Effect of protein kinase C signal pathway on resistin expression in 3T3-L1 adipocytes

AIM：To investigate the effect of protein kinase C on resistin expression in 3T3-L1 adipocytes.METHODS：The differentiated 3T3-L1 adipocytes were incubated with 50 nmol/L phorbol 12-myristate 13-acetate (PMA) or 5 μmol/L Ro-31-8220 for 24 h.Expression of resistin mRNA was detected by RT-PCR and expression of resistin protein was detected by Western blotting.RESULTS：Compared with control,PMA increased the expression of resistin mRNA and protein in 3T3-L1 adipocytes significantly (P<0.01),while Ro-31-8220 decreased the expression of resistin mRNA and protein in 3T3-L1 adipocytes obviously (P<0.01).CONCLUSION：Protein kinase C signal pathway may regulate resistin expression in 3T3-L1 adipocytes.     

Effect of lentivirus-mediated DKK3 overexpression on apoptosis of hypertrophic scar fibroblasts

AIM:To investigate the effect of lentivirus-mediated DKK3 overexpression on the apoptosis of hypertrophic scar fibroblasts. METHODS:Human hypertrophic scar fibroblasts were isolated and cultured in vitro. The cells were divided into control group, vector (negative control lentivirus infection) group and DKK3 (pcDNA3.1-DKK3 lentivirus infection) group. The overexpression effect was determined by RT-qPCR and Western blot. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. The protein levels of cleaved caspase-9, collagen type Ⅱ (COL Ⅱ), COL I and cleaved caspase-3 in the cells, and cytochrome C in the cytoplasm and mitochondrion were detected by Western blot. RESULTS:After transfection with pcDNA3.1-DKK3, the expression of DKK3 at mRNA and protein levels was increased in the hypertrophic scar fibroblasts (P<0.05). The viability of hypertrophic scar fibroblasts in DKK3 group was decreased, and the apoptotic rate was increased. The protein levels of cleaved caspase-9 and cleaved caspase-3 were increased in the cells, and the protein levels of COL Ⅱ and COL I were decreased. The protein level of cytochrome C was increased in the cytoplasm, while the protein level of cytochrome C in the mitochondrion decreased. Compared with vector group, these differences were statistically significant (P<0.05). CONCLUSION:Lentivirus-mediated DKK3 overexpression induces apoptosis and reduces collagen synthesis in the fibroblasts from hypertrophic scars.     

Effect of hypoxia on the expression of proliferating cell nuclear antigen and phenotype of cardiac fibroblasts

AIM:To investigate effect of hypoxia on the expression of proliferating cell nuclear antigen(PCNA) and phenotype of cardiac fibroblasts(CFs). METHODS:The purified cardiac fibroblasts were cultured and divided randomly into there groups :control group, moderate hypoxia(MH) group and severe hypoxia(SH) group. After 72 h, MTT method was used to investigate the proliferation of CFs, and the ultrastructure of fibroblasts were observed with transmission electron microscopy. The expression of PCNA and α-actin in cardiac fibroblasts were measured by the means of immunohistochemistry and laser scanning confocal microscopy, respectively. RESULTS: MTT A_{490 nm}value of MH group was significantly higher than that of control group by (18.4±25.0)% (P＜0.05), whereas MTT A_{490 nm} value of MH group was significantly lower than that of control group by (15.8±25.0)% (P＜0.05).The immunohistochemistry experiment showed that there was basal PCNA expression in control CFs, and it was increased in MH CFs(P＜0.05 vs control group), but there was no significant difference between SH and control CFs. Observed with transmission electron microscopy, the control CFs had typical phenotype of fibroblasts: shuttle-shaped, abundant rough endoplasmic reticulums (RER) and golgi complexes, but few mitochondria or micromyofilaments in cytoplasm. After MH for 72 h, there appeared lots of micromyofilaments in cytoplasm, but there were little micromyofilaments in SH CFs. Observed with laser confocal scanning microscopy, there was lower expression of α-actin in CFs of control group, while many regular bundles of micromyofilaments and the expression of α-actin were signifiantly increased (P＜0.05 vs control group) in CFs treated with MH. The α-actin expression in severe hypoxia CFs was not significantly different from control group. CONCLUSION:MH made CFs have the characteristics of myofibroblasts phenotype and enhanced PCNA expression.     

苹果乙烯响应因子基因MdERF11对脱落酸的敏感性分析

以‘嘎拉’苹果(Malus×domestica Borkh.)为试材,克隆了乙烯响应因子基因MdERF11(序列号MDP0000756341)。测序发现,该基因包含全长为483 bp的完整开放阅读框,编码161个氨基酸。系统进化树分析表明,这一乙烯响应因子与拟南芥AtERF11蛋白同源序列相似性最高。利用PlantCare数据库进行基因启动子顺式作用元件预测,MdERF11启动子序列中含有与脱落酸(ABA)、乙烯及干旱信号相关的顺式作用元件。荧光定量PCR分析表明,MdERF11在苹果的各组织中均有表达,在叶柄和果实中表达量相对较高;并且MdERF11的表达明显受到ABA的诱导。在外源ABA的处理下,MdERF11过量表达的苹果愈伤组织的生长势明显比野生型强,表明MdERF11降低了苹果愈伤组织对ABA的敏感性。     

Transfection and expression of murine interleukin-12 gene in B16F10 melanoma cells

AIM:To study the expression of murine interleukin-12(mIL-12) gene in B16F10 melanoma cells.METHODS:mIL-12 gene was inserted into pcDNA3.1 eukaryotic expression vector by DNA recombination and then transfected into B16F10 melanoma cells by electroporation. The integration and expression of mIL-12 gene in B16F10 melanoma cells were detected by PCR, RT-PCR and Western blot after positive cell clones had been screened.RESULTS:mIL-12 gene was confirmed to have been transfected and expressed in B16F10 cells on three levels of DNA, mRNA and protein.CONCLUSION:mIL-12 gene can successfully be transfected and expressed in B16F10 melanoma cells in vitro, which lays a foundation for the further investigation of mIL-12 gene tumor vaccine.     

Comparative study on intraocular transplatation of three B16 melanoma cell lines in mice

AIM:To establish an animal model for studying the development and metastasis of melanoma.METHODS:C57BL/6 mice were used as host to receive melanoma cell transplantation. Three kinds of melanoma cell lines, B16F0, B16F1 and B16F10, cultured to prepare the cell suspensions, were transplanted into the mouse anterior chamber (AC) of the eye. The time of eyeball diabross, time of survival and metastasis of lymph node and lung were observed.RESULTS:The time of eyeball diabross in F10 group was earlier than that in other groups. The time of eyeball diabross was no difference between F0 and F1 groups. Metastasis was developed 18 days after transplantation in F1 and F10 groups, where the tumor cells was found in ipsilateral cervical lymph nodes. The melanoma cells metastasized to lung in all three groups 28 days after transplantation. The survival time in F0 group was longer than F1 and F10 groups. There was no difference in survival times between F1 and F10 group.CONCLUSION:The differences of three kinds of melanoma cell lines in tumor development and metastasis provided the evidence that was useful for choosing suitable animal model further to study the eye melanima.     

CTRP3 increased insulin sensitivity of insulin resistant 3T3-L1 adipocytes via decreasing expression of inflammatory factors

AIM:To investigate the effects of C1q/TNF related protein 3 (CTRP3) on the insulin sensitivity of insulin resistant 3T3-L1 adipocytes. METHODS:The insulin resistance model of 3T3-L1 adipocytes was induced by palmic acid cultivation. The adipocytes were treated with different concentrations of recombinant CTRP3 protein (10, 50, 250,1 250 μg/L) for 12 h, and for different times (2, 6, 12, 24 h) at the concentration of 250 μg/L. The glucose consumption was detected by the glucose oxidase method. The glucose transport ratio was measured by 2-deoxidation-［3H］-glucose intake method. The contents of TNF-α and IL-6 in the supernatant were detected by ELISA. The mRNA expression of TNF-α, IL-6 and glucose transporter-4 (GLUT-4) was measured by real-time PCR. The protein expression of GLUT-4 was detected by Western blotting. RESULTS:Compared with normal control (NC) group, the glucose consumption and glucose intake ratio of insulin resistance (IR) group was decreased by 50.6% and 57.9%, respectively. Compared with IR group, with the increase in CTRP3 (10, 50, 250,1 250 μg/L) in intervention groups, the glucose consumptions were increased by 22.1%, 42.9%, 76.6% and 80.5%, respectively, and the glucose intake ratios were increased by 39.0%, 68.0%, 108.0% and 111.0%, respectively. With the increased duration (2, 6, 12 and 24 h) of CTRP3 treatment at the concentration of 250 μg/L, the glucose intake ratio was increased by 23.0%, 79.0%, 109.0% and 114.0%, respectively. The contents of TNF-α and IL-6 in the supernatant were decreased by 17.4% and 17.1% respectively as treated with CTRP3 at the concentration of 250 μg/L for 12 h, and the mRNA expression of TNF-α and IL-6 was decreased by 26.0% and 18.9% respectively, while the mRNA and protein expression of GLUT-4 was increased by 61.5% and 55.6% respectively. CONCLUSION: CTRP3 may increase the insulin sensitivity of insulin resistant 3T3-L1 adipocytes by down-regulating the expression of inflammatory factors, improving the insulin signal transduction and increasing the expression of GLUT-4.     

Chlamydia pneumoniae infection induces vascular smooth muscle cell migration through stimulating Rac1 activity via PI3K activation

AIM: To determine the role of Rac1 activation in the migration of vascular smooth muscle cells (VSMCs) induced by Chlamydia pneumoniae (C.pn) infection and to investigate the effect of phosphatidylinositol 3-kinase (PI3K) on Rac1 activation during the process. METHODS: The recombinant plasmid encoding glutathione S-transferase (GST)-p21-binding domain of p21-activated kinase 1(PBD) fusion protein was transformed into the competent bacteria to induce the expression of the fusion protein. The fusion protein was purified. GST-pull down assay was performed to evaluate the activity of Rac1 in C.pn-infected VSMCs pretreated with or without PI3K inhibitor LY294002 (25 μmol/L). Wound-healing assay and Transwell assay were performed to observe the changes of C.pn infection-induced VSMCs migration with or without pre-incubation of Rac1 inhibitor NSC23766 (50 μmol/L). RESULTS: Enough and biologically active GST-PBD fusion protein was obtained after purification. The activity of Rac1 in VSMCs infected with C.pn for 24 h increased significantly and was higher than that in control group. Rac1 activation induced by C.pn infection was inhibited by the pretreatment of VSMCs with LY294002. The migration ability of C.pn-infected VSMCs pre-incubated with NSC23766 was significantly reduced and was lower than that in C.pn infection group. CONCLUSION: C.pn infection induces the migration of VSMCs possibly through stimulating Rac1 activity via PI3K activation.     

Knockdown of HMGA2 gene inhibits TGF-β1-induced human embryonic lung fibroblast growth and collagen synthesis

AIM: To investigate the effects of high mobility group A2(HMGA2) gene knockdown on the cell viability, apoptosis, collagen synthesis and oxidative stress of human embryonic lung fibroblast (HELF) induced by transforming growth factor-β1 (TGF-β1). METHODS: The HELF were divided into blank group, TGF-β1 group,negative control (NC) group and HMGA2 siRNA(si-HMGA2) group. The protein levels of HMGA2, AKT and p-AKT were determined by Western blot. The cell viability and apoptotic rate was analyzed by MTT assay and flow cytometry,respectively. The mRNA expression of collagen I (COL-Ⅰ) and COL-Ⅲ was detected by RT-qPCR. DCFH-DA was used to detect the content of reactive oxygen species (ROS). RESULTS: Compared with blank group, the protein levels of HMGA2 and p-AKT, the cell viability, the mRNA expression of COL-Ⅰ and COL-Ⅲ in TGF-β1 group were significantly increased, but the apoptotic rate and ROS level were significantly decreased (P<0.05). Compared with TGF-β1 group, the protein levels of HMGA2 and p-AKT, the cell viability, the mRNA expression of COL-Ⅰ and COL-Ⅲ in si-HMGA2 group were significantly decreased, but the apoptotic rate and ROS level were significantly increased (P<0.05). CONCLUSION: Knockdown of HMGA2 gene expression decreases the viability and collagen synthesis, and promotes apoptosis and ROS production of human embryonic lung fibroblasts induced by TGF-β1. The mechanism may be related to down-regulation of PI3K/AKT signaling pathway.     

lncRNA-MALAT1 targets and downregulates miR-570-3p expression to promote proliferation of gastric cancer cells

AIM:To investigate whether long non-coding RNA MALAT1 (lncRNA-MALAT1) targets and down-regulates microRNA-570-3p (miR-570-3p) expression to further promote the proliferation of gastric cancer cells. METHODS:Gastric cancer cell line SGC7901 was cultured in vitro and divided into 3 groups:blank control, si-MALAT1 and si-MALAT1 NC. The si-MALAT1 and si-MALAT1 NC groups were transfected with MALAT1 siRNA and its negative control, respectively. The cell proliferation was evaluated by MTS assay. The expression of miR-570-3p was detected at different time points in the pure SGC7901 gastric cancer cell line, and the expression of lncRNA-MALAT1 and miR-570-3p in different groups was detected by RT-qPCR. The potential complementary binding sites of lncRNA-MALAT1 and miR-570-3p were predicted by RegRNA. The MALAT1 gene and its mutant fragment were cloned into luciferase reporter vector psiCHECK-2. Restriction enzyme analysis and sequencing were used to identify whether the recombinant plasmids carrying MALAT1 or MALAT1-Mut were successfully constructed. miR-570-3p mimic, miR-570-3p inhibitor, miR-570-3p mimic negative control and miR-570-3p inhibitor negative control were co-transfected into the 293T cells with the luciferase repor-ters containing MALAT1 or MALAT1-Mut. Dual-luciferase reporter assay was performed to detect luciferase activity in different groups in order to verify the relationship between lncRNA-MALAT1 and miR-570-3p. RESULTS:Compared with blank control group and si-MALAT1 NC group, the A₄₉₀ value in si-MALAT1 group was significantly decreased (P<0.01). The expression of miR-570-3p presented an obvious declining trend over time. The expression of lncRNA-MALAT1 in si-MALAT1 group was remarkably decreased, whereas the expression of miR-570-3p was obviously increased. The dual-luciferase reporter assay indicated that the MALAT1 reporter luciferase activity decreased significantly in miR-570-3p mimic group compared with mimic negative control (P<0.01), and the luciferase activity of MALAT1 reporter was obviously up-regulated in miR-570-3p inhibitor group compared with miR-570-3p mimic group (P<0.01). However, miR-570-3p mi-mic, miR-570-3p inhibitor, miR-570-3p mimic negative control and miR-570-3p inhibitor negative control showed no effect on the luciferase activity of MALAT1-Mut reporter. CONCLUSION:lncRNA-MALAT1 targets and down-regulates miR-570-3p expression to further promote the proliferation of gastric cancer cells.     

Antagonistic effect of thalidomide on TGF-β1-induced change of CTGF gene promoter-binding proteins in HELF cells

AIM:To investigate the antagonistic effect of thalidomide (THD) on the activation of connective tissue growth factor (CTGF) gene promoter induced by transforming growth factor-β₁ (TGF-β₁) in human embryonic lung fibroblasts (HELF). METHODS:Luciferase reporter vector driven by CTGF gene promoter was used to detect the effects of TGF-β₁ and THD on the activity of CTGF gene promoter, and DNA pull-down assay with CTGF gene promoter as a probe was used to analyze the changes of CTGF promoter-binding proteins under different conditions. RESULTS:TGF-β₁ increased the activity of reporter driven by CTGF gene promoter (P<0.01), while THD significantly inhibited TGF-β₁-induced increase in the reporter activity dose-dependently (P<0.01). At the same time, THD had inhibitory effect on the TGF-β₁-induced change of CTGF gene promoter-binding proteins (P<0.05). CONCLUSION:Regulation of CTGF gene promoter-binding proteins takes part in TGF-β₁-induced activation of CTGF gene promoter, while THD has antagonistic effect on this process.     

LC3 protein expression and localization in mouse follicular granulosa cells

AIM: To investigate the expression and localization of autophagy related protein microtublule associated protein 1 light chain 3 (LC3) at various stages of follicular development and atresia in the mice.METHODS: On 0,1,2,3,4 and 5 day after intraperitoneal injection of pregnant mare serum gonadotropin (PMSG), expression and positioning situation of autophagy related protein LC3 and apoptosis related protein cleaved caspase-3 were examined by the me-thod of immunohistochemical staining. The protein levels of cleaved caspase-3 and LC3 were determined by Western blot in cultured mouse granulosa cells after incubation under serum-free conditions in the absence or presence of FSH. LC3 subcellular localization in granulosa cells were studied by the method of immunofluorescence.RESULTS: The LC3 protein expressed in granulosa cells during all developmental stages mainly. Granulosa cells of atretic follicles that showed intense staining of cleaved caspase-3 and LC3. The protein levels of cleaved caspase-3 and LC3-Ⅱ in the granulosa cells significantly decreased at 1 d and 2 d after intraperitoneal injection of PMSG (P<0.05). The protein levels of cleaved caspase-3 and LC3-Ⅱ in the granulosa cells increased in turn on 3, 4 and 5 day after intraperitoneal injection of PMSG. The positive correlation between LC3-Ⅱ and cleaved caspase-3 protein levels was observed (r²=0.8299, P<0.05). The LC3-Ⅱ protein expressed with punctuate structures in granulosa cell cytoplasm cultured under serum-free conditions in the presence of FSH.CONCLUSION: LC3 is expressed in the follicular granulosa cells with cell specificity and regional specificity. Autophagy is induced mainly in granulosa cells during folliculogenesis and shows positive correlation with apoptosis. Ovarian granulosa cell autophagy and apoptosis are gonadotropic hormone dependent.     

Mutation of Npc1 gene causes abnormal lipid metabolism to induce apoptosis of renal cells and promote fibrosis

AIM: To investigate the dysfunction of renal cell and tissue in Npc1 mutant mice, in order to provide support for the treatment of Niemann-Pick disease type C1 (NPC1) patients.METHODS: The kidneys of wild-type (Npc1^+/+) and Npc1 mutant (Npc1^-/-) mice on postnatal day 60 were isolated. HE staining was performed to examine the morphological changes of the renal tissues. Oil red O staining was used to examine the lipid deposition in the renal tissues. The apoptosis of the renal cells was detected by TUNEL staining. The expression of apoptosis-related proteins in the renal tissue was determined by Western blot, and immunofluorescence was performed to examine the expression of α-smooth muscle actin (α-SMA) and vimentin in the renal tissues. RESULTS: Compared with Npc1^+/+ mice, the morphological observation showed obvious vacuoles and no lipid deposition in the renal tissue of Npc1^-/- mice. Subsequently, TUNEL staining showed significant increase in the apoptotic cells in the renal tissue of Npc1^-/- mice (P<0.01), and the expression levels of Bax and Bad were up-regulated in the renal tissues of Npc1^-/- mice (P<0.01), but Bcl-2 was down-regulated (P<0.05). Furthermore, the expression of α-SMA and vimentin was significantly up-regulated in the renal tissues of Npc1^-/- mice (P<0.01). CONCLUSION: Npc1 gene mutation causes abnormal lipid metabolism in the renal cells, which induces the apoptosis of renal cells and promotes the fibrosis of renal tissue.     

Effect of down-regulation of X-box binding protein 1 gene expression on viability and apoptosis of glioma cells

AIM: To investigate the effect of down-regulation of X-box binding protein 1 (XBP1) expression on the viability and apoptosis of glioma cells. METHODS: The mRNA expression of XBP1 in the glioma tissues was detected by qPCR. Small interfering RNA (siRNA) interfering with XBP1 expression (XBP1-siRNA) was transfected into human brain glioma U251 cells. At the same time, control group (the cells without special treatment) and negative control (NC-siRNA) group (transfected with siRNA without any interference) were set up. The mRNA expression of XBP1 in the 3 groups 48 h after transfection was detected by qPCR. The protein levels of XBP1, proliferating cell nuclear antigen (PCNA), B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), cyclin D1 (cyclin D1), phosphatidylinositol 3-kinase (PI3K) and phosphorylated Akt (p-Akt) were determined by Western blot. The cell viability was measured by CCK-8 assay. The cell cycle distribution and apoptosis were analyzed by flow cytometry. RESULTS: The expression level of XBP1 in the glioma tissues was significantly higher than that in the tumor adjacent tissues (P<0.05). The XBP1 expression at mRNA and protein levels was significantly decreased in the cells transfected with XBP1-siRNA (P<0.05). No statistically significant difference of the cell viability, cell cycle, apoptotic rate and the protein levels of PCNA, Bcl-2, Bax, cyclin D1, PI3K and p-Akt between NC-siRNA group and control group was observed. Compared with control group, the cell viability, S-phase cells and the protein levels of PCNA, Bcl-2, cyclin D1, PI3K, and p-Akt in XBP1-siRNA group were decreased significantly, and the apoptotic rate, G₀/G₁-phase cells and Bax protein expression were significantly increased (P<0.05). CONCLUSION: Down-regulation of XBP1 gene expression in brain glioma cells reduces the viability of cancer cells, blocks the cells in G₁ phase and promote apoptosis. The mechanism is related to the inhibition of PI3K/Akt signaling pathway.     

Clinical significance of 4-1BBL protein expression in human glioma tissues

AIM: To investigate the expression of 4-1BBL(CD137L) protein in human glioma tissues, and to analyze its clinical significance. METHODS: The expression levels of 4-1BBL protein in 82 human glioma specimens were measured by the method of immunohistochemistry. The correlation of 4-1BBL protein level with histopathologic features and survival time was analyzed. RESULTS: 4-1BBL protein positive cells were observed in human brain astrocytic tumors, which were not found in meningioma and normal brain tissues. In our study, the expression of 4-1BBL protein in 3 cases of normal brain tissues was negative. The expression of 4-1BBL protein in 31 cases of glioma specimens were positive, and total positive rate was 37.8% (31/82). The expression of 4-1BBL protein was related to the survival time and age of the patients, but not related to the pathological grade of glioma. Statistically, the patients with positive expression of 4-1BBL protein had longer survival time (P<0.05). CONCLUSION: Expression of 4-1BBL protein may have reference value to predict the prognosis of patients with glioma.     

Effects of Mage-H1 expression on PC12 cell differentiation and cell cycle

AIM: To explore the effects of melanoma-associated antigen H1 (Mage-H1) on cell proliferation and differentiation. METHODS: A phase contrast microscope was used to observe the morphological changes of PC12 cells treated with or without nerve growth factor (NGF). The expression of Mage-H1 in pre-and post-differentiated PC12 cells was detected by RT-PCR and Western blotting, and its potential effects on the cell cycle were analyzed by flow cytometry. RESULTS: After induced by NGF for 8 days, over 92% of PC12 cells were differentiated. The relative levels of Mage-H1 mRNA and protein in the differentiated PC12 cells were 4.6 times and 2.6 times higher than those in control cells,respectively. Moreover, the PC12 cells transiently expressed Mage-H1 were significantly arrested in G₀-G₁ phase as compared to the cells transfected with an empty vector.CONCLUSION: Mage-H1 inhibits the proliferation of PC12 cells and promotes the differentiation of the cells.     

Effect of RYR1 gene expression knock-down by siRNA on mitochondria in C2C12 cells

AIM: To investigate the effect of ryanodine receptor 1 (RYR1) down-regulation on mitochondria in mouse myoblast C2C12 cell line and to explore the possible mechanism. METHODS: The expression of RYR1 in the C2C12 cells was knocked down by the targeted small interfering RNA (siRNA). The mitochondrial number and morpholo-gical changes were evaluated by transmission electron microscopy and the stereoscopic analysis. Real-time PCR and Western blot were used to determine the mRNA and protein levels of mitofusin 2 (Mfn2), peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and extracellular signal-regulated kinase 1/2 (ERK1/2), respectively. RESULTS: The expression of RYR1 was significantly down-regulated by siRNA transfection (P<0.01), with fragmentized mitochondria in the C2C12 cells in knock-down (KD) group. Although no statistical difference of the mitochondrial number was observed, the mitochondria area and circumference were significantly lowered in KD group (P<0.05). In KD group, the mRNA and protein expression of Mfn2 was significantly reduced (P<0.01). The mRNA level of PGC-1α was also reduced (P<0.01), but no significant change at protein level was observed. No change of ERK1/2 expression and phosphorylated ERK1/2 level was detected. CONCLUSION: Knock-down of RYR1 expression leads to morphological changes of mitochondria, and down-regulation of Mfn2 expression may be involved in the underlying mechanism. While PGC-1α and ERK1/2-associated oxidative stress pathway may not play an important role in the process.