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Objective::To explore the effect of Zuoguiwan on the bone mineral density (BMD) and the expressions of Ca2+ transport-associated protein in ovariectomized rats. Method::The 48 female SD rats were randomly divided into six groups: normal group, model group, sham operation group, estrogen group(0.167 mg·kg-1) and low and high-dose Zuoguiwan groups(9.6, 38.4 g·kg-1), with 10 rats in each group. Except for the sham-operated group, the ovariectomized rats in the other groups received the bilateral ovariectomy. Therapeutic intervention was given in each group for 3 months after the establishment of the model. After 12 weeks, BMD was measured using dualenergy X-ray absorptiometry. Tartrated presistant acid phosphatse(TRACP) and serum calcium were detected by biochemical kits.Protein expression in Ca2+ transport (Bone tissue) was detected by Western blot. Result::Compared with the normal group, the serum calcium of the model group was decreased(P<0.01). Compared with the normal group, BMD of the model group was decreased (P<0.01). The serum calcium of rats in high-dose group and western medicine group was higher than that of model group(P<0.01). BMD in model group was lower than that of Zuoguiwan groups and estrogen group(P<0.05). There was no significant difference in TRACP among the groups. Nilestriol and Zuoguiwan can down-regulate the expressions of TRPV5, NCX1, CaBP-D28K and PMCA1b in bone tissue of castrated rats(P<0.05, P<0.01). Conclusion::Zuoguiwan can down-regulate the expressions of Ca2+ transport-associated proteins (Bone tissues) in rat osteoclasts, with an efficacy on osteoporosis.
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Objective To study the expression of plasma membrane Ca2+-ATPase isoforms 1~3 (PMCA 1~3 )in the basilar membrane (BM)of the neonatal rat cochlea by Western blot.The PMCA2 content in single BM of the neonatal rat was also examined.Methods Four rats at postnatal 2 days (P2)and 8 days (P8)were respective-ly decapitated and their BMs were isolated.The total proteins of BMs were extracted.The 20μg total proteins were respectively loaded to the gel.The expression of PMCA1-3 was detected by Western blot.Likewise,3μg total proteins from P2 and P8 rat BM were loaded.The expression of PMCA2 was detected by Western blot.Four rats at P8 were decapitated and their BM was isolated.The 5μg,10μg and 20μg total proteins of P8 rat BM were added to the gel and 100 ng,400 ng and 800 ng bovine serum albumin (BSA)were also loaded as reference.After electro-phoresis,the gel was separated into two parts.One part was used for SYPRO staining and the other part was used for PMCA2 detection by Western blot.Results In the 20μg BM total proteins of P2 and P8 rats,the expression of PMCA1 was weak (0.126±0.024,0.131±0.012,respectively),PMCA2 was strong (4.16±0.528,4.25±0.319, respectively),and PMCA3 was barely expressed (0 ).There was a statistical difference among PMCA1 ,PMCA2 and PMCA3(P<0.05).In the 3μg BM total proteins of P2 and P8 rats,the expression of PMCA2 in P8 (4.571± 0.336)was higher than P2 (3.622±0.285).There was a statistical difference(P<0.05).The PMCA2 content in the BM of a P8 rat was about 2 .5 ng.Conclusion There was a different-level expression of PMCA1~3 in the neonatal rat BM with highest expression of PMCA2 ,which could be explained that cochlear hair cells had different requirements of Ca2+ turning for these PMCA isoforms.
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Microsomal glutathione transferase 1 (MGST1) is an integral homo-trimeric membrane protein with transferase and peroxidase activities. With glutathione as a co-substrate, it scavenges toxic compounds and may exert anti-apoptotic effect. We examined the effect of suppression of plasma membrane Ca2+-ATPase isoforms — PMCA2 or PMCA3 on MGST1 in PC12 cells. GSH level was significantly higher in PMCA2-reduced line, but similar GSSG/GSH ratios in all cell lines suggested an efficient protection or absence of oxidative stress. The ATP concentration decreased in both modified lines, although in PMCA2-suppressed cells the decrease was higher. Total GSTs activity in postmitochondrial fraction increased by 30% in the cells with reduced PMCA3. After treatment with MGST1 activator N-ethylmaleimide (NEM), the activity increased in both transfected lines by 30-40%. Real-time PCR also showed a higher mRNA expression of MGST1 in these lines. Staining with antibody recognizing all cytosolic and membrane-bound GSTs revealed the difference in oligomeric forms of GSTs, and specific anti-MGST1 antibody showed the presence of MGST1 hexamers in the transfected cells. Formation of similar hexamers was detected in the control line after treatment with peroxynitrite. Modification of MGST1 under reduced PMCAs amount may represent an adaptive mechanism that offers protection against the cytotoxicity mediated by increased Ca2+.