氢气通过抑制 NF-κB 激活来预防去卵巢诱导的骨质疏松症。

Hydrogen gas protects against ovariectomy-induced osteoporosis by inhibiting NF-κB activation.

机构信息

Department of Stomatology, Shanghai 85 Hospital, Shanghai, China.

Department of Stomatology, Changhai Hospital, Shanghai, China.

出版信息

Menopause. 2019 Jul;26(7):785-792. doi: 10.1097/GME.0000000000001310.

DOI:10.1097/GME.0000000000001310

PMID:31083022

Abstract

OBJECTIVES

Osteoporosis is a prevalent condition among postmenopausal women, and lacks satisfactory therapeutic options. Hydrogen (H2) has been shown to be effective in alleviating many diseases. This study aimed to investigate the effects of H2 on inhibiting osteoclastogenesis and bone loss in ovariectomized mice.

METHODS

Osteoclast differentiation from Raw264.7 cells was induced with receptor activator NF-κB ligand (RANKL) with or without 60% H2. The number and resorption activity of osteocalsts were assessed by tartrate-resistant acid phosphatase staining and pit formation assay, respectively. The expression of osteoclast markers and NF-κB phosphorylation were detected by western blot. NF-κB nuclear translocation was assessed by immunofluorescence. NF-κB transcriptional activity was analyzed by luciferase assay. Bone loss in mice was induced by ovariectomy (OVX). OVX mice were given either regular air or 60% H2. Bone structure was analyzed by micro-computed tomography and hematoxylin and eosin staining. Cytokine levels were measured by enzyme-linked immunosorbent assay. The data were analyzed with one-way or two-way ANOVA followed by Bonferroni post hoc tests.

RESULTS

H2 did not have any measurable effect on the proliferation of Raw264.7 cells. The number of osteoclasts and size of resorption pits of RANKL+H2-treated cells were 3 to 4 times less than RANKL treated cells. The expression of osteoclast marker genes of RANKL+H2-treated cells was 30% to 60% lower than RANKL-treated cells (P < 0.05). H2 markedly inhibited RANKL-induced activation, nuclear translocation, and transcriptional activity of NF-κB (P < 0.05, RANKL+H2 vs RANKL). The amount and density of trabecular bone and bone mineral density of ovariectomized mice were significantly less than sham-operated mice (P < 0.05 OVX vs sham). The amount of trabecular bone and bone mineral density of OVX mice that inhaled H2 were more than 40% higher, whereas the levels of serum proinflammatory cytokine interleukin 1β, IL-6, and tumor necrosis factor-α were more than 50% lower than those of OVX mice (P < 0.05).

CONCLUSIONS

These results demonstrated that H2 could be an effective therapeutic agent of postmenopausal osteoporosis.

摘要

目的

骨质疏松症是绝经后妇女中常见的疾病，缺乏令人满意的治疗选择。氢气（H2）已被证明在缓解许多疾病方面有效。本研究旨在探讨 H2 对抑制去卵巢小鼠破骨细胞生成和骨丢失的影响。

方法

用核因子-κB 配体（RANKL）诱导 Raw264.7 细胞分化为破骨细胞，并用或不用 60%H2 处理。通过抗酒石酸酸性磷酸酶染色和陷窝形成试验分别评估破骨细胞的数量和吸收活性。通过蛋白质印迹法检测破骨细胞标志物和 NF-κB 磷酸化的表达。通过免疫荧光法评估 NF-κB 核易位。通过荧光素酶 assay 分析 NF-κB 转录活性。通过卵巢切除术（OVX）诱导小鼠骨丢失。给予 OVX 小鼠常规空气或 60%H2。通过微计算机断层扫描和苏木精和伊红染色分析骨结构。通过酶联免疫吸附试验测量细胞因子水平。使用单因素或双因素方差分析，然后进行 Bonferroni 事后检验对数据进行分析。

结果

H2 对 Raw264.7 细胞的增殖没有任何可测量的影响。RANKL+H2 处理细胞的破骨细胞数量和吸收陷窝大小比 RANKL 处理细胞小 3 到 4 倍。RANKL+H2 处理细胞的破骨细胞标志物基因的表达比 RANKL 处理细胞低 30%至 60%（P<0.05）。H2 显著抑制 RANKL 诱导的 NF-κB 的激活、核易位和转录活性（P<0.05，RANKL+H2 与 RANKL）。去卵巢小鼠的小梁骨量和骨密度明显低于假手术组小鼠（P<0.05，OVX 与 sham）。吸入 H2 的 OVX 小鼠的小梁骨量和骨密度增加了 40%以上，而血清促炎细胞因子白细胞介素 1β、IL-6 和肿瘤坏死因子-α的水平降低了 50%以上（P<0.05）。