Revealing the Therapeutic Mechanism of Tenghuang Jiangu Capsule on Rats with Postmenopausal Osteoporosis Based on Enterogenous 5-HT Mediating NKX2.2/Lmx1α Signaling Axis

OBJECTIVE To observing the effect of Tenghuang Jiangu capsule(TJC) on the expression of homeodomain protein NK2 homeobox 2(NKX2.2), lim homeobox transcription factor 1 alfa(Lmx1α) and 5-hydroxytryptamine(5-HT) of NKX2.2/Lmx1α signaling axis of rats with postmenopausal osteoporosis, to preliminarily reveal its possible therapeutic mechanism.

METHODS Sixty female SD rats were randomly divided into sham operation group, ovariectomized model group, estradiol valerate group(0.09 mg·kg⁻¹), high, middle, low dose of TJC groups(0.36, 0.18, 0.09 g·kg⁻¹). Except for the sham operation group, rats in other groups were used to establish a rat model of postmenopausal osteoporosis by ovariectomized method. After 8 weeks of modeling, rats in each group were given above doses of drugs by oral gavage, with continuous intervention for 8 weeks. The following day after intervention ends, the levels of bone mineral density(BMD) and bone mineral content(BMC) were detected, the pathomorphological changes of femoral tissue was observed, the concentrations of bone sialoprotein(BSP) and transcription activator 4(ATF4) were checked by ELISA, immunofluorescence was used to detect the protein expressions of NKX2.2, Lmx1α and 5-HT in the duodenum of rats in each group, the expressions of NKX2.2, Lmx1α and tryptophan hydroxylase 1(Tph1) of duodenum tissue and the gene and protein expressions of 5-hydroxytryptamine receptor 1B(5-HT1BR), forkhead transcription factor-1(FOXO1) of femoral tissue were analyzed by qPCR and Western blotting, the protein expression of cAMP-response element binding protein(CREB) in the femur of each group of rats were detected by Western blotting.

RESULTS Compared with the sham operation group, the femoral tissue of rats in the ovariectomized model group had obvious pathological changes, the levels of BMD, BMC, the concentrations of BSP and ATF4 of serum, and the protein expression level of CREB of femoral tissue were all significantly decreased, whereas the gene expression levels of NKX2.2, Lmx1α and Tph1 of duodenum tissue were significantly increased, the fluorescence intensity of NKX2.2, Lmx1α and 5-HT, the protein expression levels of NKX2.2, Lmx1α and Tph1 were also significantly increased, and the gene and protein expression levels of 5-HT1BR and FOXO1 of femoral tissue were significantly increased(P<0.01). Compared with the ovariectomized model group, the pathological damage of femoral tissue was reduced in high dose of TJC group, the concentrations of BSP and ATF4 of serum and the protein expression level of CREB of femoral tissue were all significantly increased in high and middle dose of TJC groups, while the protein expression levels of NKX2.2, Tph1 of duodenum tissue were significantly decreased in high and middle dose of TJC groups, the levels of BMD and BMC of rats in high, middle and low dose of TJC groups were significantly increased, however the gene expression levels of NKX2.2, Lmx1α, Tph1, the fluorescence intensity of NKX2.2, Lmx1α and 5-HT, the protein expression level of Lmx1α of duodenum tissue were all significantly decreased in high, middle and low dose of TJC groups, the gene and protein expression levels of 5-HT1BR and FOXO1 of femoral tissue were also significantly decreased in high, middle and low dose of TJC groups(P<0.05 or P<0.01).

CONCLUSION TJC can significantly upregulate the expression of bone differentiation factors BSP and ATF4 in postmenopausal osteoporosis rats, which maybe related to the inhibition of NKX2.2/Lmx1α/5-HT signaling axis by intestinal 5-HT mediated.