Objective To explore the mechanism of ZMS regulation of M_2 muscarinic receptor mRNA expression. Methods In vitro cultured CHOm2 cells were divided into ZMS 1 group (treatment with 1 × 10~(-5) mol/L ZMS for 24 h), ZMS 2 group (treatment with 1 × 10~(-5) mol/L ZMS for 24 h and 1 μg/mL cycloheximide for 12 h) and ZMS 3 group (treatment with 1 μg/mL cycloheximide for 4 h and 1 × 10 ~(-5) mol/L ZMS for 24 h), and their corresponding control groups were also established (substitution of ZMS by DMSO). Actinomycin D was added to cultured CHOm2 cells of each group to inhibit the synthesis of mRNA. CHOm2 cell samples were taken at different time points, the relative expression of M_2 receptor mRNA was detected by Real-time PCR, and half life of M_2 receptor mRNA was calculated. Results Compared with corresponding control groups, the half life of M_2 receptor mRNA of CHOm2 cells in ZMS 1 group and ZMS 2 group was significantly prolonged [(4.75h± 0.54) h vs (2.13 ±0.23) h, P<0.05; (5.43 ±1.13) h vs (2.46 ±0.09) h, P<0.05]. There was no significant difference in half life of M_2 receptor mRNA of CHOm2 cells between ZMS 3 group and its corresponding control [ ( 3.06 ±0.23) h vs (3.00 ± 0.20) h, P > 0.05]. Conclusion De novo protein synthesis is required for the enhancement of M_2 receptor mRNA stability regulated by ZMS.

AIM To study the effect of ZMS, an active component of traditional Chinese herb Zhimu, on two neurotrophins in brains of aged rats. METHODS The function of learning and memory was detected by Y-maze. BDNF and NGF in brain were determined by ELISA. Rats were divided into three groups, young, aged, and aged treated with ZMS for two months at a dose of 18mg/kg/d which is equivalent to the dose recommended for clinical use. RESULTS The learning and memory ability are impaired in aged animals and ZMS is effective in improving the learning and memory ability of such animals. BDNF content of whole brain is lowered in aged rats when compared with young rats. ZMS is able to raise the BDNF content to a higher level. The NGF contents of young, aged and aged treated with ZMS groups were not significantly different from each other. CONCLUSIONS ZMS is able to raise brain BDNF in aged rats and thus to protect cholinergic neurons from degeneration. It is very probable that this action is one of the mechanisms of ZMS in improving the ability of learning and memory.

Aim To investigate the effect of catalpol from Radix Rehmanniae on M_2 receptor density in CHO cells transfected with gene of M_2 receptors(CHO m2).Methods Cultured CHOm2 cells were randomly divided into 4 groups:three concentrations of catalpol :10-6、10-5、10-4 mol?L-1,and saline control.After addition of catalpol and saline for 72 h,M_2 Receptor density was measured by single point 3H-NMS binding assay,the content of protein was measured with Lowry's method.Competitive binding assay using the binding system of 3H-NMS was performed to address the question about whether catalpol could occupy the M receptor binding site.Addition of catapol to brain homogenate and measuring the enzyme activity with the Ellman's colorimetric method were performed to address the question about whether catalpol could in-hibit acetylcholinesterase activity.Results Catalpol can elevate the M_2 receptor density in CHO m2 cells significantly at the doses of 10-5、10-4 mol?L-1(P

Aim To observe the effect of ZDY102, a C25 stereo-isomer of ZMS, the active component of Zhimu, on brain M receptor density of dementia model animals and the correlation with its effect on learning/memory ability. Methods The rats were randomly divided into five groups: control group, model group, model given orally for 2 months with 3.6 mg?kg -1?d -1 of ZDY102 treatment, model treated with 9.0 mg?kg -1?d -1 of ZDY102, and model treated with 18.0 mg?kg -1?d -1 of ZDY102. Dementia model was produced by single unilateral injection of 4 ?l of normal saline containing 4 ?g of ?-amyloid (25～35) and 1 ?g of ibotenic acid into right basal ganglion region with the aid of a stereotaxic equipment. The brain muscarinic receptor density was analyzed with single-site binding assay using 3H-quinuclidinyl benzilae(QNB). The learning/memory ability was measured by Y-maze performance. Results Two months after model production, the learning and memory ability as well as the density of muscarinic receptor in brain were significantly decreased in model rats compared with those in control rats. Parallel models treated with daily oral administration of ZDY102 for two months improved in learning and memory ability and their muscarinic receptor density was significantly increased when compared with model rats. The correlation coefficient between total M receptor densities and the learning/memory ability was significant when examined with linear regression. Conclusion ZDY102 can significantly improve the learning and memory ability and increase the brain muscarinic receptor density of the model. Since brain muscarinic receptors are closely correlated to learning and memory, up-regulation of M receptor density might play a very important role in the therapeutic effect of ZDY102.

Aim To observe the effect of three steroidal saponins on the M-cholinoceptor density of cultured rat myocardiac cells. Methods The time course of M-cholinoceptor density was observed and diosgenin (DIO),timosaponin aglycone (ZMS,S-configuration) and XMS,a stereoisomeric compound of ZMS in C-25 methyl group,R-configuration) were added to the culture medium from the 12th day of culture at three final concentration of 10~(-7),10~(-6) and 10~(-5) mol?L~(-1),and the M-cholinoceptor density was measured on the 16th day with radioligand binding assay. Results The density of M-cholinoceptor increased gradually at the beginning of culture,reached a plateau at 4～10 days,and then dropped gradually. On the 16th day of culture,the M-cholinoceptor density was about 60% of the plateau value.The up-regulation effect of ZMS on the density of cultured rat myocardiac cells on the 16 th day was only significant at a final concentration of 10~(-5) mol?L~(-1). On the contrary,XMS was effective even when its final concentration was as low as 10~(-7) mol?L~(-1). DIO showed no effect on the M-cholinoceptor density at any of its three concentration. Conclusion The above results indicate that XMS with lower concentration showed similar effect on the M-cholinoceptor density of cultured mylcardiac cells as that of ZMS with more higher concentration.