OBJECTIVE To explore the effects of homeodomain protein NK2 homeobox 2(NK2 homeobox 2, NKX2.2)/LIM homeobox transcription factor 1B(LMX1B)/5-hydro xytryptamine(5-HT) signaling pathway on brain neurotransmitter changes in postmenopausal osteoporotic(PMOP) rats, and to reveal the possible intervention mechanism of Tenghuang Jiangu capsule.
METHODS Sixty SPF SD female rats were randomly divided into sham surgery group, model group, estradiol valerate group(0.09 mg·kg−1), and high, medium, and low dose group of Tenghuang Jiangu capsule(0.36, 0.18, 0.09 g·kg−1), with 10 rats in each group. Except for the sham surgery group, the other groups used bilateral ovariectomy to establish PMOP models. After the model was successfully prepared, the sham surgery group and the model group were given equal volume of distilled water by gavage, and the stradiol valerate group and the intervention groups of Tenghuang Jiangu capsule were given corresponding dose by gavage. After 8 weeks of intervention, the rats in each group were killed by femoral artery, and brain and femur tissues were collected for reservation. The general condition and weight of rats were observed in each group. HE staining was used to observe the morphological change of rat femoral tissue. Immunofluorescence staining was used to detect the expression intensity of NKX2.2, LMX1B, and 5-HT of rat brain. The expression of NKX2.2, LMX1B, tryptophan hydroxylase 2(TPH2), 5-hydro xytryptamine 2C receptor(5-HT2CR) and femoral tissue β2AR genes were detected in the brain tissues of rats in each group by RT-PCR. Western blotting were used to detect the protein expressions of NKX2.2, LMX1B, TPH2, 5-HT2CR of rat brain tissue and protein expressions of β2AR of rat femoral tissue.
RESULTS Compared with the sham surgery group, the weight of rats in the model group and the Tenghuang Jiangu capsule groups increased significantly after modeling for 8 weeks. The rats in the model group had sparse fractures, and significantly reduced numbers of bone trabeculae in the femoral tissue, with more gaps and inability to connect into a network; the bone marrow ratio was significantly reduced, and the relative area of bone marrow adipose tissue(BMAT) was significantly increased(P<0.01); the expression of β2AR gene significantly increased in femoral tissue(P<0.01); the average fluorescence intensity of NKX2.2, LMX1B, 5-HT, the gene expression of NKX2.2, LMX1B, TPH2 and 5-HT2CR, and the protein expression of NKX2.2, LMX1B, TPH2 and 5-HT2CR as well as the gray value of the protein expression in the brain tissues were all significantly reduced(P<0.01). Compared with the model group, the weight decreased significantly in the high dose group of Tenghuang Jiangu capsule after 8 weeks of intervention, the number, arrangement and connection of bone trabeculae in the femoral tissue were close to those in the sham surgery group, and the ratio of beam to pulp was significantly increased(P<0.01). The relative area of BMAT of rat femoral tissue decreased significantly in middle and high dose groups of Tenghuang Jiangu capsule(P<0.01). The average fluorescence intensity of NKX2.2 in brain tissue, as well as the gene expressions of NKX2.2, LMX1B, TPH2, the protein expressions and protein expression grayscale value of LMX1B, TPH2, 5-HT2CR were all significantly increased(P<0.05 or P<0.01). The average fluorescence intensity of LMX1B and 5-HT, the expression of 5-HT2CR gene, the protein expression and protein expression grayscale value of NKX2.2 in brain tissue of Tenghuang Jiangu capsule groups were significantly increased, however the gene, protein expressions and protein expression grayscale value of β2AR in the femoral tissue were significantly reduced(P<0.05 or P<0.01).
CONCLUSION Tenghuang Jiangu capsules improve the pathological morphology of the femur in PMOP rats by activating the NKX2.2/LMX1B/5-HT signaling pathway, thereby exerting a therapeutic effect on PMOP.
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