Mechanism of Yi Ceng Application in Alleviating Synovial Fibrosis Induced by Transforming Growth Factor-β1 in Rats with Knee Osteoarthritis Based on PI3K/AKT/HIF-1α Signal Pathway
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摘要:
目的 探讨易层敷贴对膝骨关节炎(KOA)大鼠滑膜纤维化TGF-β1、α-SMA、COL1A1表达的影响以及对PI3K/AKT/HIF-1α信号通路的调控作用。 方法 将30只SPF级SD大鼠随机分为空白组、模型组和易层组, 膝关节腔注射200 ng转化生长因子-β1重组蛋白(TGF-β1)建立膝关节滑膜纤维化动物模型, 2 d 1次, 持续3次, 造模14 d后给予易层敷贴外用治疗28 d后取滑膜组织。HE和Masson染色观察滑膜病理变化; 免疫组化检测滑膜p-PI3K、p-AKT、HIF-1α、TGF-β1、α-SMA、COL1A1的表达水平。提取雄性SD大鼠膝关节成纤维样滑膜细胞(FLSs), 以10 ng·mL-1 TGF-β1诱导24 h建立KOA滑膜纤维化细胞模型, 易层冻干粉干预24 h, Western blot检测PI3K、p-PI3K、AKT、p-AKT、HIF-1α、TGF-β1、α-SMA、COL1A1的蛋白表达, qPCR检测HIF-1α、TGF-β1、α-SMA、COL1A1 mRNA表达。 结果 与空白组相比, 模型组HE染色滑膜炎加重(P < 0.01), Masson染色滑膜纤维化占比显著增加(P < 0.01), 免疫组化中p-PI3K、p-AKT、HIF-1α、TGF-β1、α-SMA、COL1A1表达均增加(P < 0.01);滑膜细胞中p-PI3K/PI3K、p-AKT/AKT、HIF-1α、TGF-β1、α-SMA、COL1A1蛋白表达均升高(P < 0.05, P < 0.01), HIF-1α、TGF-β1、α-SMA、COL1A1 mRNA表达均上升(P < 0.01)。与模型组相比, 易层组滑膜炎和滑膜纤维化状况改善, p-PI3K、p-AKT、HIF-1α、TGF-β1、α-SMA、COL1A1的表达均减少(P < 0.05, P < 0.01);滑膜细胞中p-PI3K/PI3K、p-AKT/AKT、HIF-1α、TGF-β1、α-SMA、COL1A1蛋白表达均降低(P < 0.05), HIF-1α、TGF-β1、α-SMA、COL1A1 mRNA表达均下调(P < 0.05, P < 0.01)。 结论 易层敷贴通过调控PI3K/AKT/HIF-1α信号通路, 降低TGF-β1、α-SMA、COL1A1的表达, 有效改善TGF-β1诱导的KOA大鼠滑膜纤维化。 -
关键词:
- 膝骨关节炎 /
- 滑膜纤维化 /
- PI3K/AKT/HIF-1α信号通路 /
- 易层敷贴
Abstract:OBJECTIVE To investigate the effect of Yi Ceng application on the expression of TGF-β1, α-SMA and COL1A1 in synovial fibrosis of rats with knee osteoarthritis (KOA) and the regulation of PI3K/AKT/HIF-1α signal pathway. METHODS 30 SPF SD rats were randomly divided into blank group, model group and Yi Ceng group. The animal model of synovial fibrosis of knee joint was established with 200 ng transforming growth factor-β1 (TGF-β1) recombinant protein, once every 2 days, lasting for 3 times. After 14 days of modeling, the synovial tissue was obtained after the external application of Yi Ceng for 28 days. HE and Masson staining were used to observe the pathological changes of synovium. The expression of p-PI3K, p-AKT, HIF-1α, TGF-β1, α-SMA and COL1A1 in synovium was detected by immunohistochemistry. Fibroblast-like synoviocytes (FLSs) were isolated from the knee joint of male SD rats and induced with 10 ng·mL-1 TGF-β1 for 24 h to establish KOA synovial fibrosis cell model, then intervened with Yi Ceng freeze-dried powder for 24 h. PI3K, p-PI3K, AKT, p-AKT, HIF-1α, TGF-β1, α-SMA and COL1A1 protein expressions in synoviocytes were detected by Western blot. HIF-1α, TGF-β1, α-SMA and COL1A1 mRNA expressions in synoviocytes were detected by qPCR. RESULTS Compared with the control group, the synovitis and synovial fibrosis of the model group were significantly aggravated (P < 0.01) in HE and Masson staining, and the expressions of p-PI3K, p-AKT, HIF-1α, TGF-β1, α-SMA and COL1A1 were increased (P < 0.01). The protein expressions of p-PI3K/PI3K, p-AKT/AKT, HIF-1α, TGF-β1, α-SMA and COL1α1 in synoviocytes were increased (P < 0.05, P < 0.01), and the mRNA expressions of HIF-1α, TGF-β1, α-SMA and COL1A1 in synoviocytes were increased (P < 0.01). Compared with the model group, synovitis and synovial fibrosis were improved, and the expression levels of p-PI3K, p-AKT, HIF-1α, TGF-β1, α-SMA and COL1A1 were decreased in the Yi Ceng group (P < 0.05, P < 0.01).The protein expressions of p-PI3K/PI3K, p-AKT/AKT, HIF-1α, TGF-β1, α-SMA and COL1A1 were decreased (P < 0.05), and mRNA levels of HIF-1α, TGF-β1, α-SMA and COL1A1 were downregulated (P < 0.05, P < 0.01). CONCLUSION Yi Ceng application can effectively improve the synovial fibrosis induced by recombinant TGF-β1 in KOA rats by decreasing the expression of TGF-β1, α-SMA and COL1A1 via the PI3K/AKT/HIF-1α signal pathway. -
图 1 各组大鼠滑膜HE染色和Masson染色结果(×200)
注: 与空白组比较, * *P < 0.01;与模型组比较, ##P < 0.01。x±s, n=4。
Figure 1. HE staining and Masson staining results of synovium in each group (×200)
图 2 各组大鼠滑膜免疫组化结果(×200)
注: 与空白组比较, * *P < 0.01;与模型组比较, #P < 0.05, ##P < 0.01。x±s, n=3。
Figure 2. Immunohistochemistry results of synovium in each group (×200)
图 3 易层冻干粉对大鼠滑膜细胞的活性检测(A)和FLSs鉴定(B)
注: 与对照孔(0 μg·mL-1)比较, *P < 0.05。
Figure 3. Detection of activity of Yi Ceng freeze-dried powder on synovial cells in rats (A) and FLSs identification (B)
图 4 各组大鼠滑膜细胞蛋白Western blot检测结果
注: 与空白组比较, * *P < 0.01;与模型组比较, #P < 0.05。x±s, n=3。
Figure 4. Western blot results of synovial cell proteins of rats in each group
图 5 各组大鼠滑膜细胞mRNA表达情况
注: 与空白组比较, * *P < 0.01;与模型组比较, #P < 0.05, ##P < 0.01。x±s, n=3。
Figure 5. mRNA expression in synovial cells of rats in each group
表 1 目的基因序列
Table 1. Sequence of target genes
基因 序列(5′→3′) HIF-1α Forward
ReverseAACCCATCAACTCAGTAATC
CTACAAGAAACCGCCTATTGF-β1 Forward
ReverseCTCAGGCGTATCAGTGGG
ATGGTGGACCGCAACAACα-SMA Forward
ReverseGCTGTTATAGGTGGTTTCG
ACTGCTGCTTCCTCTTCTTCOL1A1 Forward
ReverseCCAGGGAGACCGTTGAGT
ACAAGGTGACAGAGGCATAAAβ-actin Forward
ReverseGAGAGGGAAATCGTGCGT
GGAGGAAGAGGATGCGG
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