[關鍵詞]
[摘要]
目的 探討蒼艾揮發(fā)油(Cangai volatile oil,CAVO)干預哮喘小鼠經(jīng)典瞬時受體電位通道1(transient receptor potential canonical channel 1,TRPC1)信號通路介導內(nèi)質(zhì)網(wǎng)應激對氣道重塑的影響。方法 將36只Balb/c小鼠隨機分對照組、模型組、地塞米松(1.50 mg/kg)組及CAVO高、中、低劑量(3.86、1.93、0.97 mg/kg)組。除對照組外,其余各組小鼠采用卵清蛋白(ovalbumin,OVA)致敏、激活建立小鼠哮喘模型,各給藥組分別ig相應藥物(20 mL/kg),對照組、模型組ig等體積生理鹽水,1次/d,連續(xù)給藥6周后,處死小鼠剖取肺組織,Western blotting檢測蛋白激活轉(zhuǎn)錄因子6(activating transcription factor 6,ATF6)、蛋白激酶RNA樣內(nèi)質(zhì)網(wǎng)激酶(protein kinase RNA-like endoplasmic reticulum kinase,PERK)、C/EBP同源蛋白(C/EBP homologous protein,CHOP)、I型膠原蛋白(Collagen type I,Collagen I)、Collagen III的表達水平。選取SD大鼠20只制備含藥血清,按照體表面積計算給藥劑量,2次/d,連續(xù)給藥7 d,于末次給藥1 h后處死大鼠,取血、處理備用;收集哮喘小鼠肺泡灌洗液并純化分離肺泡巨噬細胞,隨機分為對照組、5% CAVO含藥血清組、5% CAVO含藥血清+空載組、5% CAVO含藥血清+TRPC1過表達組,與人支氣管上皮樣細胞16HBE共培養(yǎng),收集細胞,采用Western blotting檢測各組細胞ATF6、PERK、CHOP、Collagen I、Collagen III、TRPC1的蛋白表達,采用定量逆轉(zhuǎn)錄聚合酶鏈反應(quantitative reverse transcription polymerase chain reaction,qRT-PCR)比較各組Collagen I、Collagen III、TRPC1的表達。結果 體內(nèi)實驗發(fā)現(xiàn),與對照組比較,模型組的ATF6、PERK、CHOP、Collagen I及Collagen III的表達顯著上升(P<0.01);與模型組比較,CAVO高、中、低劑量組ATF6、PERK、CHOP、Collagen Ⅰ、Collagen Ⅲ蛋白表達水平顯著下降(P<0.05)。體外實驗發(fā)現(xiàn),與對照組比較,5% CAVO含藥血清組、5% CAVO含藥血清+空載組中的ATF6、PERK、CHOP、Collagen I、Collagen III、TRPC1的表達水平顯著下降(P<0.01);與5% CAVO含藥血清+空載組比較,5% CAVO含藥血清+TRPC1過表達組中的ATF6、PERK、CHOP、Collagen I、Collagen III、TRPC1的表達水平顯著上升(P<0.05)。結論 CAVO對哮喘小鼠氣道重塑和氣道炎癥有改善作用,這可能與調(diào)節(jié)TRPC1信號通路,影響內(nèi)質(zhì)網(wǎng)應激反應,改善哮喘氣道重塑有關。
[Key word]
[Abstract]
Objective To investigate the effect of Cangai volatile oil (CAVO) on airway remodelling mediated by endoplasmic reticulum stress in asthmatic mice by intervening in the classical transient receptor potential canonical channel 1 (TRPC1) signalling pathway.Methods A total of 36 Balb/c mice were randomly divided into control group, model group, dexamethasone (1.50 mg/kg) group, and CAVO high-, medium- and low-dose (3.86, 1.93, 0.97 mg/kg) groups. In addition to control group, the rest of the groups of mice were sensitised by ovomucoid protein (OVA) to establish an asthma model. Each group ig corresponding drug (20 mL/kg) respectively, and control group and model group ig an equal amount of saline, once a day, for six weeks. After six weeks of administration, lung tissues were dissected for Western blotting to observe protein activating transcription factor 6 (ATF6), protein kinase RNA-like endoplasmic reticulum kinase (PERK), C/EBP homologous protein (CHOP), Collagen type I (Collagen I), Collagen III expression levels. A total of 20 SD rats were selected to prepare drug-containing serum, and the dosage of drug administered to SD rats was calculated according to the body surface area, and the drug was administered by gavage for 7 d, twice a day, and the rats were executed 1 h after the last administration, and the blood was taken and processed for spare parts. The alveolar lavage fluid of asthmatic mice was collected and purified to isolate alveolar macrophages, which were co-cultured with human bronchial epithelial cells 16HBE, and randomly divided into control group, 5% CAVO-containing serum group, 5% CAVO-containing serum + null-loaded group, and 5% CAVO-containing serum + TRPC1-overexpression group. Cells were collected and protein expression of ATF6, PERK, CHOP, Collagen Ⅰ, Collagen Ⅲ and TRPC1 were detected by Western blotting, the expression of Collagen Ⅰ, Collagen Ⅲ and TRPC1 in each group were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results In vivo experiments revealed that the expression of ATF6, PERK, CHOP, Collagen I and Collagen III in the model group was significantly increased compared with the control group (P < 0.01), compared with the model group, the protein expression levels of ATF6, PERK, CHOP, Collagen I, and Collagen III in the CAVO high-, medium-, and low-dose group were significantly decreased (P < 0.05). In vitro experiments revealed that the expression levels of ATF6, PERK, CHOP, Collagen I, Collagen III, and TRPC1 were significantly decreased in the 5% CAVO-containing serum group and the 5% CAVO-containing serum + null group compared with the control group (P < 0.01), and in the 5% CAVO-containing serum + TRPC1-overexpression group, the expression levels of expression levels of ATF6, PERK, CHOP, Collagen I, Collagen III, and TRPC1 were significantly increased compared with 5% CAVO-containing serum + null-loaded group (P < 0.05).Conclusion CAVO treatment of asthma may be related to regulating the TRPC1 signaling pathway, affecting the endoplasmic reticulum stress response, and improving asthma airway remodeling.
[中圖分類號]
R285.5
[基金項目]
國家自然科學基金資助項目(82060884);云南省基礎研究計劃重點項目(202401AS070809);云南省興滇英才支持計劃-醫(yī)療衛(wèi)生人才專項(XDYC-YLWS-2023-0092);云南省萬人計劃青年拔尖人才專項(YNWR-QNBJ-2019-196);國家中醫(yī)藥管理局高水平中醫(yī)藥重點學科建設項目(ZYYZDXK-2023190);熊磊全國名老中醫(yī)藥專家傳承工作室項目