Effects and mechanism of astragalus polysaccharide on wound healing of deep partial-thickness burns in rats
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摘要:
目的 探讨黄芪多糖对深Ⅱ度烧伤大鼠创面愈合的影响及其机制。 方法 采用实验研究方法。取50只雄性7周龄SD大鼠，按照随机数字表法分为正常组、单纯烧伤组、黄芪多糖组、抑制剂组和抑制剂+黄芪多糖组，每组10只。除正常组外，其余4组大鼠均在背部制作1个深Ⅱ度烧伤创面。正常组、单纯烧伤组大鼠腹腔注射生理盐水，其余3组大鼠分别注射黄芪多糖和/或整合素连接激酶（ILK）抑制剂。注射7 d后，计算4组烧伤大鼠创面愈合率，采用酶联免疫吸附测定（ELISA）法检测5组大鼠血清中γ干扰素、白细胞介素2（IL-2）和肿瘤坏死因子α（TNF-α）含量。取正常组大鼠正常皮肤组织及4组烧伤大鼠创面组织，测定水分并计算水分占比，采用ELISA法检测γ干扰素、IL-2和TNF-α的含量，采用实时荧光定量反转录PCR法检测表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）以及ILK mRNA表达量，采用蛋白质印迹法检测ILK、蛋白激酶B（Akt）、磷酸化Akt（p-Akt）、糖原合成激酶-3β（GSK-3β）及磷酸化GSK-3β（p-GSK-3β）蛋白表达量。对数据行单因素方差分析、LSD检验。 结果 注射7 d后，黄芪多糖组大鼠创面愈合率为（67±5）%，明显高于单纯烧伤组的（52±4）%和抑制剂+黄芪多糖组的（59±5）%（ P值均<0.05）；抑制剂+黄芪多糖组大鼠创面愈合率明显高于抑制剂组的（48±4）%（ P<0.05）。注射7 d后，与正常组大鼠血清或正常皮肤组织比较，单纯烧伤组大鼠血清中γ干扰素、TNF-α、IL-2含量和创面组织水分占比均明显升高（ P<0.05）；与黄芪多糖组比较，单纯烧伤组和抑制剂+黄芪多糖组大鼠血清中γ干扰素、TNF-α、IL-2含量及创面组织水分占比均明显升高（ P<0.05）；与抑制剂组比较，抑制剂+黄芪多糖组大鼠血清中γ干扰素、TNF-α、IL-2含量及创面组织水分占比均明显降低（ P<0.05）。注射7 d后，与正常组大鼠正常皮肤组织比较，单纯烧伤组大鼠创面组织中γ干扰素、TNF-α和IL-2含量均明显升高（ P<0.05）；与黄芪多糖组比较，单纯烧伤组和抑制剂+黄芪多糖组大鼠创面组织中γ干扰素、TNF-α和IL-2含量均明显升高（ P<0.05）；与抑制剂组比较，抑制剂+黄芪多糖组大鼠创面组织中γ干扰素、TNF-α和IL-2含量均明显降低（ P<0.05）。注射7 d后，与正常组大鼠正常皮肤组织比较，单纯烧伤组大鼠创面组织中EGF、bFGF、ILK mRNA表达量及ILK、p-Akt、p-GSK-3β蛋白表达量均明显升高（ P<0.05）；与黄芪多糖组比较，单纯烧伤组和抑制剂+黄芪多糖组大鼠创面组织中EGF、bFGF和ILK mRNA表达量及ILK、p-Akt、p-GSK-3β蛋白表达量均明显降低（ P<0.05）；与抑制剂组比较，抑制剂+黄芪多糖组大鼠创面组织中EGF、bFGF和ILK mRNA表达量以及ILK、p-Akt、p-GSK-3β蛋白表达量均明显升高（ P<0.05）。正常组大鼠正常皮肤组织以及4组烧伤大鼠创面组织中Akt和GSK-3β蛋白表达量比较，差异均无统计学意义（ P>0.05）。 结论 黄芪多糖可能通过激活ILK/Akt/GSK-3β信号通路，减轻深Ⅱ度烧伤大鼠全身和局部炎症反应，减轻组织水肿，促进愈合因子表达，从而促进创面愈合。 Abstract:Objective To investigate the effects and mechanism of astragalus polysaccharide (APS) on wound healing of deep partial-thickness burns in rats. Methods The experimental study method was used. Fifty 7-week-old male Sprague-Dawley rats were divided into normal group, simple burn group, APS group, inhibitor group, and inhibitor+APS group according to the random number table, with 10 rats in each group. Except for normal group, rats in the other 4 groups were inflicted with a deep partial-thickness burn wound on the back. Rats in normal group and simple burn group were intraperitoneally injected with normal saline, and rats in the other three groups were injected with APS and/or integrin-linked kinase (ILK) inhibitor, respectively. After 7 days of injection, the wound healing rate of rats with burns in the four groups was calculated, and the serum content of interferon-γ, interleukin-2 (IL-2), and tumor necrosis factor α (TNF-α) in rats in 5 groups was determined by enzyme-linked immunosorbent assay (ELISA). The normal skin tissue of rats in normal group and wound tissue of rats with burns in the four groups were taken, the water content was determined and the water ratio was calculated, the content of interferon-γ, IL-2, and TNF-α was detected by ELISA, the mRNA expressions of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and ILK were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction, and the protein expressions of ILK, protein kinase B (Akt), phosphorylated Akt (p-Akt), glycogen synthetic kinase-3β (GSK-3β), and phosphorylated GSK-3β (p-GSK-3β) were detected by Western blotting. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results After 7 days of injection, the wound healing rate of rats in APS group was (67±5)%, which was significantly higher than (52±4)% in simple burn group and (59±5)% in inhibitor+APS group (with all the P values <0.05). The wound healing rate of rats in inhibitor+APS group was significantly higher than (48±4)% in inhibitor group ( P<0.05). After 7 days of injection, compared with those in serum or normal skin tissue of rats in normal group, the serum content of interferon-γ, TNF-α, IL-2 and the water ratio of wound tissue of rats in simple burn group were significantly increased ( P<0.05); compared with those in APS group, the serum content of interferon-γ, TNF-α, IL-2 and the water ratio of wound tissue of rats in simple burn group and inhibitor+APS group were significantly increased ( P<0.05); compared with those in inhibitor group, the serum content of interferon-γ, TNF-α, IL-2 and the water ratio of wound tissue of rats in inhibitor+APS group were significantly decreased ( P<0.05). After 7 days of injection, compared with that in normal skin tissue of rats in normal group, the content of interferon-γ, TNF-α, and IL-2 in wound tissue of rats in simple burn group was significantly increased ( P<0.05); compared with that in APS group, the content of interferon-γ, TNF-α, and IL-2 in wound tissue of rats in simple burn group and inhibitor+APS group was significantly increased ( P<0.05); compared with that in inhibitor group, the content of interferon-γ, TNF-α, and IL-2 in wound tissue of rats in inhibitor+APS group was significantly decreased ( P<0.05). After 7 days of injection, compared with those in normal skin tissue of rats in normal group, the mRNA expressions of EGF, bFGF, ILK and protein expressions of ILK, p-Akt, p-GSK-3β in wound tissue of rats in simple burn group were significantly increased ( P<0.05); compared with those in APS group, the mRNA expressions of EGF, bFGF, ILK and protein expressions of ILK, p-Akt, p-GSK-3β in wound tissue of rats in simple burn group and inhibitor+APS group were significantly decreased ( P<0.05); compared with those in inhibitor group, the mRNA expressions of EGF, bFGF, ILK and protein expressions of ILK, p-Akt, p-GSK-3β in wound tissue of rats in inhibitor+APS group were significantly increased ( P<0.05). There were no statistically significant differences in the protein expressions of Akt and GSK-3β in normal skin tissue of rats in normal group and wound tissue of rats with burns in the four groups ( P>0.05). Conclusions APS can alleviate systemic and local inflammation, alleviate tissue edema, and promote the expressions of healing factors in rats with deep partial-thickness burns, thus to promote the wound healing, possibly by activating ILK/Akt/GSK-3β signaling pathway. -
Key words:
- Burns /
- Inflammation /
- Wound healing /
- Astragalus polysaccharide /
- Integrin-linked kinase
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参考文献
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1 正常组大鼠及4组深Ⅱ度烧伤大鼠注射7 d后血清中γ干扰素、TNF-α和IL-2含量比较（样本数均为10，
注：正常组和单纯烧伤组注射生理盐水，其余3组分别注射黄芪多糖和/或整合素连接激酶抑制剂QLT0267；5组γ干扰素、肿瘤坏死因子α（TNF-α）和白细胞介素2（IL-2）含量总体比较，差异均有统计学意义（F值分别为230.27、54.97、69.60，P<0.001）；与正常组比较，aP<0.05；与单纯烧伤组比较，bP<0.05；与黄芪多糖组比较，cP<0.05；与抑制剂组比较，dP<0.05
2 蛋白质印迹法检测的注射7 d后正常组大鼠正常皮肤组织及4组深Ⅱ度烧伤大鼠创面组织中ILK/Akt/GSK-3β信号通路相关蛋白表达量比较（样本数均为10，
注：正常组和单纯烧伤组注射生理盐水，其余3组分别注射黄芪多糖和/或整合素连接激酶抑制剂QLT0267；ILK为整合素连接酶，Akt为蛋白激酶B，p-Akt为磷酸化Akt，GSK-3β为糖原合成激酶-3β，p-GSK-3β为磷酸化GSK-3β；图2A中1、2、3、4、5分别指示正常组、单纯烧伤组、黄芪多糖组、抑制剂组和抑制剂+黄芪多糖组；5组ILK、p-Akt、p-GSK-3β蛋白表达量总体比较，差异均有统计学意义（F值分别为302.47、443.27、651.13，P<0.001）；5组Akt、GSK-3β蛋白表达量总体比较，差异均无统计学意义（F值分别为0.87、1.43，P值分别为0.491、0.240）；与正常组比较，aP<0.05；与单纯烧伤组比较，bP<0.05；与黄芪多糖组比较，cP<0.05；与抑制剂组比较，dP<0.05
表1 采用实时荧光定量反转录PCR法检测创面愈合因子mRNA表达的引物序列及产物大小
基因名称 引物序列（5′→3′） 产物大小（bp） EGF 上游 ： ATCCGGCTCATTAGGTTA155 下游 ： ACCACAACTCTCTACGCAbFGF 上游 ： GCAAGTGTGCAACCTCAA184 下游 ： CCACTGAGCAATCCTATTILK 上游 ： ACGACATTTTCACTCAGTGCC199 下游 ： TCATCCCCACGATTCATCACATGAPDH 上游 ： TCCAGTATGACAAGATCT105 下游 ： GGAACCACTATTGCGGCA注：EGF为表皮生长因子，bFGF为碱性成纤维细胞生长因子，ILK为整合素连接激酶，GAPDH为3-磷酸甘油醛脱氢酶 
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表2 注射7 d后正常组大鼠正常皮肤组织及4组深Ⅱ度烧伤大鼠创面组织中炎症因子含量比较（pg/mL，
组别 样本数 γ干扰素 TNF-α IL-2 正常组 10 12±3 48±6 82±9 单纯烧伤组 10 87±5 a 137±9 a 190±18 a 黄芪多糖组 10 34±4 b 82±9 b 106±12 b 抑制剂组 10 94±4 201±11 232±19 抑制剂+黄芪多糖组 10 52±6 cd 121±10 cd 163±16 cd F值 534.71 384.58 160.76 P值 <0.001 <0.001 <0.001 注：正常组和单纯烧伤组注射生理盐水，其余3组分别注射黄芪多糖和/或整合素连接激酶抑制剂QLT0267；TNF-α为肿瘤坏死因子α，IL-2为白细胞介素2；与正常组比较， a P<0.05；与单纯烧伤组比较， b P<0.05；与黄芪多糖组比较， c P<0.05；与抑制剂组比较， d P<0.05
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表3 注射7 d后正常组大鼠正常皮肤组织及4组深Ⅱ度烧伤大鼠创面组织中愈合因子mRNA表达量比较（
组别 样本数 EGF bFGF ILK 正常组 10 0.35±0.10 0.12±0.09 0.44±0.12 单纯烧伤组 10 0.47±0.10 a 0.38±0.11 a 0.63±0.11 a 黄芪多糖组 10 0.69±0.12 b 0.58±0.13 b 0.84±0.14 b 抑制剂组 10 0.26±0.14 0.24±0.13 0.35±0.11 抑制剂+黄芪多糖组 10 0.57±0.13 cd 0.45±0.14 cd 0.71±0.13 cd F值 20.98 15.16 26.39 P值 <0.001 <0.001 <0.001 注：正常组和单纯烧伤组注射生理盐水，其余3组分别注射黄芪多糖和/或整合素连接激酶抑制剂QLT0267；EGF为表皮生长因子，bFGF为碱性成纤维细胞生长因子，ILK为整合素连接激酶；与正常组比较， a P<0.05；与单纯烧伤组比较， b P<0.05；与黄芪多糖组比较， c P<0.05；与抑制剂组比较， d P<0.05
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