Study Of Molecular Mechanisms Of Semen Sojae Preparatum Isoflavone On Anti-osteoporosis

Objective: Our earlier researches show that Semen Sojae Preparatum cancorrect abnormalities of bone histomorphometry parameters, improve the finestructure of bone and the bone biomechanical properties, and increase bonedensity of ovariectomized rats with osteoporosis. It has an obviousanti-osteoporosis activity. But its mechanism is not yet clear. To make it clear,in our study, we take SSPI (Semen Sojae Preparatum Isoflavone, SSPI) as theresearch object, which is the main active component of SSP, and utilize theprimary culture osteoblasts to observe the effect of SSPI on osteoblasts fromthe cellular and molecular levels. The aim of the study is to investigate theregulation mechanism of SSPI through estrogen receptor pathway with a viewto enlarge the clinical use of SSP as well as anti-osteoporosis drugsdevelopment in the future.Methods:1The effects of SSPI on the proliferation and differentiation of rat osteoblasts1.1Primary culture and identification of rat osteoblastsThe primary culture OB of SD rat (24h) by the modified tissue stickingmass method was used for the study; osteoblasts were identified by alkalinephosphatase staining, cell morphology and the OB staining rate was detectedby inverted phase contrast microscope.1.2Detection of the cytotoxicity of SSPI on OBConcentration gradient of SSPI was set to1,100,1000,5000,10000,50000,100000,500000μg/L, and the action time was24h; persistence of each holewas detected by MTT method, denoting by optical density.1.3Determination of the optimal concentration and action time of SSPIInterference gradient concentrations was set as follows:1,10,50,100,200,500,1000,5000,10000,50000,100000μg/L; the incubation times were24h, 48h,72h； the optimal concentration and time were measured by MTTmethod.1.4The effects of SSPI on the proliferation and differentiation of OBThe experiment was divided into control group (control), the SSPI group(SSPI):1,10,100,1000, μg/L, E2(10-9mol/L), soy isoflavone group (ISO):1000μg/L, and the estrogen receptor antagonist ICIl82780group: SSPI (1000μg/L)+ICIl82780(10-8mol/L) was established to preliminarily observe thatwhether SSPI regulated osteoblasts through the estrogen receptor pathway.The ICIl82780was incubated for2h before the SSPI. The action times were48h and72h.1.4.1Detection of the proliferation on OBMTT assay was used to detect the proliferation on osteoblasts and the actiontimes were48h and72h.1.4.2Detection of ALP activity on OBALP kit was used to detect ALP activity on osteoblasts and the action timeswere48h and72h. It detected the effect of the intervention on the earlydifferentiation of rat osteoblasts.1.4.3Detection of hydroxyproline content on OBDigestion method was used to detect hydroxyproline content on osteoblastsat48h and72h. It detected the effect of the intervention on the collagencontent of rat osteoblasts.2The effects of SSPI on the expression of ERα and ERβ mRNA and proteinin OB2.1The effect of SSPI on Erα and ERβ mRNA expression in OBThe experiment was divided into control group (control), the SSPI group(SSPI):1,10,100,1000μg/L, and incubated for72h. Whole RNA wasextracted by Trizol, expression of ERα or ERβ mRNA in OB was detected byRT-PCR method.2.2Comparison of the expression of ERα and ERβ mRNA in OB treated withSSPI and ISOThe experiment was divided into control group (control), SSPI group (SSPI 1000μg/L), E2(10-9mol/L), soy isoflavonegroup (ISO):1000μg/L, andICIl82780group: SSPI (1000μg/L)+ICIl82780(10-8mol/L), and ICIl82780were incubated for2h before the SSPI. The action time was72h. Whole RNAwas extracted by Trizol, expression of ERα or ERβ mRNA in OB was detectedby RT-PCR method.2.3The effect of SSPI on ERα and ERβ protein expression in OBExperimental groups and drug interference were the same as2.1. Wholeprotein was extracted by RIPA lysate. ERα and ERβ protein expressions weredetected by Western blot technology.2.4Comparison of the expression of ERα and ERβ protein in OB treated withSSPI and ISOExperimental groups and drug interference were the same as2.2. Wholeprotein was extracted by RIPA lysate. ERα and ERβ protein expressions weredetected by Western blot technology.Results:1The effects of SSPI on the proliferation and differentiation of rat osteoblasts1.1Cell morphology observationUnder the inverted phase contrast microscope, we can see the primarycultured osteoblasts remove from the skull fragments24h~36h later, whichis globular at first and then floats in culture medium, the growth of cellsarranged in non-directional and around the skull fragments. The osteoblastswhich are inoculated are globular at first and then float in culture medium.Subsequently it is adherent, it will expand in24h. The shape of expanded cellis irregular with an appearance of triangle. After3days, osteoblast shows onlong fusiform and trabs shape and mixes together and the boundary is vaguebetween cells. Osteoblast shows slabstone shape after5days. If we keep onculturing cell, the cells will gradually gathere to form cell nodule.1.2Identification of rat osteoblastsAlkaline phosphatase staining: After the osteoblasts stained with cAKP, thepurple-blue precipitate can be seen in the cells, which is the typical positivestaining reaction of ALP. The positive rate of alkaline phosphatase staining is 93%under the microscope.1.3The cytotoxicity of SSPIOD value was decreased significantly (P<0.01) when the drug concentrationexceeded50000μg/L and obvious cytotoxicity emerged (P<0.01). Most of thecells were shrinked to round shape and a few of dead cells were floating in theholes.1.4Determination of the optimal concentration and action time of SSPISSPI of different concentrations enhanced the proliferation of OB indifferent degree. According to the MTT results, the optimal concentrations ofSSPI that enhanced OB proliferation were1,10,100,1000μg/L and theoptimal times were48h and72h.1.5The effect of SSPI on the proliferation of osteoblastsCompared with control group, the OD values of each group exceptICI182780group, were significantly increased by drugs intervention for48hor72h (P<0.05), and the improvement related to prolong exposure to SSPI,but no relation with concentration of SSPI. Moreover, the effect of SSPI1000μg/L in72h was the strongest, stronger than that of ISO. The effect wasinhibited by ICI182780antagonist.1.6The effect of SSPI on the differentiation of osteoblastsCompared with control group, the ALP activity of each group exceptICI182780group, were increased by drugs intervention for48h or72h(P<0.05), and the effect of SSPI and that of E2showed no significantdifference (P>0.05). Moreover, the effect of SSPI1μg/L was the strongest(P<0.05) followed by the ISO group (P<0.05). ICI182780group showed nosignificant difference with control group, but the OD values was lowercompared with the SSPI1000μg/L group with no significant difference(P>0.05).1.7The effect of SSPI on the hydroxyproline content of osteoblastsCompared with control group, the hydroxyproline of each group exceptICI182780group, was increased by drugs intervention for48h or72h(P<0.05), and the effect of SSPI1000μg/L and that of E2showed no significant difference (P>0.05), stronger than that of ISO at the same period inthe same concentration with no significant difference (P>0.05). Moreover, theeffect was inhibited by ICI182780antagonist. The effects of SSPI and that ofE2showed no significant difference (P>0.05).2The effects of SSPI on the expression of ERα and ERβ mRNA and proteinin OB2.1The effect of SSPI on ERα and ERβ mRNA expression in OBCompared with the control group, the expressions of ERβ mRNA wasenhanced by different concentrations of SSPI (P<0.01); the expression of SSPI1000μg/L was the highest, followed by the SSPI10μg/L, and there were nodifference between the rest two groups (P>0.05). The expression of ERαmRNA was not detected in the experiment.2.2Comparison of the expression of ERα and ERβ mRNA in OB treated withSSPI and ISOCompared with the control group, the expressions of ERβ mRNA wassignificantly enhanced by SSPI and E2intervention (P<0.01); the expressionof ISO1000μg/L group was increased with no significant difference (P>0.05).The effect of SSPI was weaker than that of E2. Moreover, the effect wasinhibited by ICI182780antagonist. The expression of ERα mRNA was notdetected in the experiment.2.3The effect of SSPI on ERα and ERβ protein expression in OBCompared with the control group, the expressions of ERβ protein wasenhanced by different concentrations of SSPI; the expression of SSPI1000was the highest, and there were no difference among the rest three groupsSSPI1,10,100μg/L (P>0.05). The result showed that the improvement hadno relation with concentration of SSPI. The expression of ERα protein was notdetected in the experiment.2.4Comparison of the expression of ERα and ERβ protein in OB treated withSSPI and ISOCompared with the control group, the expressions of ERβ protein wassignificantly enhanced by SSPI, E2and ISO intervention (P<0.01or P<0.05).The effect of SSPI was the strongest, stronger than that of ISO at thesame period in the same concentration. Moreover, the effect was inhibited byICI182780antagonist. The expression of ERα protein was not detected in theexperiment.Conclusion:1SSPI (1to1000μg/L) can significantly promote the proliferation,differentiation and the formation of bone matrix of osteoblasts. The effect ofSSPI1000μg/L was the strongest. Moreover, the effect was inhibited byICI182780antagonist, which suggested the effect that SSPI promotesproliferation and differentiation of OB may relate to the estrogen receptorpathway.2SSPI can up-regulate the of expression of ERβ mRNA and ERβ protein inOB, but without effects on ERα mRNA or ERα protein, which indicated thatERβ may be the major ER subtype expressed by osteoblasts. Thus we inferSSPI may exert estrogenic activity to OB and prevent osteoporosis through theERβ pathway.3SSPI can significantly promote the proliferation, differentiation and theformation of bone matrix of osteoblasts, and up-regulate the expression of ERmRNA and ER protein. And the effect of SSPI is stronger than that of ISO atthe same period in the same concentration, which indicated that SSPI mayhave stronger anti-osteoporosis activities than ISO.