杨梅苷对细菌性骨髓炎大鼠骨缺损的改善作用

doi:10.11958/20230188

摘要/Abstract

摘要：

目的探究杨梅苷通过血管内皮生长因子A（VEGFA）/基质细胞衍生因子-1（SDF-1）/C-X-C型趋化因子受体4（CXCR4）通路对细菌性骨髓炎（OM)大鼠骨缺损的改善作用。方法将大鼠按随机数字表法分为假手术组、OM组、杨梅苷组[50 mg/（kg?d）杨梅苷]、PX-478组[25 mg/（kg?d） VEGFA/SDF-1/CXCR4信号通路抑制剂PX-478]、杨梅苷+PX-478组[50 mg/（kg?d）杨梅苷和25 mg/（kg?d）PX-478]，每组18只。采用双侧胫骨近端骨缺损来构建细菌性OM大鼠模型。连续治疗12周后，观察大鼠创口愈合程度,测量大鼠肛温；酶联免疫吸附试验（ELISA）法检测血清炎性因子白细胞介素（IL）-6、IL-1β、IL-17以及骨代谢指标骨碱性磷酸酶（BALP）、骨钙素（BGP）、Ⅰ型胶原C端肽（CTX-Ⅰ）水平；检测病灶周围组织细菌负荷情况；HE染色检测大鼠病灶周围组织病理变化；免疫荧光染色检测CD31阳性表达；Western blot检测VEGFA/SDF-1/CXCR4信号通路相关蛋白表达。结果与假手术组相比，OM组达到甲级创口愈合程度的大鼠比例，BALP、BGP水平,CD31阳性区域面积,VEGFA、SDF-1、CXCR4蛋白水平下降（P<0.005或P<0.05）；肛温，细菌负荷，血清IL-6、IL-1β、IL-17、CTX-Ⅰ水平升高（P<0.05）。与OM组相比，杨梅苷组BALP、BGP水平，CD31阳性区域面积，VEGFA、SDF-1、CXCR4蛋白水平升高；肛温，细菌负荷，血清IL-6、IL-1β、IL-17水平，CTX-Ⅰ水平降低（P<0.05）；而PX-478组结果趋势相反。PX-478逆转了杨梅苷对细菌性OM大鼠骨缺损的改善作用。结论杨梅苷可能通过激活VEGFA/SDF-1/CXCR4信号通路改善OM大鼠骨缺损。

关键词: 骨髓炎, 血管内皮生长因子A, 趋化因子CXCL12, 受体, CXCR4, 杨梅苷, 骨缺损, 血管生成

Abstract:

Objective To explore the improvement effect of myricitrin on bone defect in rats with bacterial osteomyelitis (OM) through vascular endothelial growth factor A (VEGFA)/stromal cell-derived factor (SDF-1)/C-X-C chemokine receptor 4(CXCR4) pathway. Methods Rats were randomly divided into the sham operation group, the OM group, the myricitrin group [50 mg/(kg?d)], PX-478 group [25 mg/(kg?d) VEGFA/SDF-1/CXCR4 signaling pathway inhibitor PX-478], and the myricitrin+PX-478 group [50 mg/(kg?d) myricitrin and 25 mg/(kg?d) PX-478], 18 mice per group. Bilateral proximal tibia bone defects were used to construct bacterial OM rat model. After 12 weeks of continuous treatment, the wound healing degree of rats was observed, and anal temperature of rats was measured. The serum inflammatory factors interleukin (IL)-6, IL-1β, IL-17 and bone metabolism indexes bone alkaline phosphatase (BALP), osteocalcin (BGP) and C-terminal telopeptide of type Ⅰ collagen (CTX-Ⅰ) were measured by enzyma-linked immunosorbent assay (ELISA). The bacterial load of tissue around the lesion was detected. HE staining was used to detect the pathological changes of the tissue around lesions. The expression of CD31 was detected by immunofluorescence staining. Western blot assay was used to detect the expression of VEGFA/SDF-1/CXCR4 signal pathway related proteins. Results Compared with the sham operation group, the number of rats with grade A wound healing, BALP and BGP levels, CD31 positive area, VEGFA, SDF-1 and CXCR4 protein levels were decreased in the OM group (P<0.005 or P<0.05), and the anal temperature, bacterial load, IL-6, IL-1β, IL-17 and CTX-Ⅰ levels were increased (P<0.05). Compared with the OM group, the BALP and BGP levels, CD31 positive area, VEGFA, SDF-1 and CXCR4 protein levels were increased, and the anal temperature, bacterial load, IL-6, IL-1β, IL-17 and CTX-Ⅰ levels were decreased in the myricitrin group (P<0.05). Results of PX-478 group showed the opposite trend. PX-478 reversed the effect of myricitrin on the improvement of bone defects in bacterial OM rats. Conclusion Myricitrin may improve bone defect of OM rats by activating the VEGFA/SDF-1/CXCR4 signal pathway.

Key words: osteomyelitis, vascular endothelial growth factor A, chemokine CXCL12, receptors, CXCR4, myricitrin, bone defect, angiogenesis

中图分类号:

R681.21

图/表 7

表1 各组大鼠创口愈合以及肛温比较

Tab.1 Comparison of wound healing and anal temperature between the five groups of rats

组别	甲级创口愈合（n=18）/只	肛温（n=18）/℃	细菌负荷（n=6）/（×10⁵ CFU/mL）
假手术组	18	35.39±0.38	0.00±0.00
OM组	9^a	37.35±0.44^a	2.04±0.28^a
杨梅苷组	14	35.69±0.36^b	0.65±0.04^b
PX-478组	5	38.73±0.65^b	3.32±0.36^b
杨梅苷+PX-478组	10	37.46±0.54^c	2.33±0.25^c
χ²或F	23.351^＊＊	144.827^＊＊	196.369^＊＊

表2 各组大鼠血清IL-6、IL-1β、IL-17的比较

Tab.2 Comparison of serum levels of IL-6, IL-1β and IL-17 between the five groups of rats （n=18，ng/L，$\bar{x}±s$）

组别	IL-6	IL-1β	IL-17
假手术组	47.32±6.34	34.23±8.87	1.89±0.25
OM组	73.84±9.56^a	69.43±16.98^a	4.38±0.52^a
杨梅苷组	53.43±6.13^b	44.97±6.56^b	2.15±0.36^b
PX-478组	97.87±11.32^b	93.86±14.87^b	5.98±0.65^b
杨梅苷+PX-478组	69.65±8.43^c	57.56±6.57^c	3.91±0.45^c
F	95.779^＊＊	71.118^＊＊	235.371^＊＊

表3 各组大鼠血清骨代谢指标的比较

Tab.3 Comparison of serum bone metabolism indexes between the five groups of rats （n=18，μg/L，$\bar{x}±s$）

组别	BALP	BGP	CTX-Ⅰ
假手术组	8.47±0.94	15.86±1.69	0.55±0.04
OM组	5.68±0.68^a	9.57±0.94^a	1.39±0.15^a
杨梅苷组	7.94±0.84^b	14.86±1.57^b	0.65±0.07^b
PX-478组	3.31±0.25^b	6.43±0.72^b	1.97±0.23^b
杨梅苷+PX-478组	5.98±0.78^c	10.53±1.76^c	1.22±0.13^c
F	139.029^＊＊	138.927^＊＊	306.237^＊＊

图1 各组大鼠病灶周围骨组织病理变化（HE染色，×100）

Fig.1 Pathological changes of bone tissue around lesion in rats of each group (HE staining, ×100)

图2 各组大鼠病灶周围骨组织CD31阳性表达（免疫荧光染色，×200）

Fig.2 Positive expression of CD31 in bone tissue around lesion of rats in each group (immunofluorescence staining, ×200)

图3 各组大鼠病灶周围骨组织中VEGFA、SDF-1、CXCR4蛋白表达 A：假手术组；B：OM组；C：杨梅苷组；D：PX-478组；E：杨梅苷+PX-478组。

Fig.3 Expressions of VEGFA, SDF-1 and CXCR4 proteins in bone tissue around lesion of rats in each group

表4 各组大鼠病灶周围骨组织中VEGFA、SDF-1、CXCR4蛋白表达的比较

Tab.4 Comparison of VEGFA, SDF-1 and CXCR4 protein expression in bone tissue around lesion of rats between the five groups （n=6，$\bar{x}±s$）

组别	VEGFA	SDF-1	CXCR4
假手术组	1.69±0.19	1.22±0.14	1.47±0.15
OM组	0.91±0.10^a	0.68±0.07^a	0.86±0.09^a
杨梅苷组	1.52±0.13^b	1.13±0.11^b	1.37±0.13^b
PX-478组	0.60±0.06^b	0.33±0.03^b	0.39±0.04^b
杨梅苷+PX-478组	0.97±0.09^c	0.75±0.07^c	0.90±0.09^c
F	82.357^＊＊	92.101^＊＊	135.945^＊＊

参考文献 21

[1]	TALHA K M, BADDOUR L M, ISHAQ H, et al. Native vertebral osteomyelitis in patients with staphylococcus aureus bacteremia[J]. Am J Med Sci, 2022, 363(2):140-146. doi:10.1016/j.amjms.2021.06.018.
[2]	GIMZA B D, CASSAT J E. Mechanisms of antibiotic failure during staphylococcus aureus osteomyelitis[J]. Front Immunol, 2021, 12:638085. doi:10.3389/fimmu.2021.638085.
[3]	BEENKEN K E, CAMPBELL M J, RAMIREZ A M, et al. Evaluation of a bone filler scaffold for local antibiotic delivery to prevent Staphylococcus aureus infection in a contaminated bone defect[J]. Sci Rep, 2021, 11(1):10254. doi:10.1038/s41598-021-89830-z.
[4]	SONG X, TAN L, WANG M, et al. Myricetin:A review of the most recent research[J]. Biomed Pharmacother, 2021, 134:111017. doi:10.1016/j.biopha.2020.111017.
[5]	RAMESH P, JAGADEESAN R, SEKARAN S, et al. Flavonoids:classification, function,and molecular mechanisms involved in bone remodelling[J]. Front Endocrinol (Lausanne), 2021, 12:779638. doi:10.3389/fendo.2021.779638.
[6]	XI Y, WANG W, XU N, et al. Myricetin loaded nano-micelles delivery system reduces bone loss induced by ovariectomy in rats through inhibition of osteoclast formation[J]. J Pharm Sci, 2022, 111(8):2341-2352. doi:10.1016/j.xphs.2022.03.014.
[7]	ZHENG S, ZHOU C, YANG H, et al. Melatonin accelerates osteoporotic bone defect repair by promoting osteogenesis-angiogenesis coupling[J]. Front Endocrinol (Lausanne), 2022, 13:826660. doi:10.3389/fendo.2022.826660.
[8]	XIANG Y, YAO X, WANG X, et al. Houshiheisan promotes angiogenesis via HIF-1α/VEGF and SDF-1/CXCR4 pathways:in vivo and in vitro[J]. Biosci Rep, 2019, 39(10):BSR20191006. doi:10.1042/BSR20191006.
[9]	HUANG X, LIANG P, JIANG B, et al. Hyperbaric oxygen potentiates diabetic wound healing by promoting fibroblast cell proliferation and endothelial cell angiogenesis[J]. Life Sci, 2020, 259:118246. doi:10.1016/j.lfs.2020.118246.
[10]	郭剑栋, 封江标, 陈芹, 等. 抑制CX3C趋化因子受体1对细菌性骨髓炎大鼠骨缺损的改善作用[J]. 中华医院感染学杂志, 2020, 30(14):2129-2133.
	GUO J D, FENG J B, CHEN Q, et al. Effect of inhibiting CX3C chemokine receptor 1 on bone defect in rats with bacterial osteomyelitis[J]. Chinese Journal of Nosocomiology, 2020, 30(14):2129-2133. doi:10.11816/cn.ni.2020-191967.
[11]	YING X, CHEN X, WANG T, et al. Possible osteoprotective effects of myricetin in STZ induced diabetic osteoporosis in rats[J]. Eur J Pharmacol, 2020, 866:172805. doi:10.1016/j.ejphar.2019.172805.
[12]	PAN X, LV Y. Effects and Mechanism of action of PX-478 in oxygen-induced retinopathy in mice[J]. Ophthalmic Res, 2020, 63(2):182-193. doi:10.1159/000504023.
[13]	唐旭, 陈帅, 田鹏, 等. 万古霉素人工骨联合外固定支架治疗慢性胫骨骨髓炎的疗效及其对膝关节功能、踝关节功能和炎性因子的影响[J]. 临床和实验医学杂志, 2019, 18(15):1650-1653.
	TANG X, CHEN S, TIAN P, et al. Effects of Vancomycin artificial bone combined with external fixation on chronic tibial osteomyelitis and its effects on knee function,ankle function and inflammatory factors[J]. Journal of Clinical and Experimental Medicine, 2019, 18(15):1650-1653. doi:10.3969/j.issn.1671-4695.2019.15.025.
[14]	PIMENTEL DE ARAUJO F, MONACO M, DEL GROSSO M, et al. Staphylococcus aureus clones causing osteomyelitis:a literature review (2000-2020)[J]. J Glob Antimicrob Resist,2021, 26:29-36. doi:10.1016/j.jgar.2021.03.030.
[15]	MARRIOTT I. Apoptosis-associated uncoupling of bone formation and resorption in osteomyelitis[J]. Front Cell Infect Microbiol, 2013, 3:101. doi:10.3389/fcimb.2013.00101.
[16]	HU L, LIU R, ZHANG L. Advance in bone destruction participated by JAK/STAT in rheumatoid arthritis and therapeutic effect of JAK/STAT inhibitors[J]. Int Immunopharmacol, 2022, 111:109095. doi:10.1016/j.intimp.2022.109095.
[17]	WANG T, HE C. Pro-inflammatory cytokines:the link between obesity and osteoarthritis[J]. Cytokine Growth Factor Rev, 2018, 44:38-50. doi:10.1016/j.cytogfr.2018.10.002.
[18]	YANG X, YU K, WANG H, et al. Bone impairment caused by AlCl3 is associated with activation of the JNK apoptotic pathway mediated by oxidative stress[J]. Food Chem Toxicol, 2018, 116(Pt B):307-314. doi:10.1016/j.fct.2018.04.057.
[19]	MA L, ZHAO X, LIU Y, et al. Dihydroartemisinin attenuates osteoarthritis by inhibiting abnormal bone remodeling and angiogenesis in subchondral bone[J]. Int J Mol Med, 2021, 47(3):04855. doi:10.3892/ijmm.2021.4855.
[20]	SHEN T, WU Y, CAI W, et al. LncRNA Meg3 knockdown reduces corneal neovascularization and VEGF-induced vascular endothelial angiogenesis via SDF-1/CXCR4 and Smad2/3 pathway[J]. Exp Eye Res, 2022, 222:109166. doi:10.1016/j.exer.2022.109166.
[21]	DING Q, LIU H, LIU L, et al. Deletion of p16 accelerates fracture healing in geriatric mice[J]. Am J Transl Res, 2021, 13(10):11107-11125.