Effect of IL-1β and IL-6 on expression of nerve growth factor in human nucleus pulposus cells

AIM: To determine the effect of interleukin-1β(IL-1β)and IL-6 on the expression of nerve growth factor (NGF) by human nucleus pulposus (NP) cells. METHODS: The NP cells from the normal disc of operative patients with scoliosis were isolated, cultured and identified. After 7 days preculture, the NP cells were treated with IL-1β (10 μg/L, 50 μg/L) or IL-6 (10 μg/L, 50 μg/L) for 48 h in the experimental groups and 0.3% PBS was used in the control groups. The expression of NGF was detected by the methods of immunohistochemistry, semi-quantitative RT-PCR and Western blotting.RESULTS: The NP cells were chondrocyte-like cellular morphology with positive staining of toluidine blue, safranine O and anti-collagen II antibody. The NP cells cultured in monolayer showed immunoreactivity to NGF either in control condition or in experimental group. IL-1β and IL-6 up-regulated the mRNA expression of NGF and the protein production of NGF. The effect of this up-regulation was higher by treating with IL-6 than by treating with IL-1β in the same concentration.CONCLUSION: Our results demonstrate that the proinflammatory cytokines IL-1β and IL-6 stimulate the production of NGF in NP cells. The effect of IL-6 is more significant than that of IL-1β.     

Effects of leptin on hypoxia-reoxygenation induced apoptosis in human L02 cells and expression of Fas and FasL

AIM: To observe the effect of leptin (LEP) on hypoxia-reoxygenation induced apoptosis in L02 cells.METHODS: In the experiment, L02 cell injury was induced by hypoxic air (95%N2 and 5% CO2). The cultured L02 cells were divided into hypoxic 12 h group (IR group) alone, normal control group and the hypoxic plus leptin (100 μg/L, 200μg/L, 400 μg/L, 800 μg/L and 1 600 μg/L) treatment groups in vitro. Flow cytometry, terminal dUTP nick-end labeling (TUNEL) assay and fluorescent quantitative PCR were used to measure the changes of apoptosis in L02 cells and expression of Fas/FasL mRNA.RESULTS: (1) The percentage of L02 cells apoptosis and positive TUNEL cells significantly increased in IR group compared to control group (P<0.01). When L02 cells were treated with LEP, the percentage of cell apoptosis and positive TUNEL cells were decreased compared to IR group. (2) Compared to control group, the Fas/FasL mRNA expression significantly increased in IR group (P<0.01). When L02 cells were treated with LEP, the Fas/FasL mRNA expression decreased compared to IR group, the effect of LEP at concentration of 400 μg/L was obviously (P<0.05). CONCLUSION: LEP decreases the apoptosis of hypoxic-reoxygenation L02 cells by down-regulation of Fas/FasL mRNA expression in L02 cells.     

Effect of cinobufagin on L-type voltage-gated calcium channel currents in dorsal root ganglion cells of rats with bone cancer pain

AIM: To investigate the modulation effect of cinobufagin on L-type voltage-gated calcium channel (L-VGCC) currents in dorsal root ganglion (DRG) cells of rats with bone cancer pain. METHODS: A rat model of bone cancer pain was constructed by intratibial injection of Walker 256 breast cancer cells. The rat DRG cells were isolated and the DRG neurons were identified by cellular immunofluorescence technique. The effect of cinobufagin on the L-VGCC current of DRG neurons in the model rats was recorded by the technique of whole-cell patch clamp. RESULTS: The size of DRG neurons obtained from primary culture was basically the same, and the purity was over 95%. Compared with the normal group, the L-VGCC current density of DRG cells in the model rats was increased (P<0.05), while cinobufagin significantly inhibited the L-VGCC current density of DRG cells in the model rats and shifted the inactivation curve to the right. CONCLUSION: In the rat model of bone cancer pain, the intrinsic electrophysiological characteristics of the membrane of DRG neurons are changed, the excitability is increased, and the calcium current is increased. Cinobufagin may exert pharmacological effects by regulating the current characteristics of L-VGCC in rat DRG cells.     

Effect of nicotine against apoptosis of rat cortical neurons induced by colchicines

AIM:To explore the mechanism of nicotine against the apoptosis induced by colchicines in rat cortical neurons. METHODS:Cortical neurons were cultured from newborn Sprague-Dawley (SD) rats (less than 12 h). The rate of apoptosis was measured by Hoechst33258 fluorescence staining in the neurons, and the activity of Akt473 was analyzed by assay kit Akt473. RESULTS:The apoptosis of cortical neurons can be induced by 0.1 μmol/L colchicine. The phosphorlation of Akt 473 decreased significantly (1/3 times of the control group, P<0.01). However, when cortical neurons pretreated with 10 μmol/L nicotine for 2 h were cultured with 0.1 μmol/L colchicine for 24 h, the rate of apoptosis decreased from 62% to 38%. The phosphorlation of Akt473 increased significantly in a bell-shape time-dependent manner, which was respectively 1.3, 3.7, 2.4, 2.1 and 1.9 times compared with the control group (P<0.01). CONCLUSION:By activating the signal pathway of Akt473, nicotine may attenuate the apoptosis of cortical neurons induced by colchicines.     

Inhibition of HMGB1 expression and release by nicotine in RAW264.7 cells

AIM: To investigate that nicotine inhibits HMGB1 expression and release in RAW264.7 cells.METHODS: (1) RAW264.7 cells were cultured in 6 wells plate, treated with 250 μg/L LPS and 1 μmol/L or 10 μmol/L nicotine, in which the cells treated with or without 250 μg/L LPS were regarded as nicotine 1 group (N1), nicotine 2 group (N2), LPS group (LPS) and control group (C), respectively. HMGB1 protein in the cell culture media and in cell nuclear was examined by Western blotting and the cellular HMGB1 mRNA level was detected by RT-PCR. (2) Transfected with antisense RNA or sense RNA of α7 subunit-containing nicotinic receptor (α7nAChR), RAW264.7 cells were treated with 250 μg/L LPS and 10 μmol/L nicotine, HMGB1 protein in the culture media was also tested by Western blotting.RESULTS: (1) HMGB1 mRNA level in C group was low (1 659.20±121.05) and no significant statistical difference among groups of N1, N2 and LPS was observed (P>0.05). (2) Higher HMGB1 accumulation in the cell culture media was detected in LPS group (445.34±28.52) than that in C group. Compared to LPS group, both N1 and N2 groups distinctly attenuated HMGB1 accumulation in culture media (P<0.05). (3) Nuclear HMGB1 accumulation was lower in LPS group than that in C group, and two different nicotine concentrations markedly increased the nuclear HMGB1 accumulation compared to LPS group (P<0.05). (4) No significant difference of HMGB1 levels in culture media between antisense RNA group and LPS group was observed (P>0.05). In sense RNA group, however, HMGB1 level was observably reduced compared to antisense group (P<0.05).CONCLUSION: The present results suggest that nicotine dramatically inhibits RAW264.7 cell nuclear HMGB1 translocation and extracellular release, and this effect relies on α7nAch receptor expression.     

Effect of rhEGF activating extracellular signal-regulated kinase on the expression of Bcl-2 and p53 protein in human amniotic cells

AIM: To explore the effect of recombinant human epidermal growth factor (rhEGF) on the ERK1/2 pathway and its safety in use when the proliferation of cultured FL cells (human amnion epithelial cells) were promoted by rhEGF.METHODS: FL cells were stimulated by rhEGF at various concentrations.The proliferation rate of FL cells was evaluated by MTT assay.Western blotting was used to detect phosphorylation of ERK1/2 (p-ERK1/2) and the change of Bcl-2 and P53 protein.RESULTS: The proliferation rate FL cells reached the maximum when the concentration of rhEGF was 10 μg/L (42.4％，P<0.01).Western blotting showed that the activity of p-ERK1/2 increased significantly when rhEGF ranged in 10 μg/L to 60 μg/L.Level of Bcl-2 was unchanged in each groups.Level of P53 decreased significantly when the concentration of rhEGF was 60 μg/L.CONCLUSION: It is safe when rhEGF presents in optimization concentration of 10 μg/L.The capability of apoptosis may be depressed when rhEGF is in larger dose.     

Effect of SnoN protein on high-glucose-induced expression of fibronectin in primary cultured rat renal tubular cells

AIM: To investigate the effect of Ski-related novel protein N(SnoN) on high-glucose-induced expression of fibronetin (FN) in primary cultured rat renal tubular cells (RTECs). METHODS: The primary renal cells were cultured, and the cell types were indentified to be RTECs. The cells were divided into 3 groups: normal-glucose group (DMEM+2% FBS), high-glucose group (19.5 mmol/L D-glucose+DMEM+2% FBS) and high-osmotic group (19.5 mmol/L D-mannitol+DMEM+2% FBS). The cells were harvested at 30 min, 2 h, 12 h, 24 h, 48 h, 72 h and 96 h. SnoN expression in primary cultured RTECs was knocked down by RNA interference, then the cells were divided into 4 groups: normal-glucose group, high-glucose group, control siRNA group and SnoN siRNA group. The protein expressions of SnoN and FN in RTECs was examined by the methods of Western blotting, immunocytochemistry staining and immunofluorescence cytochemistry. RT-PCR was used to examine the mRNA expression of FN and SnoN. RESULTS: The RTECs constituted the major cell type of cultured cells. SnoN protein was decreased in a time-dependent manner in RTECs under high-glucose condition. The FN protein and mRNA levels raised in high-glucose group and sustained through entire experiment. Moderate reduction of SnoN in RTECs was observed by RNAi strategy, which greatly up-regulated the expression of FN (P<0.05). CONCLUSION: The down-regulation of SnoN participates high-glucose-induced expression of FN in RTECs.     

Effect of anti-Müllerian hormone on hormone secretion in cultured human luteinized granulose cells

AIM: To explore the effect of anti-Müllerian hormone (AMH) on hormone secretion of cultured human luteinized granulosa cells. METHODS: Human luteinized granulose cells were cultured with different concentrations of AMH. The levels of estrogen and progesterone in the supernatants were measured. RESULTS: The levels of estrogen and progesterone increased gradually and peaked at 72 h. The secretions of estrogen and progesterone were significantly inhibited by AMH with concentration dependence (5 μg/L to 20 μg/L). No significant difference was found in estrogen and progesterone levels in the presence of AMH at concentrations from 20 μg/L to 50 μg/L group. CONCLUSION: AMH decreases the secretion of estrogen and progesterone in cultured human luteinized granulose cells in a concentration dependent manner, suggesting that AMH negatively affect hormone secretion in granulose cells.     

H2S inhibits neuron apoptosis induced by anoxia-reoxygenation through cAMP-mediated PI3-K/Akt/P70S6K kinase cell-survival signaling pathways

AIM: To investigate the effect of hydrogen sulfide on neuron apoptosis through PI₃-K/Akt/P70S6K cell-survival signal transduction pathways after neuron anoxia-reoxygenation.METHODS: Newborn (24-48 h) Wistar rats were decapitated.The hippocampus tissue was dissected and cells were suspended.Cells were plated at 1.0×10⁸ cells/L on poly-dlysine-treated 96-well (100 μL/well) plates and 6-well (2 mL/well) plates.Cells were used after 7 days.For anoxia-reoxygenation (oxygen glucose deprivation,OGD) experiments,cells were washed three times in a glucose-free balanced salt solution (BSS).They were then placed in deoxygenated glucose-free medium and cultured under 95% N₂,5% CO₂ in an anaerobic chamber equilibrated to 37 ℃ and 100% humidity for 45 min.OGD was terminated by replacement of stored medium and by returning the cultures to a standard incubator maintained at 37 ℃ in 95% air,5% CO₂.In experimental group,cells were respectively carried out OGD,OGD+150 μmol/L NaHS,OGD+150 μmol/L NaHS+10 μmol/L triciribin,OGD+150 μmol/L NaHS+10 nmol/L rapamycin and OGD+150 μmol/L NaHS+10 μmol/L triciribin+10 nmol/L rapamycin.Control cells were cultured normally.24 h later,neuron viability and apoptosis were measured.The level of cAMP and protein expression of PI₃-K,Akt and P70S6K were detected.RESULTS: NaHS enhanced concentration of cAMP and expression of PI₃-K,Akt and P70S6K.Meanwhile,increased neuron viability and decreased neuron apoptosis (P<0.01 vs group C or group I/R) were observed.Triciribin inhibited Akt and P70S6K,as well as increased neuron apoptosis and decreased neuron viability (P<0.05,P<0.01 vs group NaHS).Rapamycin inhibited P70S6K,as well as increased neuron apoptosis and decreased neuron viability (P<0.05,P<0.01 vs group NaHS).CONCLUSION: H₂S inhibits hippocampus neuron apoptosis and protects neuron from anoxia-reoxygenation injury through cAMP-mediated PI₃-K/Akt/P70S6K kinase cell-survival signaling pathways.     

Effects of Waller degenerative sciatic nerve segments on differentiation of BMSCs into SCs in rats

AIM:To observe the effects of Waller degenerative sciatic nerve segments on the ability of proliferation and secretion in rat bone marrow mesenchymal stem cells (BMSCs) as well as the differentiation of BMSCs into Schwann cells (SCs) under co-culture condition. METHODS:BMSCs were isolated from SD rats and identified by immunofluorescence. In Transwell co-culture system, the degenerative rat sciatic nerve segments and BMSCs were co-cultured in the Transwell inserts and well plates, respectively. The experiments were divided into 3 groups: group A, degenerative sciatic nerve segments co-cultured with BMSCs; group B, normal sciatic nerve segments co-cultured with BMSCs; group C, BMSCs cultured alone. The morphological changes of co-cultured BMSCs were observed under inverted phase-contrast microscope. The expression of S-100 in the BMSCs was tested by immunofluorescence staining after 7 d of co-culture. At 0, 1, 4, 7, 11 and 14 d after co-culture, BMSCs growth curve in each group were drawn by the method of cell counting, the expression of nerve growth factor (NGF) was detected by ELISA, and the mRNA expression of S-100 in the BMSCs was determined by real-time PCR. RESULTS:BMSCs were isolated and cultured successfully. The identification of BMSCs showed positive expression of CD29, CD44, and CD90. At 7 d after co-culture, the BMSCs in group A began retraction, became tapered with the processes and had SCs-like morphology, but most BMSCs in groups B and C showed no obvious morphological changes under inverted phase contrast microscope. The positive expression rates of S-100 in groups A, B, and C were 31.1%±2.9%, 16.2%±1.7%, and 0.42%±0.07%, respectively. There were significant differences of S-100 positive expression rates among 3 groups. The BMSCs growth curve of each group was similar to an "S" shape. From 4 d after co-culture, the BMSCs proliferation in group A were significantly faster than that in group B and group C. NGF in the supernatant of group A was increased in a time-dependent manner and reached the peak at 7 d after co-culture, then decreased gradually. NGF was also also increased in group B and group C, but the content of NGF was lower than that in group A. At 4, 7, 11 and 14 d of co-culture, NGF content was higher in group A than that in group B and group C, and a significant difference was also observed between group B and group C. The S-100 mRNA expression was significantly higher in group A than that in group B and group C at 4, 7, 11 and 14 d after co-culture (P<0.05). CONCLUSION: Waller degenerative sciatic nerve segments can effectively promote rat BMSCs proliferation and induce BMSCs to differentiate into SCs in vitro, and the growth factors secreted by degenerative sciatic nerve segments may be involved in the regulation of inducing BMSCs to differentiate into SCs.     

Effect of low molecular weight heparin on hypoxia-induced apoptosis in primary cultured neonate rat cerebral cortical neurons

AIM:To investigate the mechanism by which low molecular weight heparin (LMWH) inhibits apoptosis of primary cultured cerebral cortical neurons caused by hypoxia. METHODS:The anti-apoptosis effect of LMWH on primary cultured neurons was observed by methods of the primary culture of cerebral neurons of postnatal rats in free-serum with neurobasal medium supplied with 2% B27 supplement, hypoxic culture of neurons, Hoechst 33342 staining and immunocytochemistry. RESULTS:At concentrations of 250-500 U/L, LMWH reduced apoptosis rate of cerebral cortical neurons induced by hypoxia (P<0.05) and apoptosis rate was lower in LMWH groups than that in BDNF (50 μg/L) group (P<0.05). LMWH (500 U/L) increased Bcl-2 protein expression (P<0.05) and decreased Bax protein expression (P<0.01) in the cerebral cortical neurons induced by hypoxia, the ratio of Bcl-2/Bax was improved (P<0.01). The ratio of Bcl-2/Bax in LMWH (500 U/L) group was higher than that in normal control group, BDNF group and apoptosis positive group (P<0.05). CONCLUSION:LMWH at concentrations of 250-500 U/L is able to prevent cerebral cortical neurons from apoptosis in primary culture under hypoxia. The effect of anti-apoptosis may be related to up-regulation of Bcl-2 protein expression, down-regulation of Bax-2 protein expression, and increase in the ratio of Bcl-2/Bax.     

Modulating action of substance P on H+-gated current in rat dorsal root ganglion neurons

AIM：To investigate the effects of substance P (SP) on the modulation of H+-gated current in neurons. METHODS：Whole-cell patch-clamp technique was used to record the H+-gated current in the acutely isolated rat dorsal root ganglion (DRG) neurons at pH 4.0, pH 5.0, pH 6.0, and both SP and H+. The modulating action of SP on H+-gated current in the acutely isolated rat DRG neurons was studied by intracellular dialysis. RESULTS：H+-gated currents recorded at pH 5.0 were divided into 4 types: transient inward (T-type),sustained inward (S-type),biphasic inward (B-type) and opposite (O-type). SP exhibited obvious enhancing effect on the sustained component of S-type and B-type H+-gated currents, and the enhancing current amplitude was (85.53±22.93)%. However, the enhancing effect was not blocked by the SP receptor NK1 antagonist in about 81.8% DRG neurons. The enhancing effect was blocked by nonpeptidic SP receptor antagonist in about 75% DRG neurons. SP also exhibited obvious inhibitory effect on the sustained component of S-type and B-type H+-gated currents, and the inhibitory current amplitude was (48.46±4.45)%. However, the inhibitory effect was not blocked by the SP receptor antagonist in about 88.9% DRG neurons. After intracellular dialysis, GDP-β-S could not block the modulating action of SP on H+-gated currents. CONCLUSION：There are 4 types of H+-gated currents in acutely isolated rat DRG neurons: T-type, S-type, B-type and O-type. The modulating action of SP on H+-gated current exhibits both enhancement and inhibition. The regulatory mechanism includes G-protein-coupled receptor pathway and the binding of SP to a locus of H+-gated ion channels to exert modulating action on the H+-gated current.     

Effects of FHL1 on proliferation and migration of rat pulmonary arterial smooth muscle cells stimulated by cigarette smoke

AIM:To explore the role of four-and-a-half LIM domain 1 (FHL1) in the proliferation and migration of rat distal pulmonary arterial smooth muscle cells (PASMCs) stimulated by cigarette smoke extract (CSE). METHODS:Rat distal PASMCs were isolated and primarily cultured. The cells were divided into 6 groups: blank control group, negative transfection group, FHL1 siRNA transfection group, CSE group, CSE + negative transfection group and CSE + FHL1 siRNA transfection group. The mRNA and protein expression of FHL1 was detected by real-time PCR and Western blotting, respectively. Cell proliferation and migration were determined by CCK-8 and Transwell assays, respectively. RESULTS:CSE promoted the proliferation and migration of PASMCs, and increased the expression of FHL1 protein (P<0.01), but did not change the expression of FHL1 mRNA (P>0.05). FHL1 siRNA transfection attenuated the proliferation and migration of PASMCs induced by CSE (P<0.01). CONCLUSION: FHL1 protein is involved in CSE-induced proliferation and migration of rat PASMCs.     

Role of cyclooxygenase-2 in injury induced by hypoxia/reoxygenation in cultured rat cortical neurons

AIM: To observe the role of cyclooxygenase-2 (COX-2) in injury induced by hypoxia and reoxygenation in cultured rat cortical neurons and protective effects of COX-2 specific inhibitor NS398.METHODS: Primary rat cortical neuronal cells were cultured. Experiments were divided into control group, hypoxia/reoxygenation group and hypoxia/reoxygenation with COX-2 inhibitor group. Cell viability was measured by MTT assay. COX-2 protein expression was examined by Western blotting. Apoptosis was measured by DNA agarose electrophoresis.RESULTS: The expression levels of COX-2 increased significantly after neurons were treated with hypoxia and reoxygenation, compared with control group and hypoxia/reoxygenation with COX-2 inhibitor group (P＜0.05). COX-2 specific inhibitor NS398 protected neurons from death (P＜0.05 and P＜0.01), DNA fragmentation analysis showed DNA fragmentation was inhibited significantly by NS398.CONCLUSION: COX-2 is involved in the pathogenesis of neuron apoptosis induced by hypoxia/reoxygenation. COX-2 specific inhibitor significantly protects cortical neurons against hypoxia/reoxygenation injury and inhibits apoptosis induced by hypoxia.     

RNA interference of endophilin A2 inhibits synaptic vesicle endocytosis in rat hippocampal neurons

AIM: To investigate the effect of endophilin A2 on synaptic vesicle endocytosis in rat hippocampal neurons.METHODS: The siRNAs for endophilin A2 isoform were designed, and the efficacy and specificity of these siRNAs were determined by Western blot. Immunostaining was performed to verify the interference efficiency of the siRNA for endogenous endophilin A2 in the hippocampal neurons. Excitatory postsynaptic currents (EPSCs) were recorded on cultured hippocampal neurons using dual whole-cell recordings by evoking the transfected presynaptic neurons in various stimulation patterns. RESULTS: The result of immunostaining in cultured hippocampal neurons demonstrated that the siRNA of endophilin A2 inhibited the expression of endogenous endophilin A2 significantly (P<0.05). The results of dual whole-cell recordings showed that no significant difference in the EPSCs amplitude evoked between endophilin A2 knockdown neurons and control was observed when stimulated at low frequency (0.1 Hz). The amplitude of normalized EPSCs evoked in endophilin A2 knockdown neurons decreased significantly for both single-train and multiple-train stimulations (P<0.05).CONCLUSION: An effective siRNA of endophilin A2 was screened out successfully. Knockdown of endophilin A2 isoform does not affect synaptic vesicle exocytosis, but inhibits synaptic vesicle endocytosis in rat hippocampal neurons.     

Nerve growth factor promotes proliferation of hepatocytes in vitro via TrkANGFR signal pathway

AIM: To observe the expression and distribution of nerve growth factor (NGF), its high-affinity tyrosine kinase A receptor (TrkA^NGFR) and low-affinity p75 neurotrophin receptor (p75^NTR) in hepatocytes(L02 cells), and to investigate the effects of exogenous recombinant human NGF-β on L02 cells. METHODS: L02 cell line was used in the experiment. The expression and intracellular distribution of NGF, TrkA^NGFR and p75^NTR were detected by the methods of immunocytochemistry and fluorescent quantitative PCR. The proliferation of L02 cells after exposed to exogenous NGF-β, anti-NGF, anti-TrkA^NGFR and anti-p75^NTR was detected by XTT {2,3-bis(2-methoxy-4-nitro-5- sulfophenyl)-5- -2H-tetrazolium hydroxide} assay. The cell apoptosis and the change of cell cycle of L02 cells after exposed to exogenous NGF-β were determined by flow cytometry with Annexin V-FITC and PI staining. RESULTS: The expression of NGF was mainly localized in the cytoplasm and nuclei of L02 cells. The expression of TrkA^NGFR was highly and the expression of p75^NTR was weakly detected in the cell membrane and cytoplasm of L02 cells. Exogenous NGF-β promoted the expression of NGF and TrkA^NGFR in L02 cells. Low dose of exogenous NGF-β (12.5-200 μg/L) promoted L02 cell proliferation and inhibited the cell apoptosis by affecting the cell cycle in S-phase. High dose of NGF-β (>400 μg/L) did not promote L02 cell proliferation. Specific neutralizing antibodies of NGF and TrkA^NGFR decreased the NGF-β-induced proliferation of L02 cells. However, blocking the binding of NGF to p75^NTR by anti-p75^NTR did not affect the NGF-β-induced proliferation of L02 cells. CONCLUSION: L02 cells express NGF, TrkA^NGFR and p75^NTR. Exogenous recombinant human NGF-β at optimal dose promotes L02 cell proliferation in a dose-dependent manner possibly via NGF/TrkA^NGFR signal pathway.     

Role of CRIF1 in cardiomyocyte apoptosis induced by palmitic acid

AIM: To investigate the effects of CR6-interacting factor 1 (CRIF1) on the apoptosis of mouse cardiomyocytes (MCMs) induced by palmitic acid. METHODS: The MCMs cultured with medium containing palmitic acid at 0 and 300 μmol/L for 24 h were divided into control group and palmitic acid group, respectively. In order to explore the effects of CRIF1 on MCMs injuries induced by high fat, MCM exposed to palmitic acid at 300 μmol/L for 24 h were divided into vehicle group, scrambled (Scra) siRNA group and CRIF1 siRNA group. The cells in vehicle group were only treated with transfection reagent, the cells in Scra siRNA group were given a treatment with transfection reagent and scrambled RNA, and the cells in CRIF1 siRNA group were given a treatment with transfection reagent and CRIF1 specific siRNA. In order to further confirm the specific mechanism of CRIF1 in high fat-induced MCM injuries, MCMs in CRIF1 siRNA group were divided into DMSO group and N-acetyl cysteine (NAC) group, and were given the same intervention of palmitic acid. RT-qPCR, Western blot and immunofluorescence staining were used to detect the mRNA and protein expression of CRIF1. The apoptotic rate was analyzed by flow cytometry, and the level of intracellular reactive oxygen species (ROS) was tested by DHE staining and ELISA. RESULTS: The expression of CRIF1 was significantly increased after exposure to palmitic acid (P<0.05). Compared with control group, the apoptotic rate was increased significantly in vehicle group and Scra siRNA group (P<0.05). The apoptotic rate was significantly increased in CRIF1 siRNA group (P<0.05). Compared with control group, the intracellular ROS content was significantly increased in vehicle group and Scra siRNA group (P<0.05). Compared with vehicle group and Scra siRNA group, the intracellular ROS content was significantly increased in CRIF1 siRNA group (P<0.05). Compared with DMSO group, the intracellular ROS content and the apoptotic rate were remarkably decreased in NAC group (P<0.05). CONCLUSION: With the inhibition of oxidative stress, CRIF1 may reduce the apoptosis of MCMs induced by high fat.