| ObjectiveThis study is to estimate the therapeutic effect of kidney yang-supplementing Chinese medicines, including Semen Astragali Complanati decoction (SACD), Rhizoma Cibotii decoction (RCD), Herba Cistanches decoction (HCD), and Semen Allii Tuberosi decoction (SATD), on ovariectomized (OVX) rats. The effective mechanism of the four Chinese medicine on OVX rats is revealed by focusing on the osteoclast differentiation and regulatory factors and hormones and cytokines related to bone metabolism closely, which provide further experimental evidence for osteoporosis and the traditional Chinese medicine theory of" The kidney dominating bone"Material and MethodsNinety-four virgin Wistar clean grade female rats (weight 250±20.0g) were randomly divided into three groups, including a normal control group (n=12), a sham control group (n=12), and a bilaterally ovariectomized (OVX, n=70) group. OVX rats were randomly divided into six groups, including OVX group (n=12), positive control group (n=12), SACD group (n=12), RCD group (n=12), HCD group (n=11), and SATD group (n=11). Diethylstilbestrol 0.008mg/mL dissolved in distilled water was administered intragastrically to the rats in the positive control group. The rats of the SACD group, the RCD group, the HCD group, and the SATD group were treated with SACD 5.6mL/(kg body weight day), RCD 5.6mL/(kg body weight day), HCD 5.6mL/(kg body weight day), and SATD 5.6mL/(kg body weight day), respectively. At the same time, the rats of the normal control group, the sham control group, and the OVX group were administered with the same volume of distilled water. The treatment started four weeks after the surgery and continued for 12 weeks.On the fifteenth and third days before sacrifice all the rats received tetracyclin 30mg/kg by intraperitoneal injection in order to mark the bone with fluorescence. Then all of them were taken blood for testing the serum contents of IL-1,1L-6, BGP, CT, and PTH by means of enzyme linked immunosorbent. After sacrifice, we estimated the proximal right tibiae by measuring TBV%, TRS%, TFS%, MAR, mAR, and OSW of the undecalcified tibia slides. Proximal left tibiae were sliced into 5μm sections by freezing microtome. Frozen sections were prepared for immunohistochemistry and in situ hybridization.All values were expressed as means±standard(x±s) deviations. The difference between the groups regarding the evaluated parameters was tested using the ANOVA test followed by the q test.Results1. In contrast to the OVX group, TBV% in both SACD and RCD groups increased significantly, while TRS%, TFS%, MAR, and mAR decreased significantly in the SACD group, and only TRS% decreased significantly in the RCD group. No changes of these parameters above had been observed in either HCD or SATD group.2. Compared with the OVX group, the protein expression of OPG in OB and MSC of the SACD group and the RCD group significantly increased, but that of the HCD group and the SATD group was not significantly different compared to the OVX group. The protein expression of OPG in the RCD group, the HCD group, and the SATD group was below the sham control group, but there were no significant changes in the SACD group.The protein expression of RANKL in the SACD group was significantly lower than that of the OVX group. However, there were no significant changes in the RCD group, the HCD group, the SATD group, and the OVX group. Compared with the sham control group, the protein expression of RANKL in OB of the RCD group, the HCD group, and the SATD group significantly increased, and the protein expression of RANKL in MSC of the SACD group, the RCD group, the HCD group, and the SATD group also significantly increased, but there was no significant difference in the protein expression of RANKL in OB of the SACD group.3. Compared with the OVX group, OPG mRNA expression in OB and MSC of the SACD group and the RCD group significantly increased, but that of the HCD group and the SATD group was not significantly different compared to the OVX group. OPG mRNA expression in OB and MSC of the RCD group, the HCD group, and the SATD group was below the sham control group, but there were no significant changes in the SACD group.RANKL mRNA expression in OB and MSC of the SACD group was significantly lower than that of the OVX group. However, there were no significant changes in the RCD group, the HCD group, the SATD group, and the OVX group. Compared with the sham control group, RANKL mRNA expression in OB and MSC of the RCD group, the HCD group, and the SATD group was significantly increased. There were no significant changes in the SACD group and sham control group.4. In contrast to the OVX group, the level of IL-1 in the peripheral blood of the SACD group decreased significantly, but that of RCD group, the HCD group, and the SATD group was not significantly different compared to the OVX group. However, IL-1 in the SACD group, the RCD group, the HCD group, and the SATD group was higher than that in the sham control group.The level of IL-6 in the SACD group, and the RCD group was significantly lower than that of the OVX group. However, there were no significant changes in the HCD group, the SATD group, and the OVX group. Compared with the sham control group, IL-6 in the RCD group, the HCD group, and the SATD group was significantly increased. There were no significant changes in the SACD group and sham control group.5. The level of BGP in the peripheral blood of the SACD group was below the OVX group, but there were no significant changes in the RCD group, the HCD group, and the SATD group. However, BGP of the SACD group, the RCD group, the HCD group, and the SATD group was higher than that in the sham control group.Compared with the OVX group, CT of the SACD group significantly increased, but that of the RCD group, the HCD group and the SATD group was not significantly different compared to the OVX group. CT of the RCD group, the HCD group, and the SATD group was below the sham control group, but there were no significant changes in the SACD group.No changes of PTH had been observed in the SACD group, the RCD group, the HCD group, the SATD group, the OVX group, or the sham control group. ConclusionsThe effective mechanism of the four kidney yang-supplementing Chinese medicines on OVX rats is revealed by focusing on the osteoclast differentiation and regulatory factors and hormones and cytokines related to bone metabolism closely. We found that the OVX rat was a high-transformational model of osteoporosis, and SACD and RCD enhanced overall bone quality of OVX rats significantlyOur experiments implied that the protein expression of OPG in OB and MSC was promoted by SACD, and the protein expression of RANKL in OB and MSC was inhibited by SACD. At the same time, SACD could reduce the level of IL-1, IL-6 and enhance the level of CT in the peripheral blood. We also found that the protein expression of OPG in OB and MSC was promoted by RCD, and the level of IL-6 was inhibited by RCD. However, no change of RANKL had been observed in the RCD group. |
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