Fig 2: The levels of osteocalcin (OCN) in the serum or cerebrospinal fluid (CSF) of control and 6-hydroxydopamine (6-OHDA)-induced Parkinson’s disease (PD) rat models. The CSF OCN levels in PD rats dramatically decreased compared to control rats (A, n = 17). No difference in the serum OCN levels was found between control and PD rats (B, n = 19). Expression of GPR158 and GPRC6A in various brain-derived tissues of 8-week-old rats (C, n = 3). Coimmunoprecipitation of 8-week-old mice striata and proteins were then subjected to western blot analysis using anti-Gpr158 and anti-GPRC6a antibodies (D, n = 3). Values were obtained from three independent experiments and results are given as the mean ± SEM. *P < 0.05.

Fig 3: The effect of OCN on the proliferation of astrocytes and microglia. 6-OHDA significantly increased the number of GFAP-positive astrocytes in the lesioned side compared to the intact side both in the SN (A) and striatum (B), which was inhibited by OCN injection. The same function of OCN on microglial proliferation was also observed, which was stained with Iba1 (C,D). n = 16. Green = TH, Red = GFAP or Iba1, Blue = Nucleus.

Fig 4: The protective effect of OCN on tyrosine hydroxylase (TH+) neurons and fibers in the nigrostriatal system in the unilateral 6-OHDA-lesioned PD rat model. 6-OHDA induced ~90% dopaminergic neuronal loss on the lesioned side compared to the intact side of the substantia nigra (SN), which was improved by i.p. OCN injection (A). Similarly, the decrease in TH-positive cell number in the SN of the lesioned side was also significantly alleviated by OCN injection (B). The results from western blots measuring TH were consistent with immunohistochemistry in the striatum (C). In the intra-striatum injection OCN group, 6-OHDA induced ~50% dopaminergic neuronal loss in the lesioned side compared to the intact side of the SN, which was improved by OCN injection (D). Similarly, the decrease in TH-positive cell number in the SN of lesioned side was also significantly alleviated by intra-striatum OCN injection (E). The results from western blots measuring TH were consistent with immunohistochemistry in the striatum (F). n = 16, *P < 0.05 (compared with the 6-OHDA group).

Fig 5: OCN protects PC12 cells from 6-OHDA-induced neurotoxicity. Apoptotic cells were measured by flow cytometry (A–E). The number of apoptotic cells was counted in the R2 (AV+/7-amino-actinomycin, 7-AAD+, the late phase apoptotic cells) and R4 (AV+/7-AAD-, the early phase apoptotic cells) regions, and the apoptotic rate was calculated (F). The CCK-8 test showed that OCN could attenuate the neuronal toxicity induced by 6-OHDA through activating AKT/GSK3ß, while LY294002 reversed the neuroprotective effect of OCN on PC12 cells (G) through inhibiting the phosphorylation of AKT and GSK3ß (H). Values were obtained from three independent experiments. *P < 0.05 (compared with the control group), #P < 0.05 (compared with the 6-OHDA group).