An activated mutant of SHP-2 tyrosine phosphatase causes murine myeloid abnormal proliferation

AIM: To investigate whether an activated mutant of SHP-2 tyrosine phosphatase is involved in abnormal proliferation of murine myeloid.METHODS: Wild-type (WT) and SHP-2^D61G/+mutant mice aged 8 weeks and 16 weeks were used. The number of peripheral blood leukocytes and the spleen sizes were measured by cell counting and weighing methods,respectively. The surface markers (Mac-1 and Gr-1 for myeloid, Ter119 for erythroid, CD3 for T-lymphocyte and B220 for B-lymphocyte) of hematopoitic cells in peripheral blood and bone marrow were detected by flow cytometry. The rate of Mac-1 or Gr-1 positive cells in the peripheral blood and the rate of Mac-1, Gr-1, Ter119, CD3 or B220 positive cells in bone marrow were analyzed. The ability of colony formation unit (CFU) of the bone marrow was also observed by CFU assay. Finally, the expression of p-Akt and p-ERK in the peripheral blood leukocytes induced by interleukin-3 (IL-3, 5 μg/L) was detected by Western blotting.RESULTS: The number of leukocytes in peripheral blood of 16-week-old mice was more (P<0.01) and the spleens were bigger in mutant SHP-2^D61G/+ mice than those in WT mice. The rate of Mac-1 and Gr-1 positive cells in peripheral blood leukocytes of 16-week-old SHP-2^D61G/+ mice were dramatically increased (P<0.05). Mac-1 and Gr-1 positive cell rates in bone marrow of SHP-2^{D61G / +} mice were much higher (P<0.05) than those in WT mice and no statistic significance was found in the erythroid or lymphocyte cells. The number of CFU-GM (represents myeloid) was increased in mutant mice. The expression of p-Akt and p-ERK in peripheral blood leukocytes of mutant mice was significantly enhanced after stimulated with IL-3.CONCLUSION: These results suggest that activated mutant SHP-2 results in the disorder of mouse myeloid proliferation via MAPK and PI3K activation.     

Retinoid X receptor agonists antagonize high-glucose-induced proliferation of rat vascular smooth muscle cells by inactivation of protein kinase C

AIM: To explore the effect of retinoid X receptor (RXR) agonists on high-glucose-induced proliferation of rat aortic smooth muscle cells (RASMCs). METHODS: RASMCs were cultured in DMEM containing glucose at normal concentration (5.5 mmol/L). For high glucose treatment, glucose solution was added up to a final concentration of 25 mmol/L. The proliferation of RASMCs was detected by WST-1 assay. DNA synthesis was measured by the method of BrdU incorporation. Cell cycle progression was determined by flow cytometry. Phosphorylated protein kinase C (PKC) and the expression levels of cyclin-dependent kinase 2(CDK2) and p27Kip1 were detected by immunoblotting. RESULTS: High glucose increased DNA synthesis, cell cycle progression, the expression of CDK2 and the proliferation of RASMCs. Meanwhile, the expression of p27Kip1 was decreased by high glucose. Treatment of RASMCs with RXR natural ligand 9-cis-retinoic acid (9-cis-RA) resulted in significant inhibition of high-glucose-induced proliferation, DNA synthesis, cell cycle progression and the expression of CDK2 in a concentration-dependent manner. 9-cis-RA also reversed the effect of high glucose on the expression of p27Kip1. RXR specific ligand SR11237 demonstrated the same effect as the effect of 9-cis-RA at the same concentration. PKC inhibitor showed the similar effect on high-glucose-induced proliferation and the expression of CDK2 and p27Kip1 as the RXR agonists did. Furthermore, 9-cis-RA and SR11237 rapidly inhibited high-glucose-induced activation of PKC. CONCLUSION: PKC is involved in high-glucose-induced proliferation of RASMCs. RXR agonists inhibit high-glucose-induced proliferation by depressing PKC activation in vascular smooth muscle cells.     

Protein tyrosine phosphatase SHP-2 inhibits apoptosis induced by serum withdrawal in 293T cells

AIM: To investigate the potential role of Scy homology 2 domain-containing protein tyrosine phosphatase 2(SHP-2) in apoptosis of 293T cells induced by serum deprivation.METHODS: Transfection of plasmids into 293T cells was performed by liposome protocol. The viability of 293T cells was detected by MTT assay. On day 3 after removing serum the morphological changes of 293T cells were observed by using a transmission electron microscope, apoptosis rate was detected by flow cytometry and the expression of caspase-3 was measured by immunohistochemisty.RESULTS: The apoptosis rate in wild type SHP-2 (WT) group was obviously lower than that in the SHP-2C459S catalytically inactivated group. At the same time, expression of caspase-3 showed the similar results. CONCLUSION: SHP-2 may actively participate in the signal transduction pathway of apoptosis and play a positive role in cell survival. The underlying mechanism of apoptotic inhibition may be caspase-3 dependent.     

Expression and function of calcium-sensing receptor in mouse embryonic stem cells

AIM: To observe the functional expression of calcium-sensing receptor (CaSR) in the mouse embryonic stem cells (mESCs). METHODS: The expression and distribution of CaSR were detected by Western blotting and immunofluorescence observation in 129 mouse ES-D3 cells. The intracellular concentration of free calcium (［Ca2+］i) was determined by confocal laser scanning microscopy. The cell viability was analyzed by MTT assay and flow cytometry. RESULTS: CaSR protein was expressed in mESCs. Extracellular calcium or neomycin significantly increased the expression of CaSR and ［Ca2+］i. Neomycin increased the cell viability, up-regulated the protein expression of p-ERK2. These effects of neomycin were inhibited by NPS2390. CONCLUSION: CaSR is expressed in mESCs. The activation of CaSR is involved in the proliferation of mESCs.     

Significance of TRPM8 protein expression in lung adenocarcinoma

AIM To investigate the significance of transient receptor potential cation channel subfamily M member 8 （TRPM8） protein expression in lung adenocarcinoma. METHODS The tumor samples from 112 cases of patients with lung adenocarcinoma were collected in our hospital, and 4~5 years of follow-up was conducted. The protein expression of TRPM8 was analyzed by immunohistochemical staining, and the correlations between the TRPM8 protein expression and the clinical characteristics including prognosis of the patients with lung adenocarcinoma were investigated. After TRPM8 protein expression was up-regulated in A549 lung adenocarcinoma cells by lentiviral infection, the proliferation of A549 cells was analyzed by CCK-8 assay and colony formation assay, the cell cycle and apoptosis were analyzed by flow cytometry, and the migration and invasion abilities of the cells were measured by scratch experiment and Transwell assay. The TRPM8 protein expression was stably up-regulated in H1299 cells by lentiviral infection, and then the left and right buttocks of the immunodeficient mice were subcutaneously injected with empty vector control cells and TRPM8-overexpressing cells, respectively. The effects of TRPM8 on the growth of H1299 cell-derived xenograft tumor in immunodeficient mice were evaluated. RESULTS The 4~5-year survival rate in the patients with high TRPM8 protein expression was significantly higher than that in the patients with low expression of TRPM8 protein （P=0.017）. The tumor maximum diameter in the patients with high TRPM8 protein expression was significantly smaller than that in the patients with low TRPM8 protein expression （P=0.028）. The viability, the number of colonies and the migration and invasion abilities of TRPM8-overexpressing A549 cells were significantly decreased as compared with empty vector and parental cells （P<0.01）. The results of flow cytometry analysis showed that the proportion of A549 cells at S stage was significantly increased in TRPM8 overexpression group as compared with empty vector group （P<0.01）. The growth rate and the weight of TRPM8-overexpressing H1299 cell-derived xenograft tumor in immunodeficient mice were significantly lower than those in empty vector group （P<0.01）. CONCLUSION TPRM8 is a tumor suppressor in lung adenocarcinoma, and low expression of TRPM8 protein was a poor prognositic indicator of patients with lung adenocarcinoma.     

MEK/ERK pathway activated by changing insulin receptor isoform is associated with abnormal proliferation of intestinal epithelial cells in diabe-tic mice

AIM: To investigate the role of insulin receptor (IR)-A/IR-B ratio and the downstream pathway in abnormal proliferation of intestinal epithelial cells (IECs) in diabetic mice. METHODS: Diabetes mouse models were induced by intraperitoneal streptozocin injection. The proliferating cell nuclear antigen (PCNA) proliferation rates in the small intestine tissue were evaluated by immunohistochemical methods. The expression of IR isoforms was detected by RT-PCR. To ensure that the downstream pathways of IR are involved, real-time PCR and Western blot were performed to detect the expression of MEK1/2, ERK1/2, PI3K and Akt. RESULTS: In diabetic mice, the PCNA proliferation rates were higher than those in control group (P<0.05), and a high ratio of IR-A/IR-B was detected in the IECs (P<0.05). The mRNA expression of MEK1, MEK2, ERK1/2 and their phosphorylated protein levels in the diabetic mice were significantly higher than those in control group (P<0.05). CONCLUSION: The over-proliferation of IECs in the diabetic mice is associated with high IR-A/IR-B ratio and up-regulation of IR/MEK/ERK pathway.     

Total triterpenoids from Psidium Guajava leaf improves insulin resistance in 3T3-L1 adipocytes

AIM: To investigate the effects of total triterpenoids from Psidium guajava leaf (TTPGL) on 3T3-L1 adipocyte insulin resistance (IR) and to explore the possible mechanism. METHODS: 3T3-L1 pre-adipocytes were cultured and induced to differentiate into 3T3-L1 adipocytes, then treated with TTPGL (0.3, 1, 3, 10 μg/L) for 48 h. The cells were divided into 0.1% DMSO group, positive drug sodium orthovanadate (Van, 10 μmol/L) group, model group and control group. The effect of TTPGL on the cell activity of pre-adipocytes was detected by MTT assay and its influence on the cellular differentiation was observed by oil red O staining. The IR model of the 3T3-L1 adipocytes was established successfully and then treated with different drugs for 48 h. The glucose consumption in the supernatant of IR adipocyte's culture medium was assayed by glucose oxidase-peroxidase (GOD-POD), free fatty acid (FFA) levels were measured by colorimetric method, and adipocytokines levels were assayed by ELISA. The mRNA expression of protein tyrosine phosphatase-1B (PTP1B) of IR adipocyte was detected by real-time PCR. The protein levels of phosphorylated insulin receptor substrate 1/insulin receptor substrate 1 (p-IRS-1/IRS-1) and phosphorylated protein kinase B/protein kinase B (p-Akt/Akt) were determined by Western blot. RESULTS: Compared with DMSO group, TTPGL treatment significantly promoted the cell activity of 3T3-L1 pre-adipocytes and inhibited its differentiation (P < 0.01). TTPGL (1~10 μg/L) improved glucose consumption of IR adipocytes significantly (P < 0.01), with or without insulin stimulation, and TTPGL (0.3~3 μg/L) restrained FFA production remarkably(P < 0.01). Compared with model group, TTPGL (0.3 and 3 μg/L) significantly increased the secretion of adiponectin in IR adipocytes (P < 0.05), and inhibited the secretion of tumor necrosis factor-α (TNF-α) (P < 0.01). TTPGL (3 μg/L) restrained the secretion of resistin significantly (P < 0.05), and showed no significant effect on secretion of leptin. It also down-regulated the mRNA expression of protein tyrosine phosphates 1B (PTP1B) in IR adipocytes significantly (P < 0.01), and increased the protein levels of p-IRS-1/IRS-1. TTPGL (0.3 and 3 μg/L) up-regulated the protein level of p-Akt/Akt in IR adipocytes significantly (P < 0.05).CONCLUSION: TTPGL reduces IR in 3T3-L1 adipocytes. The mechanism may be that TTPGL significantly down-regulated mRNA expression of PTP1B and increased the protein levels of p-IRS-1/IRS-1 and p-Akt/Akt in IR adipocytes.     

Changes of lung AT1R and Mas receptor protein expression in lung tissue of mice with lung injury after limb ischemia-reperfusion

AIM:To explore the role of imbalance of local renin-angiotensin system (RAS) in lung injury by observing the changes of angiotensin Ⅱ type 1 receptor (AT1R) and Mas receptor protein expression in the lung and the degree of lung injury subject to limb ischemia-reperfusion (LIR) in the mice.METHODS:Male ICR mice (n=42,8 weeks old) were randomly assigned into 7 groups (6 in each group),including control group and 6 model groups with LIR of 0.5 h,1 h,2 h,4 h,6 h and 12 h reperfusion.Tourniquets were used to block the blood flow of the hind limbs of the ICR mice and were released after 2 h ischemia to initiate reperfusion.The mice were sacrificed by eyeball blood withdrawal at different time points after reperfusion.The organ coefficient and wet/dry weight ratio (W/D) of the lung tissue were calculated.Bronchoalveolar lavage fluid (BALF) was taken for cell counting and protein concentration measurement.The histopathological changes of the lung tissues was observed,and the pathological score was calculated.The protein expression of AT1R and Mas receptor in the lung tissues was determined by Western blot.RESULTS:The organ coefficient,W/D of lung tissue,and cell number and protein concentration in BALF of model groups were significantly higher than those in control group after LIR.The pathological changes were found in the lung tissue of model mice,including alveolar capillary dilation and congestion,edema,inflammatory cell infiltration in peripheral vascular,alveolar and bronchial walls,alveolar septal thickening and inflammatory cell infiltration.The lung injury score was elevated gradually along with the extension of reperfusion time.The protein expression of AT1R began to increase at reperfusion time points of 0.5 h and 1 h.With the extension of reperfusion time,the protein expression of AT1R decreased gradually.Conversely,the protein expression of Mas receptor increased gradually with prolonged reperfusion.CONCLUSION:LIR induces acute lung injury gradually.The imbalance of AT1R and Mas receptor expression may be involved in the damage process.     

Flavonoids from stem and leaf of Scutellaria baicalonsis Georgi inhibit PHF abnormality and regulatory mechanism of protein phosphatase in rats' brain induced by okadaic acid

AIM: To investigate the effect of flavonoids from stem and leaf of Scutellaria baicalonsis Georgi (SSF) on paired helical filament (PHF) abnormality and the regulatory mechanism of protein phosphatase (PP) in rats' brain induced by okadaic acid (OA). METHODS: Male Sprague-Dawley (SD) rats were microinjected with OA (200 ng/kg) by the lateral ventricle to establish a memory impairment model. Morris water maze was used to screen the memory impairment model. The successful model rats were continuous intragastric infusion (ig) SSF for 36 days. The relative protein expression of PHF, PP1, PP2A-Cα, PP2A-Cβ, PP2CA and PP2CB in the rat cerebral cortex and hippocampus were detected by Western blot. GinKgo biloba leaf flavonoids (GLF) were used as positive control drug. RESULTS: Compared with the sham-operated rats, the relative protein expression of PHF in the cerebral cortex and hippocampus and PP1 in cortex of model rats were significantly increased (P<0.01), and the protein expression of PP2A-Cα, PP2A-Cβ in the cerebral cortex and hippocampus and PP2CB in the hippocampus were decreased (P<0.05), while the relative protein expression of PP2CA and PP2CB in the cortex were significantly increased (P<0.01). SSF reversed the abnormality in the protein expression of PHF, PP2A-Cα and PP2A-Cβ in rat cortex and hippocampus and PP1 in rat cortex induced by OA (P<0.01), which had no significant effect on the relative protein expression of PP2CA and PP2CB. GLF also showed similar results to SSF. CONCLUSION: SSF significantly reduces the abnormal formation of PHF in rats' brain induced by OA, which may be related to the regulation of PP1, PP2A-Cα and PP2A-Cβ expression, but not with PP2CA and PP2CB expression.     

Role of asiatic acid in treatment of insulin resistance in mouse adipocytes

AIM：To investigate the effects of asiatic acid, one of triterpenoids from Psidium guajava leaves, on the proliferation and differentiation of 3T3-L1 preadipocytes, and glucose and lipid metabolism of insulin-resistant adipocytes. METHODS：The proliferation of 3T3-L1 preadipocytes was tested by MTT assay, and the accumulation of lipid droplets in differentiated preadipocytes was measured by oil red O staining. The insulin-resistant cell model was established by exposure of the cells to dexamethasone. The cellular glucose uptake was determined by glucose oxidase-peroxidase assay. The free fat acid (FFA) concentration was detected by colorimetric method. Secreted adiponectin were measured by ELISA. The protein levels of peroxisome proliferator-activated receptor γ (PPARγ) and protein tyrosine phosphatase 1B (PTP1B) in insulin-resistant adipocytes were analyzed by Western blotting. RESULTS：Compared with medium group, asiatic acid increased the proliferation of 3T3-L1 preadipocytes and inhibited their differentiation at a concentration range of 10~100 μmol/L (P<0.05 or P<0.01). At concentrations of 30 μmol/L and 100 μmol/L, asiatic acid enhanced cellular glucose uptake in the insulin-resistant adipocytes both in basic and insulin-stimulation states. Asiatic acid decreased FFA production (P<0.05), and down-regulated the protein expression of PTP1B (P<0.05, or P<0.01). However, no effect on the secretion of adiponectin and the protein expression of PPARγ was observed (P>0.05). CONCLUSION：Asiatic acid enhances glucose uptake and inhibits FFA production in insulin-resistant adipocytes via down-regulating the protein expression of PTP1B, all of which play the roles of increasing insulin signaling sensitivity to improve insulin resistance.