Effects of naringin on MAPK signal pathway in rat bone marrow mesenchymal stem cells during osteogenic differentiation

AIM: To study the effects of Chinese herbal monomer naringin (NG) on the MAPK signal pathway in bone marrow mesenchymal stem cells (MSCs) derived from SD rats during the differentiation into osteoblasts in vitro . METHODS: The changes of evaluating indicators alkaline phosphatase (ALP), bone gla protein (BGP) and type I collagen (Col I) in MSCs were observed under the conditions of normal, adding p38 pathway inhibitor SB203580, adding extracellular signal-regulated kinase (ERK) pathway inhibitor PD98059, adding c-Jun N-terminal kinase (JNK) pathway inhibitor SP600125, and adding SB203580, PD98059 and SP600125 together. The protein phosphorylation of p38, ERK1/2 and JNK was measured by Western blotting. The mRNA expression levels of transforming growth factor beta 1 (TGF-β₁), bone morphogenetic protein 2 (BMP-2) and core binding factor α1 (Cbfα1) were measured by fluorescence quantitative PCR. RESULTS: The most effective concentration of NG to promote the differentiation of MSCs into osteoblasts was 10^-7 mol/L. The highest expression levels of both ALP and BGP were observed in NG group (P<0.05), while the expression of Col I did not reveal significant difference (P>0.05). Compared with NG group, the expression levels of ALP, BGP and Col I decreased differently after adding different inhibitors. Compared with control group, the protein phosphorylation of JNK was increased (P<0.05), and the phosphorylation of p38 was decreased (P<0.05), while the phosphorylation of ERK1/2 did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the protein phosphorylation of p38, ERK1/2 and JNK showed fluctuation with some increasing and others decreasing. Compared with control group, the expression of BMP-2 was increased (P<0.05), and the expression of Cbfα1 was decreased(P<0.05), while the expression of TGF-β₁ did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the mRNA expression levels of TGF-β₁, BMP-2 and Cbfα1 decreased differently after adding different inhibitors. CONCLUSION: Activation of ERK/JNK signaling and up-regulation of BMP-2 expression may be the main mechanism of NG to promote the differentiation of MSCs into osteoblasts. NG has strong impact on p38 pathway to improve the expression of BMP-2 in MSCs.     

Role of TGF-β1/Smads and ERK expression in vascular remodeling induced by high-salt diet in Wistar rats

AIM: To investigate the role of transforming growth factor β₁ (TGF-β₁)/Smads and extracellular signal-regulated kinase(ERK) expression in vascular remodeling induced by high-salt diet in Wistar rats. METHODS: Wistar rats were randomly divided into 3 groups: normal control group (n=13), high salt (8%) model group and high salt+telmisartan group (n=13). Tail-cuff arterial pressure was determined every 2 weeks. After 24 weeks, the rats in high salt model group were divided into model animals with hypertension group (MH, n=12) and model animals without hypertension group (MN, n=12). The remodeling of aorta and mesenteric artery was observed by HE and Masson staining. In addition, the techniques of immunohistochemistry and real-time PCR were applied to detect the expression of proliferating cell nuclear antigen (PCNA), TGF-β₁, p-Smad2/3, p-ERK1/2 and Smad7 at both protein and mRNA levels. RESULTS: Compared with normal control group, blood pressure in MH group was much higher, and media thickness (MT) and collagen volume fraction (CVF) of arteries in MH and MN groups were higher.The mRNA expression of TGF-β₁, Smad2 and Smad7 in the aorta was significantly increased, and the protein levels of PCNA, p-ERK1/2, TGF-β₁ and p-Smad2/3 in the aorta and mesenteric artery media were elevated, but Smad7 decreased. After telmisartan treatment, MT and CVF were much lower,and the protein levels of PCNA, TGF-β₁, p-Smad2/3 and p-ERK1/2 were significantly reduced, whereas Smad7 was increased. CONCLUSION: The abnormal expression of TGF-β₁/Smads and ERK may be involved in the mechanism of remodeling of aorta and mesenteric artery induced by high-salt diet. Telmisartan prevents the vascular remodeling via regulating TGF-β₁/Smads and ERK signal pathways mediated by angiotensinⅡ type 1 (AT₁) receptor, at least in part.     

The roles of ERK signaling pathway in osteogenic differentiation of rat MSCs promoted by quercetin

AIM:To study the roles of extracellular signal-regulated kinase(ERK) signal pathway in the process of osteogenic differentiation in rat mesenchymal stem cells(MSCs) promoted by quercetin(QUE). METHODS:The optimal concentration of QUE for promoting osteogenic differentiation of rat MSCs was determined by MTT and alkaline phosphatase(ALP) detection. The activity of ALP was detected by the ALP detection kit. The expression of bone Gla protein(BGP) and collagen typeⅠ(ColⅠ) was observed by ELISA analysis. MSCs were exposed to QUE at optimal concentration with or without ERK1/2 inhibitor PD98059. Non-phosphorylated and phosphorylated expression of ERK1/2 was analyzed by Western blotting. The mRNA expression of transforming growth factor β₁(TGF-β₁), bone morphogenetic protein 2(BMP-2) and core binding factor α1(Cbfα1) was measured by fluorescence quantitative PCR. RESULTS:QUE at concentrations of 0.1 μmol/L, 1 μmol/L and 10 μmol/L induced the expression of ALP in MSCs in a dose-dependent manner, and also promoted MSCs proliferation. The expression levels of ALP, BGP and ColⅠwere higher in QUE group, and was lower in PD89059 group than those in control group. Compared with control group, the level of phosphorylated ERK1/2, and the mRNA expression of TGF-β₁, BMP-2 and Cbfα1 increased in QUE group. The mRNA expression of TGF-β₁, BMP-2 and Cbfα1 in QUE+PD98059 group decreased as compared with QUE group. CONCLUSION:QUE promotes osteogenic differentiation of MSCs by activating ERK signaling pathway.     

Effects of Astragalus injection combined with puerarin injection on expression of BMP-7 and TGF-β1 in type 2 diabetic KKAy mouse kidney

AIM: To observe the effects of Astragalus injection combined with puerarin injection on the expression of transforming growth factor beta 1 (TGF-β₁) and bone morphogenetic protein 7 (BMP-7) in the kidney of type 2 diabetic KKA^y mouse. METHODS: The male KKA^y mice of 14 weeks old were randomly divided into model group and Astragalus injection combined with puerarin injection treatment (Astragalus+puerarin) group. The age-matched male C57BL/6J mice were selected as normal group. The general conditions and body weight of the mice were observed. Blood glucose (BG), triglyceride (TG), cholesterol (TC) and serum creatinine (SCr) were examined at the 20th, 24th and 28th week. The protein expression of renal TGF-β₁ was determined by immunohistochemical method. The mRNA expression of BMP-7 and TGF-β₁ was detected by RT-PCR. RESULTS: Compared with normal group, the body weight, BG, TG, TC and SCr increased significantly in model group. TGF-β₁ expression at protein and mRNA levels was increased, while mRNA expression of BMP-7 was decreased in KKA^y mice. Compared with model group, the body weight, BG, TG, TC and SCr reduced in Astragalus+puerarin group. The mRNA expression of BMP-7 in the renal tissues was higher, and TGF-β₁ expression at mRNA and protein levels was significantly lower in Astragalus+puerarin group than those in model group. CONCLUSION: Astragalus injection combined with puerarin injection has renal protective effects on type 2 diabetic KKA^y mice. The mechanism may be related to restoring BMP-7 expression and reducing the overexpression of TGF-β₁ in renal tissues.     

Influence of losartan potassium on renal expression of TGF-β1, CD68 and MCP-1 in type 2 diabetic nephropathy rats

AIM: To observe the effects of losartan potassium on renal expression of transforming growth factor beta 1(TGF-β₁), CD68 and monocyte chemoattractant protein-1 (MCP-1) in type 2 diabetic nephropathy rats for exploring the protective mechanism of losartan potassium on type 2 diabetic rat kidney. METHODS: Thirty Sprague-Dawley rats were randomized into 3 groups: normal control group, model group and treatment group. The morphology of kidney tissues, the renal function, and the change of 24 h urinary protein quantitative index were measured after 15 weeks of treatment, while TGF-β₁, CD68 and MCP-1 expression in kidney cortex was observed by immunohistochemistry. RESULTS: Compared with the normal control rats, the body weight of the rats was lower in other groups, but the levels of blood glucose, triglyceride and cholesterol were higher.The expression of CD68, MCP-1 and TGF-β₁, 24 h urinary protein quantitative index and serum creatinine were higher in model group than those in normal control rats. However, compared with model group, serum creatinine, 24 h urinary protein quantitative index and the expression of CD68, MCP-1 and TGF-β₁ were decreased in treatment group. CONCLUSION: Losartan potassium protects the kidney of diabetic nephropathy rats through inhibiting the expression of TGF-β₁ and MCP-1 in the kidney and restraining macrophage infiltration.     

Inhibitory effect of notoginsenoside monomer R1 on activation of p38 MAPK signaling pathway induced by hypoxic hypercapnia in PASMCs

AIM: To explore the mechanism of notoginsenoside monomer R₁ (R₁) against hypoxic hypercapnia-induced pulmonary vasoconstriction (HHPV) by investigating the effect of R₁ on p38 mitogen-activated protein kinase (p38MAPK) signaling pathway in pulmonary arterial smooth muscle cells (PASMCs) under the condition of hypoxia and hypercapnia. METHODS: Primary cultured PASMCs, which were isolated from Sprague-Dawley rats, were incubated in logarithmic growth phase from the 2nd to 5th generation with different concentrations (8, 40 and 100 mg/L) of R₁ under the condition of 6% CO₂ plus 1% O₂ for 24 h. The expression of p38 at mRNA and protein levels was detected by RT-PCR and Western blotting,respectively. RESULTS: The results of Western blotting and RT-PCR analysis indicated that the protein and mRNA expression levels of p-p38 MAPK were significantly higher in hypoxic hypercapnia group with DMSO control than those in normoxia control group (P<0.01). In R₁ treatment groups, the levels of p-p38 MAPK protein and p38 MAPK mRNA were markedly decreased (P<0.01) in a dose-dependent manner. CONCLUSION: p38 MAPK signaling pathway may mediate hypoxic hypercapnia pulmonary vasoconstriction in rats. Notoginsenoside monomer R₁ attenuates HHPV, which may be related to blockage of p38 MAPK signal pathway.     

Effect of salvianolic acid B on high glucose-induced phenotypic transition and extracellular matrix secretion in human glomerular mesangial cells

AIM:To investigate the effect of salvianolic acid B (Sal B) on high glucose-induced phenotypic transition and extracellular matrix (ECM) secretion in human glomerular mesangial cells (HGMCs) and the underlying mechanisms. METHODS:HGMCs were randomly divided into control group, high glucose group and high glucose plus high dose, medium dose and low dose of Sal B groups. The HGMCs except those in control group were exposed to high glucose (33.3 mmol/L) for 72 h, while those in Sal B groups were co-incubated with indicated concentrations of Sal B. The protein levels of α-smooth muscle actin (α-SMA), transforming growth factor-β₁ (TGF-β₁) and phosphorylated Smad2 and p38 mitogen-activated protein kinase (MAPK) were determined by Western blot. The secretion levels of collagen type I (Col I), collagen type Ⅲ (Col Ⅲ), fibronectin (FN) and laminin (LN) were measured by ELISA. RESULTS:Exposure to high glucose markedly increased the protein expression of α-SMA, TGF-β₁, Col I, Col Ⅲ, FN and LN in the HGMCs (P<0.01). The phosphorylation levels of Smad2 and p38 MAPK were also significantly increased (P<0.01). Co-incubation with Sal B evidently decreased the protein expression of α-SMA, TGF-β₁, Col I, Col Ⅲ, FN and LN in the HGMCs induced by high glucose (P<0.05 or P<0.01). The phosphorylated levels of Smad2 and p38 MAPK were also reduced noticeably (P<0.05 or P<0.01). CONCLUSION:Sal B significantly suppresses high glucose-induced phenotypic transition and ECM secretion in the HGMCs, which might be attributed, at least partly, to inhibition of TGF-β₁/Smad signaling pathway and p38 MAPK activation.     

Association among COX2, iNOS and p38MAPK in late phase of preconditioning in rat cardiomyocytes

AIM:To explore the association among COX₂ , iNOS and p38MAPK in late phase of preconditioning. METHODS: The expression of COX₂ , iNOS and p38MAPK activity were determined by western blotting and the injury of cardiomyocyte was assessed by LDH release and trypan blue exclusion in four groups: control group, group IPC (ischemic preconditioning), group SMT (iNOS inhibitor), group NS₃₉₈ (COX₂ inhibitor) and group SB (p38MAPK inhibitor).RESULTS:The expression of COX₂ in group IPC increased markedly in comparision with group SMT and group SB.The expression of iNOS in group SB was lower than that in group IPC and group NS₃₉₈.The difference of the amount of iNOS was not significant between group IPC and group NS₃₉₈.The difference of the amount of phospho-p38MAPK was not significant among group IPC, group SMT and group NS₃₉₈(P>0.05).The LDH was lower, and cell viability was higher in group IPC than those in control group.The LDH was higher, and cell viability was lower in group SMT, group NS₃₉₈ and group SB than those in group IPC. CONCLUSIONS:In late phase of preconditioning , the p38MAPK activity and expression of iNOS and COX₂ increase significantly in rat cardiomyocytes, activited p38MAPK mediates iNOS, then promotes COX₂ expression.     

Alteration of p38 MAPK activity in lung tissue in diabetic rats

AIM: To observe the pathologic changes in lung and the role of p38 MAPKinase signal pathways in pulmonary alteration in diabetic rats. METHODS: Diabetic rats were induced by intraperitoneally injected streptozotozin (STZ). After 4 weeks, we observed the pathologic changes in lungs, tested protein kinase C (PKC) activities by isotope in lungs of model rats, tested transforming growth factor (TGF-β₁) by Western blotting and immunohistochemical analysis, and determined the expression of p38 MAPKinase mRNA using in situ hybridization.RESULTS: After STZ administration for 4 weeks, we observed thickened pulmonary capillary basal lamina and increased number of fibre in Diabetes mellitus (DM) rats. TGF-β₁ levels, PKC and p38 MAPK activities were also found increased. CONCLUSION: The increased activities of TGF-β₁ and p38 MAPK suggeste that TGF-β₁ may play an important role in diabetic lung, and hyperglycemia-PKC-p38 MAPK signal pathways may be involved in the pathogenesis of diabetes.     

Effects of thrombospondin 1 on rat cardiac fibroblasts induced by transforming growth factor β1

AIM:To investigate the effects of thrombospondin 1 on transforming growth factor β₁ induced rat cardiac fibroblasts (CFs). METHODS: CFs of neonatal Sprague -Dawley (SD) rats were isolated with the method of digestion and differential anchoring velocity. The proliferation and collagen synthesis of rat CFs were observed with MTT and hydroxyproline. The expression of TSP-1mRNA was analyzed by RT-PCR.RESULTS: The dose and time-dependent effects of TGF-β₁ were observed. Expression of TSP-1 was increased significantly (P<0.01). Stimulation of CFs with TGF-β₁ (20 μg/L, 24 h) significantly increased CFs proliferation and collagen synthesis (P<0.01). TSP-1 antisense oligonucleotide effectively inhibited TGF-β₁ induced CFs proliferation and collagen synthesis (P<0.01).CONCLUSION:The proliferation and collagen synthesis of CFs induced by TGF-β₁ are inhibited by TSP-1 antisense oligonucleotide, which may exert helpful effect on anti-fibrosis.     

Effects of Coriaria sinica Maxim’s extract on burn wound healing and scar hyperplasia in rats based on ILK signaling pathway

AIM: To explore the mechanism of Coriaria sinica Maxim’s extract (CSME) promoting burn wound healing in the early stage and inhibiting excessive scar hyperplasia in the later stage, based on the signaling pathways, such as transforming growth factor-β₁ (TGF-β₁) regulated by integrin-linked kinase (ILK) and ILK regulated by PI3K/AKT. METHODS: Female SD rats (n=180; 180~200 g) were randomly divided into 6 groups: normal control (NC) group, vaseline (VL) group, silver sulfadiazine (SS) group and low-, medium- and high-dose of CSME (CSME-L, CSME-M and CSME-H) groups, with 30 rats in each group. Except for the rats in NC group, VL, SS, and 3 doses of CSME were applied to the wound surface of the rats in the corresponding groups every day after II° burn model was made on their waist-back in the condition of chloral hydrate anesthesia. To calculate the healing rate (HR), 10 rats in each experimental group were randomly selected to remove their wound skin for observing the pathologic change, detecting the expression of related proteins by Western blot and RT-qPCR, and checking the collagen shrinkage (SK) by fibroblast culture at the 7th, 14th, and 21st days. RESULTS: The expression of ILK, fibronectin (FN), TGF-β₁, α-smooth muscle actin (α-SMA) and integrin-β₁ (ITG-β₁) at protein and mRNA levels in wound skin of CSME groups was stronger than that in VL group and SS group at the 7th day in a dose-dependent manner, but weaker than that in VL group and SS group at the 21st day (P<0.05). Meanwhile, the protein and mRNA expression of collagen type I (Col I) in CSME groups was stronger than that in VL group and SS group from the 7th day to the 14th day, but weaker than that in VL group and SS group at the 21st day in a dose-dependent manner (P<0.05). However, the protein and mRNA expression of collagen type III (Col III) in CSME groups was weaker than that in VL group and SS group from the 7th day to the 14th day, but stronger than that in VL group and SS group at the 21st day in a dose-dependent manner (P<0.05). The SK of fibroblasts in VL group and SS group was increased continuously over time and reached its peak at 96 h. SK in CSME groups was only higher than that in VL group and SS group at 24 h and 48 h in a dose-dependent manner, but lower than that in VL group and SS group at 96 h (P<0.05). CONCLUSION: CSME promotes burn wound healing in the early stages and inhibits the scar hyperplasia in the later stages. The mechanisms may be related to its multicomponents or multiple-targets to intervene in the signaling pathways such as TGF-β₁ regulated by ILK and ILK regulated by PI3K/AKT. It may also be related to the ratio of Col I and Col III expression.     

Expression and functional role of p38 MAPK in the kidney after unilateral ureteral obstruction in rats

AIM: To investigate the expression and functional role of p38MAPK in the kidney after unilateral ureteral obstruction in rats. METHODS: Unilateral ureteral obstruction (UUO) models were induced by ligating the left ureter. Rats were sacrificed at 1 h, 3 h, 6 h, 12 h, 1, 3, 5, 7, 14, 21, and 28 days after UUO was initiated. p38MAPK activity was assayed by immunohistochemical staining and specific substrate phosphorylation with immunoprecipitation and Western blotting. TGFβ mRNA and protein expression were analyzed with in situ hybridization and immunohistochemical stainning. RESULTS: A basic p38MAPK activity was detectable in the normal kidney(0.22±0.06).p38MAPK pathway was rapidly act ivated at 1 hour(0.45±0.14 vs control,P<0.05)and this was steadily in creased by 12 hours(0.91±0.07 vs control,P<0.01)after UUO.Afterwards,the activity of p38MAPK reduced grad ually,then increased again from 3 days and this was steadily increased by 7 days(0.93±0.06 vs control,P<0.01). Upregulation of TGFβ₁ was markedly tested at 3 days(13.55±6.33 vs control,P<0.05)and this was steadily in creased by 7 days(26.78 8.77 vs control,P<0.01).The activation of p38MAPK preceded markedly the expression of TGF 1.The early activity of p38 MAPK was positively related to the amount of TGFβ₁ expression.The amount of TGFβ₁ expressed in obstructed kidney also related significant ly to the late activity of p38MAPK(r=0.84,P<0.01). CONCLUSION: The activity of p38MAPK is increased significantly in the obstructed kidney, which may cause renal fibrosis via inducing the expression of TGFβ₁.     

Dexmedetomidine reduced isoflurane-induced neuroapoptosis by inhibiting activation of p38 and JNK proteins in hippocampus of neonatal rats

AIM：To investigate the effect of dexmedetomidine (Dex) on neuronal apoptosis induced by isoflurane (Iso) and its relationship with the expression of p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinase (JNK) proteins in the hippocampus of neonatal rats. METHODS：Forty-eight neonatal SD rats at postnatal day 7 were randomly divided into control group (Con), Dex group, Iso group and Iso combined with Dex (Iso+Dex) group. Rats in Iso and Iso+Dex groups were exposed to 0.75% Iso for 6 h, while rats in Con and Dex groups were exposed to air for 6 h. Rats were intraperitoneally injected with 25 μg·kg-1 Dex (Dex and Iso+Dex groups) or 150 μL saline (Con and Iso groups) 20 min before exposure and 2 and 4 h after exposure. After the termination of anesthesia, the neuronal apoptosis in hippocampal CA1 region was detected by TUNEL staining, and the protein expression of cleaved caspase-3, phospho-p38 (p-p38), p38, phospho-JNK (p-JNK) and JNK in hippocampal tissues was detected by Western blotting. RESULTS：The number of TUNEL positive cells in hippocampal CA1 region of the rats in Iso group was increased by 447.57% (P<0.01) compared with Con group, while Dex significantly inhibited the increased TUNEL positive cells in Iso group by 75.18% (P<0.01). The expression of cleaved caspase-3 protein in Iso group was increased by 126.29% (P<0.01) compared with Con group, while Dex reversed the increased cleaved caspase-3 protein expression (P<0.01). Iso significantly increased the phosphorylation of p38 and JNK proteins (P<0.01), while Dex reversed the increased p-p38 and p-JNK proteins (P<0.01). CONCLUSION：Dex attenuates Iso-induced neuroapoptosis in the hippocampus of neonatal rats through inhibiting the phosphorylation of p38 and JNK proteins.     

Effects of cyclic tensile strain on expression of p38 MAPK and phospho-p38 MAPK in osteoarthritis chondrocytes

AIM: To observe the effect of cyclic tensile strain (CTS) on the expression of p38 MAPK and phospho-p38 MAPK in rabbit osteoarthritis (OA) chondrocytes in vitro. METHODS: The animal model of OA was induced by anterior cruciate ligament transection in New Zealand white rabbits. The animals in all groups were evaluated 10 weeks later. The rabbits in OA group were randomly divided into 3 groups, low CTS (0.5 Hz, sin10%, 6 h/d) group, high CTS (1.0 Hz, sin10%, 6 h/d) group and control group. Both CTS groups were stimulated by a Flexercell-4000 tension system. The expression of p38 MAPK and phospho-p38 MAPK of the chondrocytes was analyzed by RT-PCR and Western blotting at the time points of 24 h, 1 week and 2 weeks. RESULTS: The knee joints of the rabbits in OA group had obvious degeneration of articular cartilage. The expression of p38 MAPK in normal group was significantly lower than that in control group (P<0.01), and the difference between low CTS group and high CTS group 1 week after stimulation (P<0.05) was observed. Meanwhile, significant difference was found between low CTS group and control group 2 weeks after CTS treatment (P<0.01). The expression of phospho-p38 MAPK was decreased at different time points in low CTS group. CONCLUSION: Different cyclic tensile strains lead to different effects on the expression of p38 MAPK and phospho-p38 MAPK in the chondrocytes. p38 MAPK signaling pathway plays an important role in the development of osteoarthritis in chondrocytes.     

Effect of Akt/GSK-3β/Snail signaling pathway on EMT in A549/DDP cells mediated by TGF-β1

AIM: To observe the expression of Akt/GSK-3β/Snail signaling pathway-related molecules in cisplatin-resistant cell line A549/DDP mediated by transforming growth factor-β₁ (TGF-β₁), and to explore the association of Akt/GSK-3β/Snail signaling pathway with epithelial-mesenchymal transition (EMT). METHODS: The A549/DDP cells were divided into TGF-β₁ (+) group, TGF-β₁ (-) group and LY294002 group. The morphological changes of A549/DDP cells treated with TGF-β₁ were observed under microscope. The protein expression of E-cadherin and N-cadherin was determined by the methods of immumofluorescence and Western blot. The protein levels of Akt, p-Akt, GSK-3β, p-GSK-3β^Ser9 and Snail were also detected by Western blot. RESULTS: The A549/DDP cells in TGF-β₁ (+) group were dispersive, showed a spindle-like shape and developed pseudopodia. This transformation was conformed to classic EMT markers. Compared with TGF-β₁ (-) group, the protein expression of E-cadherin in TGF-β₁ (+) group was significantly decreased (P<0.05), and N-cadherin was significantly increased (P<0.05). The protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were also significantly increased (P<0.05). Compared with TGF-β₁ (+) group, the protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were significantly decreased in LY294002 group (P<0.05). No difference of Akt and GSK-3β expression between TGF-β₁ (-) group and TGF-β₁ (+) group was observed. CONCLUSION: The mechanism of EMT in A549/DDP cells might be related to Akt/GSK-3β/Snail signaling pathway activated by TGF-β₁.     

Effects of isoflurane and sevoflurane on apoptosis of cortical neuron in neonatal rats and the role of MAPKs pathway

AIM: To investigate the effects of isoflurane and sevoflurane at the same dose on apoptosis of cortical neuron in neonatal rats and the role of mitogen-activated protein kinases (MAPKs) pathway.METHODS: Eleven neonatal rats were selected at postnatal day 7 from 1 litter (altogether 5 litters) and assigned randomly into control group (C group), isoflurane group (Ⅰ group) and sevoflurane group (S group). The rats in Ⅰ group, S group or C group were exposed to 1.1% isoflurane, 1.8% sevoflurane and room air for 4 h, respectively. The brain of neonatal rats were perfused and embedded by paraffin. Caspase-3 positive expression in the retrosplenial cortex (RS) of the brain was observed by immunohistochemical staining. Meanwhile, the fresh cortex was separated at 0 h in C group and at 2 h and 4 h in Ⅰ group and S group. The levels of phospho-SAPK/JNK and SAPK/JNK, phospho-p38 and p38 in fresh cortex were detected by Western blotting.RESULTS: Caspase-3 positive cells in the the cortex were increased by 441% in Ⅰ group (P<0.01) and 151% in S group (P<0.01) as compared to C group, and increased by 115% in Ⅰ group (P<0.05) as compared to S group. The protein levels of phospho-SAPK/JNK in the cortex were increased by 219% at 2 h (P<0.05) and 181% at 4 h (P<0.05) in Ⅰ group, while no significant difference between S group and C group was observed. The phospho-p38 protein in the cortex was increased by 38.9% at 2 h (P<0.05) and 36.9% at 4 h (P<0.05) in Ⅰ group, and increased by 32.6% (P<0.05) at 2 h and 128.0% at 4 h (P<0.01) in S group as compared to C group.CONCLUSION: Isoflurane induces more apoptotic neurons in the cortex of the brain in neonatal rats at postnatal day 7 than sevoflurane. Isoflurane induces apoptosis mainly by activating SAPK/JNK phosphorylation, while sevoflurane induces aopotosis by activating p38 phosphorylation.     

p38 MAPK-Pim-3 signal pathway may be involved in cardiomyocyte anoxia preconditioning against anoxia/reoxygenation injury

AIM: To investigate the role of mitogen-activated protein kinases (MAPKs) pathways and the molecular mechanism by which the proto-oncogene Pim-3 protects cardiomyocyte against anoxia/reoxygenation (A/R) injury. METHODS: The primarily cultured neonatal rat ventricular cardiomyocytes were randomly divided into 4 groups: control group; A/R group; APC+A/R group; SB203850, U0126 or SP600125+APC+A/R group. The cells were pre-incubated with U0126 (ERK1/2 inhibitor), SP600125 (SAPK/JNK inhibitor), or SB203850 (p38 MAPK inhibitor) at concentration of 10 μmol/L for 30 min before the APC. The activities of p38 MAPK, JNK and ERK1/2 were detected by Western blotting. The viability of cardiomyocytes was assayed by MTT and the apoptosis of cardiomyocyte was detected by TUNEL. RESULTS: U0126, SB203850, and SP600125 abolished the increased expression of ERK1/2, p38-MAPK, and JNK proteins induced by APC+A/R or A/R, respectively. The expression level of Pim-3 protein significantly decreased when the p38 MAPK signal pathway was inhibited. Meanwhile, the activity of LDH and the apoptosis index increased, and the viability of cardiomyocytes decreased. CONCLUSION: Pim-3 expression through a p38 MAPK signaling pathway may protect cardiomyocytes from A/R injury.     

Involvement of extracellular signal regulated kinase in the regulation of amyloid precursor protein processing in PC12 cells by TPPB

AIM: To explore the signal transduction pathways involved in the regulation of amyloid precursor protein (APP) processing by protein kinase C (PKC) activator TPPB.METHODS: PC12 cells were treated with TPPB (PKC activator) for 3 h and various signal transduction inhibitors were added to the conditioned medium to investigate their effects on α-secretase form of soluble amyloid precursor protein (sAPPα) secretion after TPPB treatment via Western blotting. Extracellular signal regulated kinase (ERK, p42/44MAPK) and phospho-p42/44MAPK were also measured after TPPB treatment.RESULTS: TPPB (1 μmol/L) significantly increased sAPPα secretion as compared with control group. The increase in sAPPα secretion by TPPB was partially blocked by ERK inhibitor U0126, c-Jun N-terminal kinase (JNK) inhibitor SP600125 and protein tyrosine kinase (PTK) inhibitor genistein, but not by p38MAPK inhibitor SB203580. TPPB (1 μmol/L) increased the expression of phospho-p42/44MAPK without altering total p42/44MAPK levels.CONCLUSION: ERK, JNK and PTK may be involved in the regulation of APP processing by TPPB.     

Expression of Th cytokines in peripheral blood before and after splenectomy in immune thrombocytopenia patients

AIM: To investigate the changes of Th cytokines before and after splenectomy in immune thrombocytopenia (ITP) patients. METHODS: The QuantiGene Plex method was used to measure the mRNA expression of Th1, Th2 (IL-4, IL-5, IL-6 and IL-10), Th3 (transforming growth factor β₁,TGF-β₁), and Th17 (IL-17) cytokines in peripheral blood of ITP patients before and after laparoscopic splenectomy and those in peripheral blood of healthy controls. RESULTS: The mRNA level of IL-2 was significantly decreased in ITP patients before operation compared with the healthy controls, whereas IL-17 was obviously over-expressed. No significant difference of the other cytokines between preoperative group and the normal controls was found. After splenectomy, the expression levels of both IL-2 and TGF-β₁ were significantly higher than those in preoperative group and the normal controls. IL-2 was also significantly increased after operation, but was still lower than that in the normal controls. No significant difference of other cytokines between postoperative group and healthy controls was observed. In addition, The Th1 cytokines (IL-2 and IFN-γ) were found to be positively correlated (r=0.647, P<0.01) in preoperative patients, while no correlation was found between the other cytokines. There was a positive correlation between IL-2 and IFN-γ (r=0.787, P<0.01) in postoperative patients. IL-17 also had positive correlations with IL-2 (r=0.554,P<0.01) and IFN-γ (r=0.461,P<0.05) in ITP patients after operation, respectively. CONCLUSION: There is an imbalance of Th cytokines in ITP patients. The mechanism of splenectomy for treating ITP may be associated with the balance regulation of Th cytokines.