山姜素调节VEGF/SphK1/S1P信号通路对膝骨关节炎大鼠血管生成的影响

doi:10.11958/20230892

摘要/Abstract

摘要：

目的探讨山姜素（APT）调节血管内皮生长因子/鞘氨醇激酶1/1磷酸鞘氨醇（VEGF/SphK1/S1P）信号通路对膝骨关节炎（KOA）大鼠血管生成的影响。方法采用改良的Videman法构建KOA大鼠模型，将90只大鼠分为对照组（Control组）、模型组（Model组）、山姜素低剂量组（L-APT组）、山姜素高剂量组（H-APT组）、山姜素高剂量组+慢病毒阴性对照组（APT+NC组）、山姜素高剂量组+过表达SphK1慢病毒组（APT+SphK1组），每组15只。HE染色观察大鼠软骨组织病理变化；酶联免疫吸附试验测定软骨组织白细胞介素（IL）-1β、肿瘤坏死因子α（TNF-α）、IL-6、基质金属蛋白酶-13（MMP-13）水平；TUNEL检测软骨组织细胞凋亡情况；免疫组化检测血管内皮生长因子（VEGF）、CD31蛋白表达情况；Western blot检测血管内皮生长因子受体2（VEGFR2）、磷酸化VEGFR2（p-VEGFR2）、SphK1、S1P蛋白水平。结果与Control组比较，Model组大鼠出现病理损伤，细胞凋亡率、IL-1β、TNF-α、IL-6、MMP-13、VEGF阳性表达、CD31阳性表达和p-VEGFR2、SphK1、S1P蛋白表达水平增加（P<0.05）；与Model组比较，L-APT组、H-APT组病理损伤明显减轻，细胞凋亡率、IL-1β、TNF-α、IL-6、MMP-13、VEGF阳性表达、CD31阳性表达和p-VEGFR2、SphK1、S1P蛋白表达水平降低（P<0.05）；与APT+NC组比较，APT+SphK1组软骨组织病理损伤加重，细胞凋亡率、IL-1β、TNF-α、IL-6、MMP-13、VEGF阳性表达、CD31阳性表达和p-VEGFR2、SphK1、S1P蛋白表达水平增加（P<0.05）。结论 APT通过抑制VEGF/SphK1/S1P信号通路抑制KOA大鼠血管生成。

关键词: 骨关节炎, 膝, 新生血管化, 病理性, 血管内皮生长因子类, 山姜素, 鞘氨醇激酶1, 1磷酸鞘氨醇

Abstract:

Objective To investigate effects of alpinetin (APT) on angiogenesis in knee osteoarthritis (KOA) rats by regulating vascular endothelial growth factor/sphingosine kinase 1/sphingosine 1 phosphate (VEGF/SphK1/S1P) signaling pathway. Methods The KOA rat model was established by Videman method. Ninety rats were grouped into the control group, the model group, the low-dose kaempferol group (L-APT group), the high-dose kaempferol group (H-APT group), the high-dose kaempferol group+lentivirus negative control group (APT+NC group) and high-dose kaempferol+overexpression of SphK1 lentivirus group (APT+SphK1 group), with 15 rats in each group. Pathological changes of cartilage tissue in rats were observed by HE staining. Contents of IL-1β, TNF-α, IL-6 and MMP-13 in cartilage tissue were measured by enzyme linked immunosorbent assay. Chondrocyte apoptosis of cartilage tissue cells was detected by TUNEL. VEGF and CD31 protein positive expression levels were detected by immunohistochemistry assay. The p-VEGFR2, VEGFR2, SphK1 and S1P protein levels were detected by Western blot assay. Results Rats in the model group showed pathological damage. Compared with the control group, the apoptosis rate, IL-1β, TNF-α, IL-6, MMP-13 levels, VEGF positive expression, CD31 positive expression, p-VEGFR2, SphK1 and S1P protein expression levels were increased in the model group (P<0.05). Compared with the model group, the pathological damage was obviously reduced in the L-APT group and the M-APT group, and cell apoptosis rate, IL-1β, TNF-α, IL-6, MMP-13 levels, VEGF positive expression, CD31 positive expression, p-VEGFR2, SphK1 and S1P protein expression levels were obviously reduced (P<0.05). Compared with the APT+NC group, the pathological injury of cartilage tissue increased in the APT+SphK1 group, cell apoptosis rate, IL-1β, TNF-α, IL-6, MMP-13 levels, VEGF positive expression, CD31 positive expression, p-VEGFR2, SphK1 and S1P protein expression levels were obviously increased (P<0.05). Conclusion APT inhibits angiogenesis in knee osteoarthritis rats by inhibiting the VEGF/SphK1/S1P signaling pathway.

Key words: osteoarthritis, knee, neovascularization, pathologic, vascular endothelial growth factors, alpinetin, sphingosine kinase 1, sphingosine 1 phosphate

中图分类号:

R684.3

图/表 7

图1 各组大鼠软骨组织病理变化（HE，×200）黄色箭头示血管，红圈示炎性细胞。

Fig.1 The pathological changes of cartilage tissue in each group（HE staining, ×200）

图2 各组大鼠软骨组织细胞凋亡检测（TUNEL染色，×200）

Fig.2 Detection of apoptosis in cartilage tissue of rats in each group（TUNEL staining, ×200）

表1 各组大鼠软骨组织中IL-1β、TNF-α、IL-6、MMP-13水平比较（n=5，$\bar{x}±s$）

Tab.1 Comparison of IL-1β, TNF-α, IL-6 and MMP-13 in cartilage tissue of rats between six groups

组别	IL-1β/（ng/L）			TNF-α/（ng/L）
Control组	38.41±2.73			93.24±5.37
Model组	147.36±10.34^a			327.59±10.34^a
L-APT组	121.29±9.57^b			243.37±9.52^b
H-APT组	59.44±4.25^bc			125.46±6.25^bc
APT+NC组	55.38±4.19			120.52±6.13
APT+SphK1组	114.61±6.83^d			223.66±8.24^d
F	201.238^＊＊			667.445^＊＊
组别		IL-6/（ng/L）	MMP-13/（μg/L）
Control组		5.32±0.94	29.38±1.63
Model组		34.27±5.34^a	246.46±24.16^a
L-APT组		28.49±3.17^b	164.62±17.39^b
H-APT组		13.51±2.05^bc	105.29±12.25^bc
APT+NC组		12.26±1.65	98.57±11.34
APT+SphK1组		22.18±2.26^d	148.33±15.27^d
F		69.389^＊＊	114.780^＊＊

表2 各组大鼠组织中VEGF、CD31阳性表达的影响（n=5，%，$\bar{x}±s$）

Tab.2 Effects of APT on the positive expressions of VEGF and CD31 in each group of rats

组别	VEGF	CD31
Control组	1.78±0.24	3.21±0.24
Model组	30.36±2.47^a	14.36±0.96^a
L-APT组	21.19±2.03^b	11.15±0.73^b
H-APT组	7.41±0.84^bc	6.58±0.61^bc
APT+NC组	7.24±0.79	6.33±0.55
APT+SphK1组	18.27±1.36^d	9.69±0.68^d
F	256.424^＊＊	179.247^＊＊

图3 各组大鼠软骨组织中VEGF、CD31阳性表达（免疫组化染色，×200） A—F依次为Control组、Model组、L-APT组、H-APT组、APT+NC组、APT+SphK1组。

Fig.3 The positive expression of VEGF and CD31 in cartilage tissue of rats (immunohistochemical staining, ×200)

图4 Western blot检测软骨组织中p-VEGFR2、SphK1、S1P蛋白表达 A—F依次为Control组、Model组、L-APT组、H-APT组、APT+NC组、APT+SphK1组。

Fig.4 p-VEGFR2, SphK1 and S1P protein expression in cartilage tissue detected by Western blot assay

表3 各组大鼠组织中p-VEGFR2、SphK1、S1P表达水平比较（n=5，$\bar{x}±s$）

Tab.3 Comparison of p-VEGFR2, SphK1 and S1P expression levels in tissue of rats between six groups

组别	p-VEGFR2/VEGFR2	SphK1	S1P
Control组	0.52±0.03	0.44±0.02	0.33±0.03
Model组	1.00±0.06^a	1.04±0.07^a	0.93±0.08^a
L-APT组	0.89±0.04^b	0.91±0.05^b	0.79±0.05^b
H-APT组	0.71±0.05^bc	0.74±0.04^bc	0.65±0.04^bc
APT+NC组	0.69±0.04	0.71±0.05	0.62±0.03
APT+SphK1组	0.82±0.05^d	0.87±0.06^d	0.71±0.07^d
F	67.221^＊＊	83.052^＊＊	70.494^＊＊

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