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Objective:To investigate the changes of PERK,Runx2,osterix,RANKL and OPG in bone tissue of the female rats with experimental postmenopausal osteoporosis(PMOP)before and after treatment,and to elucidate the role of PERK signaling pathway in PMOP.Methods:The ovariectomized rats were reproduced to osteoporosis models.A total of 45 rats were divided into normal control group(the rats didn't receive any treatment,n=15),osteoporosis group(the rats were ovariectomized,n=15)and osteoporosis treatment group (the ovariectomized rats were injected with estrogen through caudal vein,n=15).The changes of serum collagenⅠ(Col Ⅰ),alkaline phosphatase(ALP)and osteocalcin(OCN)of the rats in various groups were observed. Three months after feeding,the femoral shaft of the rats in various groups were taken for pathological section.The gene expression levels of PERK,ATF4,Runx2,osterix,RANKL and OPG in bone tissue of the rats in various groups were detected by RT-PCR;the protein expression levels of PERK,ATF4,Runx2,osterix,RANKL and OPG were detected by Western blotting method.Results:Compared with control group,the levels of serum Col Ⅰ,ALP and OCN in the rats in osteoporosis group were significantly decreased(P<0.05 or P<0.01);compared with osteoporosis group,the levels of serum ColⅠ,ALP and OCN of the rats in osteoporosis treatment group were significantly increased(P<0.01).Compared with control group,the gene expression levels of PERK, ATF4,Runx2 and osterix in bone tissue of the rats in osteoporosis group were significantly decreased(P<0.01), and the gene expression level of RANKL was increased(P<0.01);compared with osteoporosis group,the gene expression levels of PERK,ATF4 Runx2 and osterix in bone tissue of the rats in osteoporosis treatment group were significantly increased(P<0.01),and the gene expression level of RANKL was significantly decreased(P<0.01).Compared with control group,the protein expression levels of PERK,Runx2 and osterix in bone tissue of the rats in osteoporosis group were significantly decreased(P<0.05 or P<0.01),and the protein expression level of RANKL were increased(P<0.05 or P<0.01);compared with osteoporosis group,the protein expression levels of PERK,Runx2 and osterix in bone tissue of the rats in osteoporosis treatment group were significantly increased(P<0.05 or P<0.01),and the protein expression level of RANKL was significantly decreased(P<0.01).The HE staining results showed that compared with control group,the bone resorption pits in bone tissue of the rats in osteoporosis group became large with the increased bone absorption,which caused bone loss;compared with osteoporosis group,the resorption in bone tissue of the rats in osteoporosis treatment group was decreased,and the bone structure returned to normal.Conclusion:After the female rats are ovariectomized and injected with estrogen,the expression trends of PERK and osteoblast transcription factors Runx2 and osterix are consistent,in contrast with the osteoclast transcription factor RANKL expression,suggesting that the osteoblast function is reduced and it is related to the decreased expression of PERK in PMOP onset.
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Objective To observe the expression of the unfolded protein response especially the inositol-requiring enzyme-1 (IREl)-Xbp1 signaling pathway, and the change trend of osteogenic markers after inhibition of IREl expression through siRNA interference in osteoblasts exposed to fluoride. Methods Proliferation activity of MC3T3-E1 cells was detected by CCK-8 assay, and 0.0, 0.1, 1.0, 2.0, 8.0, 16.0, 20.0, 32.0, 64.0 mg/L groups were set up. Then representative doses of low, medium and high fluoride (2.0, 8.0, 20.0 mg/L) were selected to treat MC3T3-E1 cells and the expression of the unfolded protein response related genes and osteogenic markers [alkaline phosphatase (ALP), osteocalcin (OCN), Runx2, osterix, binding immunoglobulin protein (Bip), protein kinase-like endoplasmin reticulum kinase ( PERK ) , activated transcription factor 6 (ATF6), Xbp1] was detected by Real-time PCR. MC3T3-E1 cells were transfected with IRE1 siRNA and then exposed to fluoride, and the expression of IRE1 signaling pathway and osteogenic markers was detected by Western blotting and real-time PCR. Results CCK-8 results showed the bidirectional effect of fluoride on the activity of osteoblasts. Compared with the 0.0 mg/L group [1.00 ± 0.01 (d 1), 1.00 ± 0.02 (d 3), 1.00 ± 0.08 (d 7)], the osteoblast activity was significantly enhanced at 2.0 mg/L [1.11 ± 0.02 (d 1), 1.29 ± 0.02 (d 3)], 8.0 mg/L [1.16 ± 0.02 (d 1), 1.44 ± 0.03 (d 3), all P<0.05], while 20.0 mg/L inhibited cell activity [0.83 ± 0.01 (d 1), 0.81 ± 0.01 (d 3), 0.96 ± 0.04 (d 7), all P< 0.05]. Compared with the 0.0 mg/L group [6.86 ± 2.13 (ALP), 4.58 ± 1.52 (OCN), 2.65 ± 0.38 (Runx2), 12.48 ± 3.96 (osterix)], 2.0 mg/L significantly induced the expression of intracellular ALP (12.80 ± 3.62), Runx2 (6.61 ± 0.48) and osterix (21.42 ± 1.56), and the differences were statistically significant (all P< 0.05), while 20.0 mg/L inhibited the expression of ALP (0.88 ± 0.17), OCN (0.16 ± 0.05) and osterix (1.35 ± 0.51), and the differences were statistically significant (all P<0.05). Compared with the 0.0 mg/L group [1.36 ± 0.58 (IRE1), 0.96 ± 0.45 (Xbp1)], the expression of endoplasmic reticulum stress related genes IRE1 [14.84 ± 2.57 (2.0 mg/L), 4.10 ± 0.52 (8.0 mg/L), 5.30 ± 0.63 (20.0 mg/L)] and Xbp1 [2.62 ± 0.66 (2.0 mg/L), 1.97 ± 0.47 (20.0 mg/L)] were significantly increased in the corresponding fluoride groups (all P<0.05). After IRE1 gene knockout, compared with the control group [gene:3.25 ± 0.48 (OCN), 5.62 ± 1.86 (Runx2), 2.67 ± 0.35 (ALP); protein: 0.16 ± 0.03 (OCN), 0.34 ± 0.27 (ALP)], the gene expression of OCN [0.63 ± 0.46 (2.0 mg/L), 0.81 ± 0.36 (8.0 mg/L), 0.62 ± 0.31 (20.0 mg/L)], Runx2 [0.18 ± 0.03 (2.0 mg/L), 0.12 ± 0.01 (8.0 mg/L), 1.09 ± 0.33 (20.0 mg/L)] and ALP [1.01 ± 0.12 (8.0 mg/L), 0.38 ± 0.09 (20.0 mg/L)] in the corresponding fluoride groups were significantly decreased (all P < 0.05), protein expression of OCN [0.06 ± 0.02 (2.0 mg/L), 0.06 ± 0.02 (8.0 mg/L), 0.07 ± 0.03 (20.0 mg/L)], and ALP [0.02 ± 0.01 (8.0 mg/L), 0 (20.0 mg/L)] were significantly decreased (all P< 0.05). Conclusion Unfolded protein response is observed under different doses of fluoride in osteoblasts, and IRE1 gene knockout has inhibited the expression of ALP, OCN, osterix and Runx2 in osteoblasts induced by fluoride, which suggests that IRE1 signaling pathway may play a key role in the differentiation of osteoblasts exposed to fluoride.
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Objective To explore the clinical effect of edaravone on acute ischemic cerebrovascular disease ,and to observe its effect on serum C‐reactive protein (CRP) and matrix metalloproteinase‐9(MMP‐9) level .Methods Eighty four patients with acute ischemic cerebral vascular disease who were treated in our hospital during 2012 May to 2013 December were selected and divided in‐to observation group and control group ,with 42 cases in each group .The control group was treated with anti‐platelet ,regulating blood pressure and blood lipid ,reducing intracranial pressure ,improve microcirculation and other comprehensive treatment ;the pa‐tients in observation group were treated with increased dose of edaravone on the basis of the control group .America national institu‐tes of health stroke scale(NIHSS) and activities of daily living scale (ADL) were used to evaluate the neurological function and ac‐tivities of daily living ability of patients before and after the treatment ,the CRP and MMP‐9 level were detected before and after treatment ,and the adverse reaction during the treatment was recorded .Results After one week and two weeks after treatment , there were significant differences between treatment group and control group in the NIHSS score and ADL score (P0 .05) .Conclusion Edaravone could improve the clinical treatment effect of acute ischemic cerebrovascular disease ,and reducing se‐rum CRP and MMP‐9 level mechanism pathway might be involved in the regulation of its pharmacodynamics .