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OBJECTIVE@#To investigate the effect of teriparatide on the differentiation of MC3T3-E1 cells in high-glucose microenvironment and explore the possible mechanism.@*METHODS@#MC3T3-E1 cells cultured in normal glucose or high-glucose (25 mmol/L) medium were treated with 10 nmol/L teriparatide with or without co-treatment with H-89 (a PKA inhibitor). CCK-8 assay was used to detect the changes in cell proliferation, and cAMP content in the cells was determined with ELISA. Alkaline phosphatase (ALP) activity and mineralized nodules in the cells were detected using ALP kit and Alizarin red staining, respectively. The changes in cell morphology were detected by cytoskeleton staining. Real-time PCR was used to detect the mRNA expressions of PKA, CREB, RUNX2 and Osx in the treated cells.@*RESULTS@#The treatments did not result in significant changes in proliferation of MC3T3-E1 cells (P > 0.05). Compared with the cells in routine culture, the cells treated with teriparatide showed significantly increased cAMP levels (P < 0.05) with enhanced ALP activity and increased area of mineralized nodules (P < 0.05). Teriparatide treatment also resulted in more distinct visualization of the cytoskeleton in the cells and obviously up-regulated the mRNA expressions of PKA, CREB, RUNX2 and Osx (P < 0.05). The opposite changes were observed in cells cultured in high glucose. In cells exposed to high glucose, treatment with teriparatide significantly increased cAMP levels (P < 0.05), ALP activity and the area of mineralized nodules (P < 0.05) and enhanced the clarity of the cytoskeleton and mRNA expressions of PKA, CREB, RUNX2 and Osx; the effects of teriparatide was strongly antagonized by co-treatment with H-89 (P < 0.05).@*CONCLUSION@#Teriparatide can promote osteoblast differentiation of MC3T3-E1 cells in high-glucose microenvironment possibly by activating the cAMP/PKA/CREB signaling pathway.
Subject(s)
Animals , Mice , Cell Differentiation , Core Binding Factor Alpha 1 Subunit , Glucose/pharmacology , Osteoblasts/drug effects , RNA, Messenger , Signal Transduction , Teriparatide , Cell LineABSTRACT
OBJECTIVE To s tudy the improvement effects of sinapic acid on Aβ42-induced injury of PC 12 cells and the mechanism. METHODS PC12 cells were divided into five groups ：control group ，model group ，sinapic acid group ，phosphoinositide- 3-kinase（PI3K）inhibitor group and extracellular signal-regulated kinase （ERK）inhibitor group. Each inhibitor group was added with LY 294002 and U 0126（10 μmol/L）for 1 h；except for control group ，other four groups were treated with 2 μmol/L Aβ42 for 24 h to replicate the Alzheimer ’s disease cell model ；except for control group and model group ，other three groups were added with 100 μmol/L sinapic acid respectively. After 24 hours of continuous culture ，survival rate of PC 12 cells was detected and the morphology of PC 12 cells was observed. The content of Aβ42，mRNA expression of cAMP response element binding protein （CREB），protein expression of cyclic adenosine monophosphate （cAMP），protein kinase A （PKA），CREB signaling pathway and phosphorylated CREB （p-CREB）were detected. RESULTS After treated with sinapic acid ，the survival rate of PC 12 cells，mRNA expression of CREB and protein expressions of cAMP ，PKA and p-CREB were increased significantly （P＜0.05），while the content of Aβ42 was decreased significantly （P＜0.05）；cell morphology was significantly improved and synapses increased. After intervened with PI 3K and ERK inhibitors ，the survival rate of PC 12 cells，above mRNA and protein expressions were reversed significantly （P＜0.05 or P＜0.01）；cell morphology was irregular ，the fragments increased ，and the synaptic connections decreased. CONCLUSIONS Sinapic acid can improve the survival rate of PC 12 cells injured by A β 42，improve cell （No.2021-KYYWF-0349） morphology and decrease the content of Aβ42，the mechanism of which may be associated with promoting the gene transcription of CREB ， and activating cAMP/PKA/CREB signaling pathway.
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OBJECTIVE: We have demonstrated that electroacupuncture (EA) of "Weizhong" (BL 40) and "Huantiao" (GB 30) could evidently relieve mechanical hyperalgesia in spared nerve injury (SNI) rats. The present study was designed to observe the effect of EA on levels of cAMP, and protein kinase A (PKA) and cAMP response element binding protein (CREB) in the lumbar spinal cord of the same pain model rats, so as to explore its mechanisms underlying improvement of neuropathic pain. METHODS: One hundred male SD rats were randomly divided into 5 groups: sham operation, model, EA, AP-5 and L-NAME groups (n＝20 in each group). The sham operation group underwent a simple separation of the sciatic nerve but without ligation and abscission. The neuropathic pain model was established by abscission of the right tibial and common peroneal nerve. EA (2 Hz, 1－3 mA) was applied to right BL 40 and GB 30 for 30 min, once a day for 7 days, starting from day 11 after surgery. For rats of the AP-5 and L-NAME groups, AP-5 (a competitive antagonist for NMDA receptor, 0.7 mg•kg－1•d－1) and L-NAME (a non-selective antagonist for nitric oxide synthase, NOS, 60 mg•kg－1•d－1) were respectively administrated by intraperitoneal injection, once daily for 7 days. The mechanical pain threshold was measured, the cAMP content of spinal cord (L 4－L 6) was determined by radioimmunoassay, and the expression of PKA, p-PKA and CREB proteins of spinal cord (L 4－L 6) was detected by Western blot. RESULTS: The content of cAMP and the expression levels of PKA, p-PKA and CREB were significantly higher in the model group than in the sham operation group (P<0.01), and considerably lower in the EA group than in the model group (P<0.01, P<0.05). Compared to the model group, the expression levels of PKA and p-PKA were significantly decreased in the AP-5 and L-NAME groups (P<0.01, P<0.05). Compared to the EA group, the content of cAMP and the expression levels of CREB were significantly higher (P<0.05), and the expression of p-PKA was significantly lower in the AP-5 and L-NAME groups (P<0.05)．. CONCLUSION: EA intervention-induced down-regulation of cAMP, PKA and CREB levels in the lumbar spinal cord may contribute to its analgesic effect in neuropathic pain rats, suggesting an involvement of reduction of cAMP/PKA/CREB signaling of spinal cord in EA analgesia.
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OBJECTIVE: To explore the protective effect of mullein glycoside polysaccharide of Cistanche deserticola Ma on PC12 nerve-cell model induced by D-galactose. METHODS: The cell survival rate was determined by MTT assays, which provided the basis for selecting mullein glycoside polysaccharide dose and estimated the dose and action time of D-galactose for inducing PC12 nerve cell damage model. After mullein glycoside polysaccharide incubation of PC12 cells, western blotting was used to detect the levels of CREB and p-CREB protein expression. ELISA Kit was used to detect the levels of cyclic adenosine monophosphate(cAMP), cAMP dependent protein kinase(PKA) and brain derived neurotrophic factor(BDNF). The content of MDA, activities of SOD and LDH were measured by their respective kits. RESULTS: (1)After the exposure of the PC12 cells to 16 g·L-1 D-galactose for 40 h, the cell survival rate was (46.67±6.59)%, which has a significant difference compared with the control group(P<0.05), indicating that successful cell aging model was established. (2)Compared with those in model group, mullein glycoside polysaccharide could significantly increase p-CREB expression in dose-dependent manner(r=0.989, P<0.01), content of PKA, cAMP, BDNF and SOD and decrease the levels of MDA and LDH(rMDA=0.875, P<0.05);(rLDH=0.834, P<0.05). However, blockers H-89 could significantly decrease p-CREB expression, PKA, cAMP, SOD and BDNF content(P<0.05), and increase the levels of MDA and LHD(P<0.05). CONCLUSION: The mullein glycoside polysaccharide of Cistanche deserticola Ma has obvious protective effect on PC12 nerve-cell damage model induced by D-galactose and its mechanism relates to the upregulation of cAMP/PKA/ CREB signaling pathways.