity in induction of cell death and apoptosis to ceramide, a lipid signaling molecule that is associated with heat-induced apoptosis, than did WKAH rat cells. SP600125, an inhibitor of JNK suppressed the induction of cell death in both heated LEC and WKAH rat cells, but SB203580, an inhibitor of p38 mapk, did not. The relative surviving fractions of heated LEC and WKAH rat cells in the presence of both SB203580 and SP600125 were higher than those of cells in the presence of SP600125 alone. The amounts of hsp70 protein in WKAH rat cells increased from 4 to 12 hr after heat treatment, but did not in LEC rat cells. These results suggest that higher thermosensitivity in the fibroblast cell line from LEC rat is due to low inducibility of hsp70 protein after heat treatment.     

Mitogen-activated protein kinases p38 and JNK mediate Actinobacillus pleuropneumoniae exotoxin ApxI-induced apoptosis in porcine alveolar macrophages

Actinobacillus pleuropneumoniae exotoxins (Apx) are major virulence factors that play important roles in the pathogenesis of pleuropneumonia in swine. A previous study has demonstrated that native ApxI at low concentrations induces apoptosis in primary porcine alveolar macrophages (PAMs) via a caspase-3-dependent pathway. However, the molecular mechanisms underlying ApxI-induced apoptosis remain largely unknown. In this study, it was shown that ApxI treatment in PAMs rapidly induced phosphorylation of both p38 and JNK, members of the mitogen-activated protein kinase family. Application of a selective p38 or JNK inhibitor significantly reduced ApxI-induced apoptosis, indicating the involvement of p38 and JNK pathways in this event. Furthermore, activation of both caspase-8 and -9 were observed in ApxI-stimulated PAMs. Inhibition of caspase-8 and caspase-9 activity significantly protected PAMs from ApxI-induced apoptosis. In addition, Bid activation was also noted in ApxI-treated PAMs, and inhibition of caspase-8 suppressed the activation of Bid and caspase-9, suggesting that ApxI was able to activate the caspases-8-Bid-caspase-9 pathway. Notably, inhibition of p38 or JNK pathway greatly attenuated the activation of caspases-3, -8, and -9. This study is the first to demonstrate that ApxI-induced apoptosis of PAMs involves the activation of p38 and JNK, and engages the extrinsic and intrinsic apoptotic pathways.     

Equine coronavirus induces apoptosis in cultured cells

Equine coronavirus (ECoV) was first isolated from a diarrheic foal and was found genetically similar to group II coronaviruses. However, its pathological characteristics were not adequately investigated. In our preliminary in vitro investigation, ECoV-induced cell death was observed in bovine kidney-derived MDBK cells. Based on this finding, we investigated whether the ECoV-induced CPE was apoptosis. Following ECoV infection, MDBK cells showed morphological changes such as cell rounding and detachment from the culture surface. Moreover, syncytium formation was observed as the other type of cytopathic effect in ECoV infection. Morphologic and biochemical features of apoptosis, such as nuclear fragmentation and DNA ladder formation, were also detected in ECoV-infected cells. Moreover, as is commonly observed in coronavirus infection in other animals, the activities of effecter caspases – caspase-3/7 – and initiator caspases – caspase-8 and caspase-9 – that are representative factors in the death receptor-mediated apoptotic pathway and mitochondrial apoptotic pathway, respectively, were increased in ECoV-infected MDBK cells. Therefore, it was suggested that ECoV can induce apoptosis in MDBK cells via a caspase-dependent pathway. Apoptotic death of infected cells is detrimental because it causes cell and tissue destruction and inflammatory responses. Although the pathological characteristics of ECoV are largely unknown, apoptosis may be the pathological basis of lesions of the digestive system in ECoV infection.     

Antiapoptotic activity of bovine herpesvirus type-1 (BHV-1) UL14 protein

Viruses have evolved different strategies to interfere with apoptotic pathways in order to halt cellular responses to infection. One previous study showed that transient transfection of bovine herpesvirus type-1 (BHV-1) UL14 protein is efficient in protecting Madin Darby kidney (MDBK) and human chronic myelogenous leukemia (K562) cells from sorbitol-induced apoptosis. This protein corresponds to a putative protein of BHV-1, which shares aminoacid sequence with a part of the peptide-binding domain conserved in human heat shock protein (HSP70) family. The pBK-CMV-UL14 plasmid transfected MDBK cells treated with sorbitol did not show caspase-3 and caspase-9 activation with respect to non-transfected MDBK cells (UL14 negative). Furthermore, we report that the expression of the full length sequence of BHV-1 UL14 is evident after 7 h of infection of BHV-1 on MDBK cells which were then treated with sorbitol. These results indicate that UL14 gene product has important implications to enhance cell survival in response to apoptotic stimuli.     

JNK抑制剂对仔猪肝细胞药物性损伤的缓解作用及其机理

【目的】研究c-Jun氨基末端激酶(JNK)抑制剂SP600125对仔猪原代肝细胞药物性损伤的缓解作用及机理。【方法】通过二步灌流法获得仔猪原代肝细胞,将肝细胞分为对照组(C)、JNK抑制剂组(SP)、模型组(M)和治疗组(T),每组6个重复。对照组细胞不添加药物,SP组用2μmol/L SP600125处理细胞,M组用80μg/mL脂多糖(LPS)+20μg/mL恩诺沙星(ENR)处理细胞,T组用80μg/mL LPS+20μg/mL ENR+2μmol/L SP600125处理细胞,处理12 h后,收集上清测定谷丙转氨酶(ALT)和谷草转氨酶(AST)活性,收集细胞测定谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)、超氧化物歧化酶(superoxide dismutase, SOD)活性、丙二醛(malondialdehyde, MDA)含量及肝细胞核因子-1(hepatocyte nuclear factor 1,HNF-1)和谷胱甘肽-S-转移酶A1(glutathione S-transferase alpha 1,GSTA1)mRNA的相...     

Transmissible gastroenteritis virus infection induces apoptosis through FasL- and mitochondria-mediated pathways

Transmissible gastroenteritis virus (TGEV) has been reported to induce apoptosis in swine testis (ST) cells. However, the mechanisms underlying TGEV-induced apoptosis are still unclear. In this study we observed that TGEV infection induced apoptosis in porcine kidney (PK-15) cells in a time- and dose-dependent manner. TGEV infection up-regulated FasL, activated FasL-mediated apoptotic pathway, leading to activation of caspase-8 and cleavage of Bid. In addition, TGEV infection down-regulated Bcl-2, up-regulated Bax expression, promoted translocation of Bax to mitochondria, activated mitochondria-mediated apoptotic pathway, which in turn caused the release of cytochrome c and the activation of caspase-9. Both extrinsic and intrinsic pathways activated downstream effector caspase-3, followed by the cleavage of PARP, resulting in cell apoptosis. Moreover, TGEV infection did not induce significant DNA fragmentation in ammonium chloride (NH(4)Cl) pretreated PK-15 cells or cells infected with UV-inactivated TGEV. In turn, block of caspases activation also did not affect TGEV replication. Taken together, this study demonstrates that TGEV-induced apoptosis is dependent on viral replication in PK-15 cells and occurs through activation of FasL- and mitochondria-mediated apoptotic pathways.     

Changes in caspase activity during the postmortem conditioning period and its relationship to shear force in porcine longissimus muscle

The objective of this study was to investigate the protease family caspases in skeletal muscle and their potential contribution to postmortem proteolysis and meat tenderization. Ten Large White gilts were slaughtered, and samples of LM were taken at 0, 2, 4, 8, 16, 32, and 192 h after slaughter and immediately snap frozen in liquid nitrogen. Samples were subsequently analyzed for caspase 3/7 and caspase 9 activity, protein levels of known caspase substrates, alpha II spectrin and poly (ADP-ribose) polymerase (PARP), as well as, at 192 h, shear force. Specific degradation products of alpha II spectrin and PARP, which are known indicators of caspase activity, and apoptosis were detected on immunoblots of muscle samples taken over the postmortem period. The relationships between the changes observed in caspase activities and protein levels of PARP and spectrin across the entire postmortem conditioning period were investigated (n = 70). Protein levels of alpha II spectrin cleavage products across the conditioning period were found to correlate positively to caspase 3/7 activity (r = 0.38, P = 0.003) and caspase 9 activity (r = 0.32, P = 0.012), indicating that caspase-mediated cleavage was occurring in situ. There was a negative relationship between shear force and the 0 to 32 h ratio of caspase 3/7 (r = -0.62, P = 0.053) and caspase 9 activities (r = -0.68, P = 0.044). In addition, there was also a negative relationship between shear force and the level of the caspase-generated alpha II spectrin 120 kDa degradation product (r = -0.75, P = 0.012). The findings of this study indicate that changes in caspase activity and caspase-mediated cleavage take place in muscle during the conditioning period, and this could be associated with the development of tender meat.     

Possible roles of intracellular cyclic AMP, protein kinase C and calcium ion in the apoptotic signaling pathway in bovine luteal cells

Structural luteolysis occurs by apoptosis of luteal cells. The present study examined the effects of activators of well-characterized second messengers on Fas and caspase-3 mRNA expression and on P4 production in luteal cells in order to trace the pro- and anti-apoptotic factors in the bovine corpus luteum (CL). Cultured bovine mid luteal cells were treated for 24 h with a cyclic AMP analogue (8-bromo cyclic AMP; 8br-cAMP; 2.5 mM), a protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate; PMA; 10 microM), or calcium ionophore (A23187; 10 microM). Fas and caspase-3 mRNA expression was inhibited by 8br-cAMP and PMA but was increased by A23187 (P<0.05). In addition, P4 production by bovine luteal cells was stimulated by 8br-cAMP and PMA, whereas it was inhibited by A23187, compared with untreated controls (P<0.05). The overall results suggest that cAMP and PKC suppress apoptosis in bovine luteal cells through inhibition of Fas and caspase-3 mRNA expression and through stimulation of P4 production. Therefore, substances that activate cAMP or PKC may act as survival factors in the bovine CL. Furthermore, substances that mobilize Ca2+ may act as apoptotic factors by stimulating Fas and caspase-3 expression in the bovine luteal cells.     

Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce inflammation and apoptosis in porcine peripheral blood mononuclear cells in vitro

Mycoplasma hyopneumoniae is the causative agent of swine enzootic pneumonia (EP), a disease that causes considerable economic losss in swine industry. Lipid-associated membrane proteins (LAMPs) of mycoplasma play important roles in causing mycoplasma diseases. The present study explores the pathogenic mechanisms of M. hyopneumoniae LAMPs by elucidating their role in modulating the inflammation, apoptosis, and relevant signaling pathways of peripheral blood mononuclear cells (PBMCs) of pig. LAMP treatment inhibited the growth of PBMCs. Up-regulation of cytokines, such as IL-6 and IL-1β, as well as increased production of nitric oxide (NO) and superoxide anion were all detected in the supernatant of LAMPs-treated PBMCs. Furthermore, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMPs of M. hyopneumoniae induced a time-dependent apoptosis in lymphocyts and monocytes from PBMCs, which was blocked by NOS inhibitor or antioxidant. In addition, LAMPs induced the phosphorylation of p38, the ratio of pro-apoptotic Bax protein to anti-apoptotic Bcl-2, activation of caspase-3 and caspase-8, and poly ADP-ribose polymerase (PARP) cleavage in PBMCs. These findings demonstrated that M. hyopneumoniae LAMPs induced the production of proinflammatory cytokines, NO and reactive oxygen species (ROS), and apoptosis of PBMCs in vitro through p38 MAPK and Bax/Bcl-2 signaling pathways, as well as caspase activation.     

No caspase activation but overexpression of Bcl-2 in bovine cells infected with noncytopathic bovine virus diarrhoea virus

Cytopathic bovine viral diarrhoea viruses (cp BVDV) induce apoptosis in permissible cell cultures via the intrinsic pathway, which involves the mitochondria as key organelles. An important event is the irreversible opening of the permeability transition pore (PTP) and the breakdown of the transmembrane potential DeltaPsi(m). The resulting release of cytochrome C from the mitochondria serves as a trigger to form the apoptosome which then leads to caspase activation and cell death. In contrast, noncytopathic (ncp) BVDV do not seem to affect cells in vivo or in vitro, suggesting that they inhibit apoptosis. Interestingly, inhibition of caspases in cells infected with cp BVDV delayed the apoptotic cascade but did not prevent the cytopathic effect (CPE). This suggests that the induction of apoptosis and the processes finally leading to the CPE may proceed separately, implying that the inhibition of apoptosis by ncp BVDV has to start earlier in the cascade. In this study we show that in fact apoptosis inhibition in cells infected with ncp BVDV must occur at the mitochondrial level, before the activation of the caspase cascade occurs. To elucidate the role of mitochondria after infection of cells with ncp BVDV, expression of Bcl-2 and Bax were analysed. It was shown that while Bax expression was not affected, the anti-apoptotic Bcl-2 protein was upregulated, presumably suppressing initiation of cell death and enabling persistent infection in vitro.     

Caprine herpesvirus-1 (CapHV-1) induces apoptosis in goat peripheral blood mononuclear cells

Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. A number of studies have shown that deregulation of apoptosis is an important feature of virus-induced immunosuppression for various viral diseases. In the present study, CapHV-1 was found to cause apoptosis in mitogen-stimulated as well as nonstimulated caprine peripheral blood mononuclear cells (PBMC). Apoptotic index, as quantified by fluorescent dyes, revealed a significant increase in the percentage of apoptotic cells at 24 and 48 h postinfection as compared to their respective noninfected controls. Apoptosis specific internucleosomal laddering in DNA from CapHV-1 infected PBMC was seen in agarose gel electrophoresis. No DNA fragmentation was observed in control noninfected PBMC. Virus-induced apoptosis was reduced by Z-VAD-FMK, an aspecific caspase inhibitor, by AC-DEVD-CHO (caspase-3-specific) and AC-VEID-CHO (caspase-6-specific) treatment. PCD in CapHV-1 infected peripheral blood mononuclear cells occurs at the G0/G1 phase of the cell cycle. However, penetration of virus particles and infection was not required for PCD, as UV-inactivated CapHV-1 induced apoptosis of mitogen-stimulated bovine peripheral blood mononuclear cells in vitro.     

Comparative study of cytotoxicity of Aeromonas spp. on four different cell lines

In vitro cytotoxicity is an important virulence property of motile mesophilic Aeromonas species. Cell-free supernatant prepared from 55 Aeromonas isolates including one A. hydrophila type strain (MTCC 646) were examined for their cytotoxic potential on four different cell lines (Vero, BHK-21, MDBK, B 95a). Results of the study revealed cytotoxic potential in 92.72% of the isolates. Analysis of data exposed significant variation among isolates in respect of their cytotoxicity. Vero cells proved to be most sensitive to aeromonal toxins and B 95a cells showed significantly (P<0.01) lower response compared to other cell lines. Sensitivities of BHK-21 and MDBK cell lines were in between Vero and B 95a.     

Assessment of calpain and caspase systems activities during ageing of two bovine muscles by degradation patterns of αII spectrin and PARP‐1

The activities of calpain and caspase systems during ageing in Longissimus lumborum (LL) and Infraspinatus (IS) muscles of Italian Simmental young bulls (Bos taurus) were assessed. Samples from 10 animals were collected within 20 min of exsanguination (T0), after 48 h (T1) and 7 days (T2) post mortem. Calpain and caspase activity were evaluated based on the formation of αII spectrin cleavage products of 145 kDa (SBDP145) and 120 kDa (SBDP120), respectively. Caspase activity was also assessed by the presence of poly (adenosine diphosphate‐ribose) polymerase‐1 (PARP‐1) cleavage product. At T0, LL showed higher levels of SBDP145 than IS (P < 0.01), while SBDP120 and PARP‐1 degradation products were similar between muscles. At T1, no difference was found in the level of SBDP145 between muscles, while SBDP120 and PARP‐1 cleavage products were not detected. At T2 neither αII spectrin nor PARP‐1 cleavage products were found. LL and IS showed different proteolysis after slaughter that was influenced more by calpain than caspase activity, which was detectable only in the early post mortem period.     

Fas/FasL通路对镉激活PC12细胞MAPK通路的影响

旨在研究Fas/FasL通路对镉激活PC12细胞MAPK通路的影响,用10 μmol L^-1醋酸镉（CdAc₂）分别处理插入非特异性序列PC12细胞与Fas基因沉默PC12细胞12 h;用10 μmol·L^-1CdAc₂与40 μmol·L^-1 Z-IETD-FMK （caspase-8特异性抑制剂）单独或联合处理PC12细胞12 h。通过Western blot检测Fas/FasL通路相关蛋白Fas、FasL、Fas相关死亡域蛋白（FADD）、Cleaved caspase-8、死亡结构域相关蛋白（Daxx）、凋亡信号调节激酶1（ASK1）的表达与MAPK通路相关蛋白ERK1/2、JNK1/2、p-ERK1/2、p-JNK1/2的表达;Hoechst33258荧光染色检测细胞凋亡形态学变化,流式细胞术检测细胞凋亡率。结果显示,Fas shRNA慢病毒极显著抑制镉引起的PC12细胞Fas/FasL通路相关蛋白表达量和凋亡率的升高（P<0.01）,缓解镉引起的PC12细胞凋亡形态学变化;Fas shRNA与Z-IETD-FMK均能够极显著抑制镉引起的PC12细胞MAPK通路相关蛋白ERK1/2与JNK1/2磷酸化水平升高（P<0.01）。综上表明,Fas/FasL通路调控MAPK通路参与镉致PC12细胞凋亡。