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[1]郭運嶺,李蕊,傅聰,等.基于Hippo-YAP信號通路探討黃芩苷干預(yù)膝骨關(guān)節(jié)炎大鼠的療效和作用機制[J].中醫(yī)正骨,2024,36(03):15-22.
　GUO Yunling,LI Rui,FU Cong,et al.Outcome and mechanism of baicalin against knee osteoarthritis in rats:a Hippo-YAP signaling pathway-based experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2024,36(03):15-22.
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基于Hippo-YAP信號通路探討黃芩苷干預(yù)膝骨關(guān)節(jié)炎大鼠的療效和作用機制()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第36卷
期數(shù):
2024年03期
頁碼:
15-22
欄目:
基礎(chǔ)研究
出版日期:
2024-03-20

文章信息/Info

Title:
Outcome and mechanism of baicalin against knee osteoarthritis in rats:a Hippo-YAP signaling pathway-based experimental study
作者:
郭運嶺1; 李蕊2; 傅聰1; 褚昆3; 李會杰3
1.秦皇島市中醫(yī)醫(yī)院,河北 秦皇島 066000; 2.秦皇島市第一醫(yī)院,河北 秦皇島 066000; 3.河北醫(yī)科大學第三醫(yī)院,河北 石家莊 050051
Author(s):
GUO Yunling1; LI Rui2; FU Cong1; CHU Kun3; LI Huijie3
1.Qinhuangdao Hospital of Traditional Chinese Medicine,Qinhuangdao 066000,Hebei,China 2.The First Hospital of Qinhuangdao,Qinhuangdao 066000,Hebei,China 3.The Third Hospital of Hebei Medical University,Shijiazhuang 050051,Hebei,China
關(guān)鍵詞:
骨關(guān)節(jié)炎; 膝;  黃芩苷;  Hippo信號通路;  YAP-信號蛋白;  大鼠;  動物實驗
Keywords:
osteoarthritis; knee;  baicalin;  Hippo signaling pathway;  YAP-signaling proteins;  rats;  animal experimentation
摘要:
目的:探討黃芩苷干預(yù)膝骨關(guān)節(jié)炎(knee osteoarthritis,KOA)大鼠的療效和作用機制。方法:將50只大鼠隨機分為假手術(shù)組、模型組、低劑量黃芩苷組、高劑量黃芩苷組、高劑量黃芩苷聯(lián)合抑制劑組,每組10只。將假手術(shù)組以外的大鼠采用切斷前交叉韌帶和切除內(nèi)側(cè)半月板的方法建立右膝關(guān)節(jié)KOA模型,假手術(shù)組大鼠于右膝關(guān)節(jié)內(nèi)側(cè)做一切口后縫合。造模成功后,低劑量黃芩苷組、高劑量黃芩苷組大鼠分別按照50 mg·kg-1、100 mg·kg-1的劑量給予黃芩苷生理鹽水溶液灌胃; 高劑量黃芩苷聯(lián)合抑制劑組大鼠按照100 mg·kg-1的劑量給予黃芩苷生理鹽水溶液灌胃,并按照1 mg·kg-1的劑量給予XMU-MP-1溶液腹腔注射; 假手術(shù)組和模型組大鼠均給予等量生理鹽水灌胃。每天給藥1次,連續(xù)給藥30 d。分別于給藥前、給藥15 d時、給藥30 d時測量大鼠雙側(cè)膝關(guān)節(jié)腫脹程度。給藥結(jié)束后處死大鼠,取大鼠右側(cè)膝關(guān)節(jié)軟骨組織,采用蘇木精-伊紅染色和番紅O-固綠染色觀察膝關(guān)節(jié)軟骨組織病理變化,采用Mankin評分標準評價軟骨退變情況,采用ELISA試劑盒檢測腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)、白細胞介素(interleukin,IL)-1β、IL-10、基質(zhì)金屬蛋白酶(matrix metalloproteinase,MMP)-13表達水平,采用Western blotting檢測滑膜組織中切割活化的半胱氨酸天冬氨酸蛋白酶(Cleaved-cysteine aspartic acid specific protease,Cleaved-Caspase)-3、B淋巴細胞瘤-2相關(guān)X蛋白(Bcl2 associated X protein,Bax)、B淋巴細胞瘤-2(B-cell lymphoma-2,Bcl-2)蛋白、Yes相關(guān)蛋白(Yes-associated protein,YAP)、Tafazzin(TAZ)蛋白的表達水平。結(jié)果:①大鼠右膝關(guān)節(jié)腫脹程度。給藥15 d時和給藥30 d時,高劑量黃芩苷組大鼠右膝關(guān)節(jié)腫脹程度均低于模型組、低劑量黃芩苷組和高劑量黃芩苷聯(lián)合抑制劑組(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000); 模型組、低劑量黃芩苷組、高劑量黃芩苷聯(lián)合抑制劑組大鼠右膝關(guān)節(jié)腫脹程度兩兩比較,差異均無統(tǒng)計學意義(P=0.063,P=0.215,P=0.399; P=0.052,P=0.261,P=0.240)。②大鼠右膝關(guān)節(jié)軟骨組織病理變化。干預(yù)結(jié)束后,模型組大鼠右膝關(guān)節(jié)軟骨萎縮、排列混亂; 低劑量黃芩苷組、高劑量黃芩苷組、高劑量黃芩苷聯(lián)合抑制劑組大鼠右膝關(guān)節(jié)軟骨萎縮、細胞排序情況均較模型組改善,且高劑量黃芩苷組大鼠的改善情況優(yōu)于低劑量黃芩苷組和高劑量黃芩苷聯(lián)合抑制劑組。③大鼠右膝關(guān)節(jié)軟骨Mankin評分。干預(yù)結(jié)束后,低劑量黃芩苷組、高劑量黃芩苷組、高劑量黃芩苷聯(lián)合抑制劑組大鼠右膝關(guān)節(jié)軟骨Mankin評分均低于模型組(P=0.000,P=0.000,P=0.000),高劑量黃芩苷組右膝關(guān)節(jié)軟骨Mankin評分低于低劑量黃芩苷組和高劑量黃芩苷聯(lián)合抑制劑組(P=0.000,P=0.000),低劑量黃芩苷組大鼠右膝關(guān)節(jié)軟骨Mankin評分低于高劑量黃芩苷聯(lián)合抑制劑組(P=0.000)。④大鼠右膝關(guān)節(jié)軟骨組織中TNF-α、IL-1β、IL-10、MMP-13表達水平。低劑量黃芩苷組、高劑量黃芩苷組、高劑量黃芩苷聯(lián)合抑制劑組大鼠右膝關(guān)節(jié)軟骨組織中TNF-α、IL-1β、MMP-13表達水平均低于模型組(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000),IL-10表達水平均高于模型組(P=0.000,P=0.000,P=0.000); 高劑量黃芩苷組大鼠右膝關(guān)節(jié)軟骨組織中TNF-α、IL-1β、MMP-13表達水平均低于低劑量黃芩苷組和高劑量黃芩苷聯(lián)合抑制劑組(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000),IL-10表達水平高于低劑量黃芩苷組和高劑量黃芩苷聯(lián)合抑制劑組(P=0.000,P=0.000)。⑤大鼠右膝關(guān)節(jié)軟骨組織細胞凋亡及Hippo-YAP信號通路相關(guān)蛋白表達水平。低劑量黃芩苷組、高劑量黃芩苷組、高劑量黃芩苷聯(lián)合抑制劑組大鼠右膝關(guān)節(jié)軟骨組織中Cleaved-Caspase-3、Bax表達水平均低于模型組(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000),BCL-2、YAP、TAZ表達水平均高于模型組(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000); 高劑量黃芩苷組大鼠右膝關(guān)節(jié)軟骨組織中Cleaved-Caspase-3、Bax表達水平均低于低劑量黃芩苷組和高劑量黃芩苷聯(lián)合抑制劑組(P=0.000,P=0.000; P=0.000,P=0.000),BCL-2、YAP、TAZ表達水平均高于低劑量黃芩苷組和高劑量黃芩苷聯(lián)合抑制劑組(P=0.000,P=0.000; P=0.000,P=0.000; P=0.000,P=0.000)。結(jié)論:黃芩苷干預(yù)KOA大鼠,能夠緩解膝關(guān)節(jié)腫脹,抑制炎癥反應(yīng)和細胞凋亡,延緩關(guān)節(jié)軟骨退變,其作用機制可能與激活Hippo-YAP信號通路有關(guān)。
Abstract:
Objective:To observe the effects of baicalin(BC)on knee osteoarthritis(KOA)in rats,and to explore its mechanism.Methods:Fifty rats were randomized into sham(SH)group,model group,low-dose BC(L-BC)group,high-dose BC(H-BC)group,and H-BC combined inhibitor group,10 rats in each group.All rats but the ones in SH group were subjected to anterior cruciate ligament transection(ACLT)and medial meniscectomy on the right knees for inducing KOA; while the ones in SH group underwent surgeries for merely making incision on the medial side of right knee,and the incision was sutured instantly after exposing the articular cavity.After successful modeling,the rats in L-BC group and H-BC group were intragastric administrated with 50 and 100 mg/kg BC physiological saline solution(PSS),respectively; the ones in H-BC combined inhibitor group with 100 mg/kg BC PSS,followed by intraperitoneal injection of 1 mg/kg XMU-MP-1 solution; while the ones in SH group and model group with the same dose of normal saline.All rats in the 5 groups were intervened once a day for consecutive 30 days.Before the drug intervention,on day 15 and 30 after the drug intervention,the degree of swelling in the bilateral knees was measured,respectively.After the end of drug intervention,the rats were sacrificed,and their right knee cartilage tissues were harvested and stained with hematoxylin-eosin(HE)and safranin O-fast green(SO-FG)for observing the histopathological changes; meanwhile,the degree of knee articular cartilage degeneration was evaluated by using Mankin scoring system.Furthermore,the expression levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-10 and matrix metalloproteinase(MMP)-13 in the right knee cartilage tissues were detected by using the enzyme-linked immunosorbent assay(ELISA)kit,and the expression levels of the Cleaved-cysteine aspartic acid specific protease(Cleaved-Caspase)-3,Bcl2-associated X protein(Bax),B-cell lymphoma-2(Bcl-2)protein,Yes-associated protein(YAP)and Tafazzin(TAZ)in the synovial tissues were detected by using Western blotting.Results:①On day 15 and 30 after the drug intervention,the degree of swelling in the right knee was lower in H-BC group compared to model group,L-BC group and H-BC combined inhibitor group(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000); further pairwise comparison among model group,L-BC group and H-BC combined inhibitor group showed no statistical significance(P=0.063,P=0.215,P=0.399; P=0.052,P=0.261,P=0.240).②After the end of drug intervention,the atrophic and disorderly arranged cartilage was observed in the right knee of rats in model group.The cartilage atrophy and cellular arrangement were improved in rats of L-BC group,H-BC group,and H-BC combined inhibitor group compared with that of model group,and the improvement was better in H-BC group in contrast to L-BC group and H-BC combined inhibitor group.③After the end of drug intervention,the Mankin score of the right knee cartilage was lower in L-BC group,H-BC group and H-BC combined inhibitor group compared to model group(P=0.000,P=0.000,P=0.000),and was lower in L-BC group compared to H-BC combined inhibitor group(P=0.000),and was lowest in H-BC group(P=0.000,P=0.000).④The expression levels of TNF-α,IL-1β,and MMP-13 in the right knee cartilage tissues were lower in L-BC group,H-BC group and H-BC combined inhibitor group compared to model group(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000),and was lower in H-BC group compared to L-BC group and H-BC combined inhibitor group(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000); while the expression level of IL-10 was higher in L-BC group,H-BC group and H-BC combined inhibitor group compared to model group(P=0.000,P=0.000,P=0.000),and was higher in H-BC group compared to L-BC group and H-BC combined inhibitor group(P=0.000,P=0.000).⑤The Cleaved-Caspase-3 and Bax were lowly expressed in the right knee cartilage tissues of rats in L-BC group,H-BC group and H-BC combined inhibitor group compared to model group(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000),and were lowly expressed in H-BC group compared to L-BC group and H-BC combined inhibitor group(P=0.000,P=0.000; P=0.000,P=0.000); while the BCL-2,YAP and TAZ were highly expressed in L-BC group,H-BC group,and H-BC combined inhibitor group compared to model group(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000),and were highly expressed in H-BC group compared to L-BC group and H-BC combined inhibitor group(P=0.000,P=0.000; P=0.000,P=0.000; P=0.000,P=0.000).Conclusion:BC can alleviate knee swelling,inhibit inflammatory response and apoptosis,and delay articular cartilage degeneration in KOA rats.It may work by activating Hippo-YAP signaling pathway.

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通訊作者:李蕊 E-mail:[email protected]
更新日期/Last Update: 1900-01-01