| Objectives In order to observe intracellular calcium homeostasis imbalances which is induced by the calcium antagonist-Tet randrine and the sarcoplasmic reticulum calcium pump inhibitors-thapsigargin,the express of type ?collagen and apoptosis of the activating mouse lung fibroblasts.Investigate apoptotic signaling pathway from the endoplasmic reticulum stress related to combat pulmonary fibrosis provide a theoretical basis for clinical practice.Methods The mouse fibroblasts cell ryopreserved-40?,recovered at 37?,and grown in culture flasks containing complete medium with 10% FBS at 37?and 5% CO2. Using an inverted microscopr to observe cell growth,and the cell conventional treatment which includes cell replacement liquid, passage and cryopreserved.The mouse fibroblasts cells were stimulated by TGF-?1 with the concentration of 5ng/ml and the time of 24 h which is actived.The activated mouse fibroblasts cell were stimulated by Tet and TG with different concentrations at 24 h.MTT proliferation to determinate the best concentration of Tet and TG.Based on the results of the MTT cell proliferation,dividing them into 4 groups:blank group,TGF-?1 control group,Calcium antagonists group,sarcoplasmic reticulum calcium pump inhibitors group.Cell proliferation was measured by MTT method.Cell morphology was observed by transmission electron microscopy.Cell cycle and apoptosis were evaluated by flow cytometry.The concentration change of calcium ion were measured by Laser scanning confocal microscope.Protein expressions of ?collagen?GRP78?calpain-2 and CHOP were detected by Western Blot.Intracellular caspse-12 and caspase-3 were measured by immunohistochemistry.Results 1 The mouse fibroblasts cell was activated by treating with TGF-?1(5ng/ml) for24 h.Different concentrations of Calcium antagonists and sarcoplasmic reticulum calcium pump inhibitors can inhibit cell proliferation.2 MTT assay:Compared with the control group,after adding the TGF-?1,the absorbance value was increased significantly in TGF-?1group(P<0.05); Compared with the TGF-?1 group,the absorbance value was reduced in Calcium antagonists and sarcoplasmic reticulum calcium pump inhibitors groups. 3 Cell morphology control group and TGF-?1 group was observed under a transmission electron microscope cell membrane, nucleus, chromatin morphology intact.Calcium antagonists,sarcoplasmic reticulum calcium pump inhibitors were two groups of cells appeared typical apoptotic morphology: cytoplasm concentrated cell shrinkage, chromatin gradually gathered crescent, etc.4 Cell cycle Compared with the blank group,the proportion of G1 and G2 phase cells were reduced significantly in TGF-?1control group,while the proportion of S phase cells were increased obviously(P<0.05).After using the intervention Calcium antagonists and sarcoplasmic reticulum calcium pump inhibitors,the proportion of G2 and S phase cells were decreased significantly and G1 phase cells were increased obviously.5Cell apoptosis Flow cytometry results showed that the blank / control/Calcium antagonists/sarcoplasmic reticulum calcium pump inhibitors groups apoptosis rate were 6.73% ?5.03%?9.60%?21.53%.6 Confocal laser light Compared with the control group,Higher fluorescence intensity of sarcoplasmic reticulum calcium pump inhibitors group,and with a stronger fluorescence(P<0.05),while Calcium antagonists group cells fluorescence intensity of intervention group was decreased significantly than control group(P<0.05).7Protein resuts Compared with the blank group,the expression of I type collagen were largely increasing in TGF-?1control group(P<0.05);Compared with the TGF-?1control group,the expression of I type collagen were dereasing in Calcium antagonists and sarcoplasmic reticulum calcium pump inhibitors groups.Compared with the control group,Calcium antagonists and sarcoplasmic reticulum calcium pump inhibitors could significantly increase the expression of GRP78 ? Capase-12 ? CHOP ? and Capase-3;Compared with the control group,the calpain-2 protein expression,Calcium antagonists group did not chang significantly(P>0.05),while sarcoplasmic reticulum calcium pump inhibitors group showed high expression(P<0.05).Conclusions 1 Calcium antagonists and sarcoplasmic reticulum calcium pump inhibitors could block mouse lung fibroblast cells cycle induce their apoptosis.2 Calcium antagonists and sarcoplasmic reticulum calcium pump inhibitors can make lung fibroblasts calcium homeostasis is broken and can enhance the expression of ERS indicators which can promote apoptosis through ERS pathway.Thereby preventing the progression of pulmonary fibrosis... |
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