Li Wang-Yang, Xiong Hui
The Second Affiliated Hospital of Hunan University of Chinese Medicine, Changsha 410000, Hunan, China.
Zhongguo Gu Shang. 2022 Apr 25;35(4):367-74. doi: 10.12200/j.issn.1003-0034.2022.04.014.
To observe the effects of Taohong Siwu Decoction(, THSWD) on the mesenchymal stem cells(MSCs) migration, homing number and cytokine expression in callus during the early process of fracture healing, and to explore the mechanism of THSWD on accelerationg fracture healing by regulating the homing of MSCs in rats.
A rat model of right femoral shaft open fracture was established. Thirty-two 5-week-old male Sprague-Dawley rats, weighting 110 to 130 g, were divided into control group, low-dose group, medium-dose group and high-dose group by using random number table. Distilled water was given to the control group, and the other groups were given Decoction. The rats were gavaged twice a day for 5 consecutive days after surgery. Bone volume/tissue volume(BV/TV) and bone mineral density(BMD) were observed using micro-computed tomography (micro-CT) at 21 days after surgery. At 5 days post-fracture, peripheral blood MSCs from THSWD treated and untreated rats were cultured in vitro. Subsequently, the migration ability of MSCs was observed by cell migration assay. The number of MSCs homing to the callus at the early stage of fracture (5 d) was detected by Immunohistochemistry (IHC). Protein chip was used to detect the expression of cytokines in callus.
Micro-CT results showed that BV/TV was higher in the high-dose group than in the medium-dose group (=0.032), and higher in the medium-dose group than in the low-dose group(=0.041), with no difference between the control and low-dose group (=0.651). In addition, there was no difference in BMD between low-dose group and the model group (=0.671), and lower in the low-dose group than in the medium-dose group(=0.018), and the medium-dose group was lower than the high-dose group(=0.008). Cell migration assay showed that THSWD promotes enhanced the migration ability of peripheral blood MSCs. IHC assay revealed that CD45, CD90, CD29 MSCs significantly increased in bone callus after THSWD intervention compared with the control group. Protein chip showed that THSWD promoted the upregulation of CINC-1(×2.91), CINC-3(×1.59), LIX(×1.5), Thymus Chemokine (×2.55), VEGF (×1.22) and the down-regulation of TIMP-1 (×2.98).
THSWD, a representative formula of "promoting blood circulation and removing blood stasis", can significantly accelerate fracture healing, and its mechanism may be related to enhancing the migration ability of peripheral blood MSCs and up-regulating CINC-1, CINC-3, LIX, Thymus Chemokine, VEGF and down-regulating TIMP-1 in bone callus, which promotes the peripheral blood MSCs homing in the early stage of fracture.
观察桃红四物汤对骨折愈合早期骨痂中间充质干细胞（MSCs）迁移、归巢数量及细胞因子表达的影响，探讨桃红四物汤通过调控大鼠MSCs归巢促进骨折愈合的机制。
建立大鼠右股骨干开放性骨折模型。将32只5周龄、体重110～130g的雄性Sprague-Dawley大鼠，采用随机数字表法分为对照组、低剂量组、中剂量组和高剂量组。对照组给予蒸馏水，其余各组给予桃红四物汤。术后大鼠连续5天每天灌胃2次。术后21天采用显微计算机断层扫描（micro-CT）观察骨体积/组织体积（BV/TV）和骨密度（BMD）。骨折后5天，将桃红四物汤处理组和未处理组大鼠的外周血MSCs进行体外培养。随后，通过细胞迁移实验观察MSCs的迁移能力。采用免疫组织化学（IHC）检测骨折早期（5天）归巢至骨痂的MSCs数量。用蛋白质芯片检测骨痂中细胞因子的表达。
Micro-CT结果显示，高剂量组BV/TV高于中剂量组（P=0.032），中剂量组高于低剂量组（P=0.041），对照组与低剂量组之间无差异（P=0.651）。此外，低剂量组与模型组BMD无差异（P=0.671），低剂量组低于中剂量组（P=0.018），中剂量组低于高剂量组（P=0.008）。细胞迁移实验表明，桃红四物汤可增强外周血MSCs的迁移能力。IHC检测显示，与对照组相比，桃红四物汤干预后骨痂中CD45、CD90、CD29阳性的MSCs显著增加。蛋白质芯片显示，桃红四物汤促进CINC-1（×2.91）、CINC-3（×1.59）、LIX（×1.5）、胸腺趋化因子（×2.55）、VEGF（×1.22）上调，TIMP-1（×2.98）下调。
“活血化瘀”代表方桃红四物汤可显著促进骨折愈合，其机制可能与增强外周血MSCs迁移能力及上调骨痂中CINC-1、CINC-3、LIX、胸腺趋化因子、VEGF，下调TIMP-1有关，从而促进骨折早期外周血MSCs归巢。
