羟基红花黄色素A促进激素诱导兔骨髓基质细胞成骨分化及其MAPK信号转导机制的研究
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摘要
目的
探讨羟基红花黄色素A(HSYA)促进激素诱导的股骨头缺血坏死模兔股骨头髓内及离体兔骨髓基质细胞(BMSCs)成骨分化及其MAPK信号转导通路的作用机制。
方法
1.健康成年新西兰大白兔48只,抽签法随机分为空白组、模型组,模型组采用臀肌注射醋酸泼尼松龙(7.5mg/kg)结合强迫立位制备激素性股骨头缺血坏死模型。造模成功后抽签法将模型组兔随机分为模型对照组、髓芯减压组、生理盐水+髓芯减压组、HSYA+髓芯减压组,髓芯减压组仅行髓芯减压术,生理盐水+髓芯减压组采用髓芯减压术配合髓腔内注射生理盐水,HSYA+髓芯减压组采用髓芯减压术配合髓腔内注射HSYA,空白组和模型对照组不作治疗。1周后兔麻醉,腹主动脉采血,采用化学比色法、氧化酶法检测各组兔血清中碱性磷酸酶、甘油三脂的含量；取出股骨头,光学显微镜、透射电子显微镜观察各组兔股骨头的形态结构；RT-PCR检测各组兔股骨头中Cbfal及Collagen-I mRNA的表达；Western Blotting检测各组兔股骨头中p-ERK. p-JNK和p-P38蛋白的表达。
2.健康成年新西兰大白兔36只,无菌条件下于胫骨上端内侧骨髓腔穿刺抽取骨髓血,分离、体外培养BMSCs,流式细胞仪检测细胞表面标记物结合成骨、成脂诱导剂诱导对其鉴定。取第3代生长良好的BMSCs,抽签法随机分为空白组,模型组及HSYA低、中、高剂量组。模型组用大剂量地塞米松诱导BMSCs成脂分化,HSYA低、中、高剂量组加入地塞米松的同时分别加入不同浓度的HSYA;空白组无特殊处理。6d后,透射电子显微镜观察各组细胞的形态结构,检测细胞内碱性磷酸酶、甘油三脂的含量,RT-PCR检测细胞中Cbfal及Collagen-I mRNA的表达,Western Blotting检测细胞中p-ERK、p-JNK和p-P38蛋白的表达。
结果
1. HSYA促进激素诱导兔BMSCs成骨分化及其MAPK信号转导机制的在体实验：
(1)血清中成骨标志物碱性磷酸酶、成脂标志物甘油三脂含量的检测：干预1周后,空白组、髓芯减压组、生理盐水+髓芯减压组和HSYA+髓芯减压组兔血清中碱性磷酸酶含量明显高于模型对照组,甘油三脂含量明显低于模型对照组(P<0.05)。其中HSYA+髓芯减压组碱性磷酸酶含量明显高于髓芯减压组和生理盐水+髓芯减压组(P<0.05),甘油三脂含量明显低于髓芯减压组和生理盐水+髓芯减压组(P<0.05)。
(2)光镜观察股骨头组织的病理改变：空白组软骨下骨小梁致密,排列规则整齐；骨细胞清晰,核位于中央,较大；髓腔内可见增生活跃的骨髓组织,造血细胞丰富。模型对照组软骨层变薄、结构紊乱,骨小梁较稀疏,部分有断裂现象,小梁中有骨细胞坏死；软骨下区骨髓内脂肪细胞增多,造血细胞数量减少。髓芯减压组、生理盐水+髓芯减压组、HSYA+髓芯减压组骨细胞结构较清晰,骨小梁较多,坏死骨细胞较模型对照组少,其中尤以HSYA+髓芯减压组最为明显。
(3)透射电镜观察股骨头组织的超微结构：空白组软骨细胞粗面内质网丰富,无明显脱颗粒；线粒体较多,嵴结构清晰,未见明显断裂；染色质分布相对均匀,细胞表面突起较多。模型对照组空泡样变较空白组明显增多,异染色质部分边聚,胞内可见较大脂滴或次级溶酶体。HSYA+髓芯减压组空泡样变明显减少,次级溶酶体明显减少,大部分线粒体嵴存在。
(4)股骨头中成骨标志物Cbfα1及Collagen-Ⅰ mRNA的表达：干预1周后，空白组、髓芯减压组、生理盐水+髓芯减压组和HSYA+髓芯减压组明显高于模型对照组(P<0.05)。其中HSYA+髓芯减压组明显高于髓芯减压组和生理盐水+髓芯减压组(P<0.05)。
(5)股骨头中与MAPK信号通路相关的p-ERK、p-JNK和p-P38蛋白的表达：干预1周后,空白组、髓芯减压组、生理盐水+髓芯减压组和HSYA+髓芯减压组p-ERK1、p-ERK2蛋白表达明显高于模型对照组(P<0.05),p-JNK、p-P38蛋白表达明显低于模型对照组(P<0.05)。其中HSYA+髓芯减压组p-ERK1、p-ERK2蛋白表达明显高于髓芯减压组和生理盐水+髓芯减压组(P<0.05),p-JNK、p-P38蛋白表达明显低于髓芯减压组和生理盐水+髓芯减压组(P<0.05)。
2. HSYA促进激素诱导兔BMSCs成骨分化及其MAPK信号转导机制的离体实验：
(1)油红“O”染色结果：空白组未发现红染的脂肪颗粒；模型组发现细胞的胞浆内可见大量染成红色的脂肪滴,呈诱导后的脂肪细胞形态；HSYA低、中、高剂量组发现有脂肪滴分布,三组红染脂肪滴明显少于模型组,HSYA高剂量组脂肪滴明显少于模型组,少于中、低剂量组。
(2)透射电镜观察BMSCs的超微结构：各组细胞干预6d后,空白组细胞呈椭圆形或不规则状,细胞质少,核大,未见明显脱颗粒现象。模型组细胞呈长梭形,未见明显核仁；细胞器中线粒体较多呈空泡,可见较多次级溶酶体或脂滴。HSYA低剂量组表面空泡不太明显,损伤的线粒体数量较模型组少,脂滴减少。HSYA中剂量组线粒体较低剂量组增多,空泡样变化减少,次级溶酶体减少,未见明显脂滴。HSYA高剂量组线粒体明显增多,空泡样变明显减少,次级溶酶体明显减少,未见明显脂滴。
(3) BMSCs中成骨标志物碱性磷酸酶、成脂标志物甘油三脂含量的检测：各组细胞干预6d后,模型组BMSCs内碱性磷酸酶含量较空白组明显下降(P<0.05),而HSYA各组较模型组均有明显升高(P<0.05),且随着剂量的加大,碱性磷酸酶含量呈上升趋势。模型组BMSCs内甘油三脂含量较空白组明显升高(P<0.05),而HSYA各组较模型组均有明显降低(P<0.05),且随着HSYA剂量的加大,甘油三脂含量呈下降趋势。
(4) BMSCs中成骨标志物Cbfα1及Collagen-I mRNA的表达：各组细胞干预6d后,模型组细胞内Cbfα1、Collagen-Ⅰ mRNA含量均明显低于空白组(P<0.05),而HSYA各组Cbfα1、Collagen-Ⅰ mRNA含量均明显高于模型组(P<0.05),且随着HSYA剂量的增加,细胞内Cbfα1、Collagen-Ⅰ mRNA含量呈上升趋势。
(5) BMSCs中与MAPK信号通路相关的p-ERK、p-JNK和p-P38蛋白的表达：各组细胞干预6d后,模型组细胞内p-ERK1、p-ERK2蛋白表达明显低于空白组(P<0.05),p-JNK、p-P38蛋白表达明显高于空白组(P<0.05),而HSYA各组p-ERK1、 P-ERK2蛋白表达明显高于模型组(P<0.05),p-JNK、p-P38蛋白表达明显低于模型组(P<0.05),且随着HSYA剂量的增加,细胞内p-ERK1、p-ERK2蛋白表达呈上升趋势,p-JNK、p-P38蛋白表达呈下降趋势。
结论
1.羟基红花黄色素A(HSYA)能促进激素性股骨头缺血坏死模兔股骨头髓内及离体兔BMSCs内成骨标志物碱性磷酸酶、Cbfα1、Collagen-Ⅰ的表达,抑制成脂标志物甘油三脂的表达,抑制激素诱导股骨头髓内及离体兔BMSCs成脂分化,促使其成骨分化。
2. HSYA通过激活MAPK信号转导通路,上调通路中与增殖关联的p-ERK蛋白的表达,下调凋亡相关联的p-JNK、p-P38蛋白的表达,促进兔股骨头髓内及离体兔BMSCs增殖,抑制其凋亡,减轻激素诱导的BMSCs凋亡的影响。
Objective
To observe the promoting effect of Hydroxy safflower yellow A (HSYA) on osteogenic differentiation of glucocorticoid-induced rabbit bone marrow stroma cells (BMSCs) in femur head marrow cavity of rabbit models with ischemic necrosis of femoral head and isolated glucocorticoid-induced rabbits BMSCs, and explore the MAPK signal transduction mechanism.
Methods
148New Zealand adult white rabbits were randomly divided into blank group and model group. Prednisolone acetate (7.5mg/kg) was injected into the rabbits of model group using intragluteal injection to induce models of ischemic necrosis of femoral head. After successful modeling, rabbits in model group were divided into model control group, core decompression group, normal saline+core decompression group and HSYA+core decompression group. Core decompression group received core decompression, normal saline+core decompression group received core decompression and intramedullary injection with normal saline, HSYA+core decompression group received core decompression and intramedullary injection with HSYA, and normal group and model control group didn't accept any treatment. After a week, the rabbits were anesthetized, and the blood of abdominal aorta was obtained. The levels of alkaline phosphatase (ALP) and triglyceride (TG) in rabbit serum were detected by chemical colorimetry and oxidase method. The femoral heads were taken off, their morphology was observed by optical microscope and transmission electron microscope, the mRNA expressions of Cbfal and Collagen-I were detected by RT-PCR assay, and the protein expressions of p-ERK, p-JNK and p-P38were detected by Western Blotting.
236New Zealand adult white rabbits were used in this experiment, and the bone marrow blood was drawn from the medial aspect of proximal tibia, then the BMSCs were isolated and cultured in vitro. The cell surface markers were detected by flow cytometry and the cells were induced by adipogenic and osteogenic inducers to identify the cells. The3rd generation of BMSCs which had good growing status were randomly divided into blank group, model group and HSYA groups with different doses. The model group was treated with high dose of dexamethasone to induce adipogenic differentiation of BMSCs, the HSYA groups received high dose of dexamethasone and different concentrations of HSYA simultaneously, and the blank group did not receive any special handling. After6days, the morphology of cells was observed by fluorescence inverted phase contrast microscope and transmission electron microscope, the levels of ALP and TG in BMSCs were detected, the mRNA expressions of Cbfal and Collagen-I were detected by RT-PCR assay, and the protein expressions of p-ERK, p-JNK and p-P38were detected by Western Blotting.
Results
1HSYA promoting osteogenic differentiation of glucocorticoid-induced rabbit BMSCs and its MAPK signal transduction mechanism through experiments in vivo
(1) Determination of levels of serum ALP and TG:after a week, the level of serum ALP in blank group, core decompression group, normal saline+core decompression group and HSYA+core decompression group was respectively higher than that of model control group (P<0.05), and the level of serum TG was respectively lower than that of model control group (P<0.05). The level of serum ALP in HSYA+core decompression group was significantly higher than that of core decompression group, normal saline+core decompression group (P<0.05), and the level of serum TG was significantly lower than that of core decompression group, normal saline+core decompression group (P<0.05).
(2) Observation of femoral head pathologic change by light microscope:the bone trabecula of cartilage in blank group was compact, and arranged in line; the osteocytes were clearly seen, and the nucleus which located in the center were big; hyperplastic myeloid tissues were found in marrow cavity, and the hematopoietic cells were present in great number.The structural disorder of cartilage in model control group was observed, bone trabecula were rare, some occurred disrupted, and necrotic osteocytes were found in trabecula; a increase of adipocytes and decrease of hematopoietic cells were seen in bone marrow. The cellular structures were clear in core decompression group, normal saline+core decompression group and HSYA+core decompression group, and the bone trabecula was more and necrotic osteocyte was less than those of model control group, and the phenomenon was the most significant in HSYA+core decompression group.
(3) Observation of femoral head ultra-structure by transmission electron microscope:more rough endoplasmic reticulums were found in chondrocytes of blank group, and the degranulation of cells was not significant; the structure of cristae was clear without significant disruption; the chromatin was uniformly distributed, and more cell surface projections were found. In model control group, more cavitations were found than those of blank group, the heterochromatin was condensed and marginated, and more lipid droplets and secondary lysosomes appeared. In HSYA+core decompression group, less vacuoles and secondary lysosomes were found, and most of crista mitochondriales still appeared.
(4) Expressions of Cbfal and Collagen-I mRNA in femur head:after a week, the mRNA expressions of Cbfal and Collagen-I in blank group, core decompression group, normal saline+core decompression group and HSYA+core decompression group were respectively higher than that of model control group (P<0.05), and the values in HSYA+core decompression group were significantly higher than those of core decompression group, normal saline+core decompression group (P<0.05).
(5) Expressions of p-ERK, p-JNK and p-P38protein related with MAPK signal pathway in femur head:after a week, the expressions of p-ERK1and p-ERK2in blank group, core decompression group, normal saline+core decompression group and HSYA+core decompression group were respectively higher, p-JNK and p-P38were respectively lower than those of model control group (P<0.05), and the values of p-ERK1and p-ERK2in HSYA+core decompression group were significantly higher, values of p-JNK and p-P38were respectively lower than those of core decompression group, normal saline+core decompression group (P<0.05).
2HSYA promoting osteogenic differentiation of glucocorticoid-induced rabbit BMSCs and its MAPK signal transduction mechanism through experiments in vitro
(1) Observation of fat drops in BMSCs by Oil Red O staining:in blank group, red lipochondria was not found; in model group, a lot of red fat drops with a shape of adipocyte appeared; fat drops also appeared in HSYA groups, the number in each HSYA group was less than that of model group, the number in HSYA high-dose group was significantly less than that of model group, and less than that of HSYA low-dose group, HSYA middle-dose group respectively.
(2) Observation of BMSCs ultrastructure by transmission electron microscope: after6days, the BMSCs in blank group had oval or irregular shape, the cell had a large nucleus with a little cytoplasm, and the phenomenon of degranulation was not evident. In model group, the cells had spindle shape, the nucleolus was not evident, more mitochondria with big vacuoles appeared in organelles, and more secondary lysosomes or fat drops were found. In HSYA low-dose group, the phenomenon of vacuoles was not evident, the number of injured mitochondria, fat drops was respectively less than that of model group. In HSYA middle-dose group, the number of mitochondria was more than that of HSYA low-dose group, the vacuolar change was decreased, the number of secondary lysosomes was decreased, and the fat drop was not evident. In HSYA high-dose group, the number of mitochondria was increased significantly, the vacuolar change and number of secondary lysosomes were decreased significantly, and the fat drop was not evident.
(3) Determination of levels of ALP and TG in BMSCs:after6days, the level of ALP in model group was significantly lower than that of blank group (P<0.05), in HSYA groups was respectively higher than that of model group (P<0.05), and showed an increasing tendency with the increase of dose. The level of TG in model group was significantly higher than that of blank group (P<0.05), in HSYA groups was respectively lower than that of model group (P<0.05), and showed an decreasing tendency with the increase of dose.
(4) Expressions of Cbfal and Collagen-I mRNA in BMSCs:after6days, the mRNA expressions of Cbfal, Collagen-I in model group was respectively lower than that of blank group (P<0.05), in HSYA groups was respectively higher than that of model group (P<0.05), and showed an increasing tendency with the increase of dose.
(5) Expressions of p-ERK, p-JNK and p-P38protein related with MAPK signal pathway in BMSCs:after6days, the protein expressions of p-ERK1, p-ERK2in model group was respectively lower than that of blank group (P<0.05), in HSYA groups was respectively higher than that of model group (P<0.05), and showed an increasing tendency with the increase of dose; the protein expressions of p-JNK, p-P38in model group was respectively higher than that of blank group (P<0.05), in HSYA groups was respectively lower than that of model group (P<0.05), and showed an decreasing tendency with the increase of dose.
Conclusions
1HSYA can promote the expressions of osteogenic markers including ALP, Cbfal and Collagen-I in BMSCs in femur head marrow cavity of rabbit models with ischemic necrosis of femoral head and isolated rabbits BMSCs. and inhibit the expression of adipogenic marker including TG to inhibit adipogenic differentiation of BMSCs in marrow cavity of femur head and isolated rabbits BMSCs induced by glucocorticoid, and promote their osteogenic differentiation simultaneously.
2HSYA can up-regulate he expression of p-ERK protein associated with proliferation and down-regulate he expression of p-JNK and p-P38protein associated with apoptosis by activating MAPK signal transduction pathway to promote the proliferation of BMSCs in femur head marrow cavity and cultured in vitro, inhibit their apoptosis, and reduce the effects of osteogenic differentiation and apoptosis of BMSCs induced by glucocorticoid.
探讨羟基红花黄色素A(HSYA)促进激素诱导的股骨头缺血坏死模兔股骨头髓内及离体兔骨髓基质细胞(BMSCs)成骨分化及其MAPK信号转导通路的作用机制。
方法
1.健康成年新西兰大白兔48只,抽签法随机分为空白组、模型组,模型组采用臀肌注射醋酸泼尼松龙(7.5mg/kg)结合强迫立位制备激素性股骨头缺血坏死模型。造模成功后抽签法将模型组兔随机分为模型对照组、髓芯减压组、生理盐水+髓芯减压组、HSYA+髓芯减压组,髓芯减压组仅行髓芯减压术,生理盐水+髓芯减压组采用髓芯减压术配合髓腔内注射生理盐水,HSYA+髓芯减压组采用髓芯减压术配合髓腔内注射HSYA,空白组和模型对照组不作治疗。1周后兔麻醉,腹主动脉采血,采用化学比色法、氧化酶法检测各组兔血清中碱性磷酸酶、甘油三脂的含量；取出股骨头,光学显微镜、透射电子显微镜观察各组兔股骨头的形态结构；RT-PCR检测各组兔股骨头中Cbfal及Collagen-I mRNA的表达；Western Blotting检测各组兔股骨头中p-ERK. p-JNK和p-P38蛋白的表达。
2.健康成年新西兰大白兔36只,无菌条件下于胫骨上端内侧骨髓腔穿刺抽取骨髓血,分离、体外培养BMSCs,流式细胞仪检测细胞表面标记物结合成骨、成脂诱导剂诱导对其鉴定。取第3代生长良好的BMSCs,抽签法随机分为空白组,模型组及HSYA低、中、高剂量组。模型组用大剂量地塞米松诱导BMSCs成脂分化,HSYA低、中、高剂量组加入地塞米松的同时分别加入不同浓度的HSYA;空白组无特殊处理。6d后,透射电子显微镜观察各组细胞的形态结构,检测细胞内碱性磷酸酶、甘油三脂的含量,RT-PCR检测细胞中Cbfal及Collagen-I mRNA的表达,Western Blotting检测细胞中p-ERK、p-JNK和p-P38蛋白的表达。
结果
1. HSYA促进激素诱导兔BMSCs成骨分化及其MAPK信号转导机制的在体实验：
(1)血清中成骨标志物碱性磷酸酶、成脂标志物甘油三脂含量的检测：干预1周后,空白组、髓芯减压组、生理盐水+髓芯减压组和HSYA+髓芯减压组兔血清中碱性磷酸酶含量明显高于模型对照组,甘油三脂含量明显低于模型对照组(P<0.05)。其中HSYA+髓芯减压组碱性磷酸酶含量明显高于髓芯减压组和生理盐水+髓芯减压组(P<0.05),甘油三脂含量明显低于髓芯减压组和生理盐水+髓芯减压组(P<0.05)。
(2)光镜观察股骨头组织的病理改变：空白组软骨下骨小梁致密,排列规则整齐；骨细胞清晰,核位于中央,较大；髓腔内可见增生活跃的骨髓组织,造血细胞丰富。模型对照组软骨层变薄、结构紊乱,骨小梁较稀疏,部分有断裂现象,小梁中有骨细胞坏死；软骨下区骨髓内脂肪细胞增多,造血细胞数量减少。髓芯减压组、生理盐水+髓芯减压组、HSYA+髓芯减压组骨细胞结构较清晰,骨小梁较多,坏死骨细胞较模型对照组少,其中尤以HSYA+髓芯减压组最为明显。
(3)透射电镜观察股骨头组织的超微结构：空白组软骨细胞粗面内质网丰富,无明显脱颗粒；线粒体较多,嵴结构清晰,未见明显断裂；染色质分布相对均匀,细胞表面突起较多。模型对照组空泡样变较空白组明显增多,异染色质部分边聚,胞内可见较大脂滴或次级溶酶体。HSYA+髓芯减压组空泡样变明显减少,次级溶酶体明显减少,大部分线粒体嵴存在。
(4)股骨头中成骨标志物Cbfα1及Collagen-Ⅰ mRNA的表达：干预1周后，空白组、髓芯减压组、生理盐水+髓芯减压组和HSYA+髓芯减压组明显高于模型对照组(P<0.05)。其中HSYA+髓芯减压组明显高于髓芯减压组和生理盐水+髓芯减压组(P<0.05)。
(5)股骨头中与MAPK信号通路相关的p-ERK、p-JNK和p-P38蛋白的表达：干预1周后,空白组、髓芯减压组、生理盐水+髓芯减压组和HSYA+髓芯减压组p-ERK1、p-ERK2蛋白表达明显高于模型对照组(P<0.05),p-JNK、p-P38蛋白表达明显低于模型对照组(P<0.05)。其中HSYA+髓芯减压组p-ERK1、p-ERK2蛋白表达明显高于髓芯减压组和生理盐水+髓芯减压组(P<0.05),p-JNK、p-P38蛋白表达明显低于髓芯减压组和生理盐水+髓芯减压组(P<0.05)。
2. HSYA促进激素诱导兔BMSCs成骨分化及其MAPK信号转导机制的离体实验：
(1)油红“O”染色结果：空白组未发现红染的脂肪颗粒；模型组发现细胞的胞浆内可见大量染成红色的脂肪滴,呈诱导后的脂肪细胞形态；HSYA低、中、高剂量组发现有脂肪滴分布,三组红染脂肪滴明显少于模型组,HSYA高剂量组脂肪滴明显少于模型组,少于中、低剂量组。
(2)透射电镜观察BMSCs的超微结构：各组细胞干预6d后,空白组细胞呈椭圆形或不规则状,细胞质少,核大,未见明显脱颗粒现象。模型组细胞呈长梭形,未见明显核仁；细胞器中线粒体较多呈空泡,可见较多次级溶酶体或脂滴。HSYA低剂量组表面空泡不太明显,损伤的线粒体数量较模型组少,脂滴减少。HSYA中剂量组线粒体较低剂量组增多,空泡样变化减少,次级溶酶体减少,未见明显脂滴。HSYA高剂量组线粒体明显增多,空泡样变明显减少,次级溶酶体明显减少,未见明显脂滴。
(3) BMSCs中成骨标志物碱性磷酸酶、成脂标志物甘油三脂含量的检测：各组细胞干预6d后,模型组BMSCs内碱性磷酸酶含量较空白组明显下降(P<0.05),而HSYA各组较模型组均有明显升高(P<0.05),且随着剂量的加大,碱性磷酸酶含量呈上升趋势。模型组BMSCs内甘油三脂含量较空白组明显升高(P<0.05),而HSYA各组较模型组均有明显降低(P<0.05),且随着HSYA剂量的加大,甘油三脂含量呈下降趋势。
(4) BMSCs中成骨标志物Cbfα1及Collagen-I mRNA的表达：各组细胞干预6d后,模型组细胞内Cbfα1、Collagen-Ⅰ mRNA含量均明显低于空白组(P<0.05),而HSYA各组Cbfα1、Collagen-Ⅰ mRNA含量均明显高于模型组(P<0.05),且随着HSYA剂量的增加,细胞内Cbfα1、Collagen-Ⅰ mRNA含量呈上升趋势。
(5) BMSCs中与MAPK信号通路相关的p-ERK、p-JNK和p-P38蛋白的表达：各组细胞干预6d后,模型组细胞内p-ERK1、p-ERK2蛋白表达明显低于空白组(P<0.05),p-JNK、p-P38蛋白表达明显高于空白组(P<0.05),而HSYA各组p-ERK1、 P-ERK2蛋白表达明显高于模型组(P<0.05),p-JNK、p-P38蛋白表达明显低于模型组(P<0.05),且随着HSYA剂量的增加,细胞内p-ERK1、p-ERK2蛋白表达呈上升趋势,p-JNK、p-P38蛋白表达呈下降趋势。
结论
1.羟基红花黄色素A(HSYA)能促进激素性股骨头缺血坏死模兔股骨头髓内及离体兔BMSCs内成骨标志物碱性磷酸酶、Cbfα1、Collagen-Ⅰ的表达,抑制成脂标志物甘油三脂的表达,抑制激素诱导股骨头髓内及离体兔BMSCs成脂分化,促使其成骨分化。
2. HSYA通过激活MAPK信号转导通路,上调通路中与增殖关联的p-ERK蛋白的表达,下调凋亡相关联的p-JNK、p-P38蛋白的表达,促进兔股骨头髓内及离体兔BMSCs增殖,抑制其凋亡,减轻激素诱导的BMSCs凋亡的影响。
Objective
To observe the promoting effect of Hydroxy safflower yellow A (HSYA) on osteogenic differentiation of glucocorticoid-induced rabbit bone marrow stroma cells (BMSCs) in femur head marrow cavity of rabbit models with ischemic necrosis of femoral head and isolated glucocorticoid-induced rabbits BMSCs, and explore the MAPK signal transduction mechanism.
Methods
148New Zealand adult white rabbits were randomly divided into blank group and model group. Prednisolone acetate (7.5mg/kg) was injected into the rabbits of model group using intragluteal injection to induce models of ischemic necrosis of femoral head. After successful modeling, rabbits in model group were divided into model control group, core decompression group, normal saline+core decompression group and HSYA+core decompression group. Core decompression group received core decompression, normal saline+core decompression group received core decompression and intramedullary injection with normal saline, HSYA+core decompression group received core decompression and intramedullary injection with HSYA, and normal group and model control group didn't accept any treatment. After a week, the rabbits were anesthetized, and the blood of abdominal aorta was obtained. The levels of alkaline phosphatase (ALP) and triglyceride (TG) in rabbit serum were detected by chemical colorimetry and oxidase method. The femoral heads were taken off, their morphology was observed by optical microscope and transmission electron microscope, the mRNA expressions of Cbfal and Collagen-I were detected by RT-PCR assay, and the protein expressions of p-ERK, p-JNK and p-P38were detected by Western Blotting.
236New Zealand adult white rabbits were used in this experiment, and the bone marrow blood was drawn from the medial aspect of proximal tibia, then the BMSCs were isolated and cultured in vitro. The cell surface markers were detected by flow cytometry and the cells were induced by adipogenic and osteogenic inducers to identify the cells. The3rd generation of BMSCs which had good growing status were randomly divided into blank group, model group and HSYA groups with different doses. The model group was treated with high dose of dexamethasone to induce adipogenic differentiation of BMSCs, the HSYA groups received high dose of dexamethasone and different concentrations of HSYA simultaneously, and the blank group did not receive any special handling. After6days, the morphology of cells was observed by fluorescence inverted phase contrast microscope and transmission electron microscope, the levels of ALP and TG in BMSCs were detected, the mRNA expressions of Cbfal and Collagen-I were detected by RT-PCR assay, and the protein expressions of p-ERK, p-JNK and p-P38were detected by Western Blotting.
Results
1HSYA promoting osteogenic differentiation of glucocorticoid-induced rabbit BMSCs and its MAPK signal transduction mechanism through experiments in vivo
(1) Determination of levels of serum ALP and TG:after a week, the level of serum ALP in blank group, core decompression group, normal saline+core decompression group and HSYA+core decompression group was respectively higher than that of model control group (P<0.05), and the level of serum TG was respectively lower than that of model control group (P<0.05). The level of serum ALP in HSYA+core decompression group was significantly higher than that of core decompression group, normal saline+core decompression group (P<0.05), and the level of serum TG was significantly lower than that of core decompression group, normal saline+core decompression group (P<0.05).
(2) Observation of femoral head pathologic change by light microscope:the bone trabecula of cartilage in blank group was compact, and arranged in line; the osteocytes were clearly seen, and the nucleus which located in the center were big; hyperplastic myeloid tissues were found in marrow cavity, and the hematopoietic cells were present in great number.The structural disorder of cartilage in model control group was observed, bone trabecula were rare, some occurred disrupted, and necrotic osteocytes were found in trabecula; a increase of adipocytes and decrease of hematopoietic cells were seen in bone marrow. The cellular structures were clear in core decompression group, normal saline+core decompression group and HSYA+core decompression group, and the bone trabecula was more and necrotic osteocyte was less than those of model control group, and the phenomenon was the most significant in HSYA+core decompression group.
(3) Observation of femoral head ultra-structure by transmission electron microscope:more rough endoplasmic reticulums were found in chondrocytes of blank group, and the degranulation of cells was not significant; the structure of cristae was clear without significant disruption; the chromatin was uniformly distributed, and more cell surface projections were found. In model control group, more cavitations were found than those of blank group, the heterochromatin was condensed and marginated, and more lipid droplets and secondary lysosomes appeared. In HSYA+core decompression group, less vacuoles and secondary lysosomes were found, and most of crista mitochondriales still appeared.
(4) Expressions of Cbfal and Collagen-I mRNA in femur head:after a week, the mRNA expressions of Cbfal and Collagen-I in blank group, core decompression group, normal saline+core decompression group and HSYA+core decompression group were respectively higher than that of model control group (P<0.05), and the values in HSYA+core decompression group were significantly higher than those of core decompression group, normal saline+core decompression group (P<0.05).
(5) Expressions of p-ERK, p-JNK and p-P38protein related with MAPK signal pathway in femur head:after a week, the expressions of p-ERK1and p-ERK2in blank group, core decompression group, normal saline+core decompression group and HSYA+core decompression group were respectively higher, p-JNK and p-P38were respectively lower than those of model control group (P<0.05), and the values of p-ERK1and p-ERK2in HSYA+core decompression group were significantly higher, values of p-JNK and p-P38were respectively lower than those of core decompression group, normal saline+core decompression group (P<0.05).
2HSYA promoting osteogenic differentiation of glucocorticoid-induced rabbit BMSCs and its MAPK signal transduction mechanism through experiments in vitro
(1) Observation of fat drops in BMSCs by Oil Red O staining:in blank group, red lipochondria was not found; in model group, a lot of red fat drops with a shape of adipocyte appeared; fat drops also appeared in HSYA groups, the number in each HSYA group was less than that of model group, the number in HSYA high-dose group was significantly less than that of model group, and less than that of HSYA low-dose group, HSYA middle-dose group respectively.
(2) Observation of BMSCs ultrastructure by transmission electron microscope: after6days, the BMSCs in blank group had oval or irregular shape, the cell had a large nucleus with a little cytoplasm, and the phenomenon of degranulation was not evident. In model group, the cells had spindle shape, the nucleolus was not evident, more mitochondria with big vacuoles appeared in organelles, and more secondary lysosomes or fat drops were found. In HSYA low-dose group, the phenomenon of vacuoles was not evident, the number of injured mitochondria, fat drops was respectively less than that of model group. In HSYA middle-dose group, the number of mitochondria was more than that of HSYA low-dose group, the vacuolar change was decreased, the number of secondary lysosomes was decreased, and the fat drop was not evident. In HSYA high-dose group, the number of mitochondria was increased significantly, the vacuolar change and number of secondary lysosomes were decreased significantly, and the fat drop was not evident.
(3) Determination of levels of ALP and TG in BMSCs:after6days, the level of ALP in model group was significantly lower than that of blank group (P<0.05), in HSYA groups was respectively higher than that of model group (P<0.05), and showed an increasing tendency with the increase of dose. The level of TG in model group was significantly higher than that of blank group (P<0.05), in HSYA groups was respectively lower than that of model group (P<0.05), and showed an decreasing tendency with the increase of dose.
(4) Expressions of Cbfal and Collagen-I mRNA in BMSCs:after6days, the mRNA expressions of Cbfal, Collagen-I in model group was respectively lower than that of blank group (P<0.05), in HSYA groups was respectively higher than that of model group (P<0.05), and showed an increasing tendency with the increase of dose.
(5) Expressions of p-ERK, p-JNK and p-P38protein related with MAPK signal pathway in BMSCs:after6days, the protein expressions of p-ERK1, p-ERK2in model group was respectively lower than that of blank group (P<0.05), in HSYA groups was respectively higher than that of model group (P<0.05), and showed an increasing tendency with the increase of dose; the protein expressions of p-JNK, p-P38in model group was respectively higher than that of blank group (P<0.05), in HSYA groups was respectively lower than that of model group (P<0.05), and showed an decreasing tendency with the increase of dose.
Conclusions
1HSYA can promote the expressions of osteogenic markers including ALP, Cbfal and Collagen-I in BMSCs in femur head marrow cavity of rabbit models with ischemic necrosis of femoral head and isolated rabbits BMSCs. and inhibit the expression of adipogenic marker including TG to inhibit adipogenic differentiation of BMSCs in marrow cavity of femur head and isolated rabbits BMSCs induced by glucocorticoid, and promote their osteogenic differentiation simultaneously.
2HSYA can up-regulate he expression of p-ERK protein associated with proliferation and down-regulate he expression of p-JNK and p-P38protein associated with apoptosis by activating MAPK signal transduction pathway to promote the proliferation of BMSCs in femur head marrow cavity and cultured in vitro, inhibit their apoptosis, and reduce the effects of osteogenic differentiation and apoptosis of BMSCs induced by glucocorticoid.
引文
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