TWI386218B - The use of the king of the line of ethanol extracted products to treat diabetic nephropathy caused by renal fibrosis - Google Patents
The use of the king of the line of ethanol extracted products to treat diabetic nephropathy caused by renal fibrosis Download PDFInfo
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本發明是有關於使用王不留行(Vaccaria segetalis (Neck.) Grack)之一經乙醇萃取的產物來治療糖尿病腎病變引發之腎纖維化。The present invention relates to the use of ethanol-extracted products of one of Vaccaria segetalis (Neck.) Grack to treat renal fibrosis caused by diabetic nephropathy.
糖尿病腎病變是一種慢性腎臟疾病,會導致腎纖維化(renal fibrosis),最終會導致出現糖尿病(diabetes mellitus,DM),目前約影響全球10%的人口。這樣的疾病損害會影響很多器官,包括眼睛,神經,血管,心臟和腎臟。在美國,糖尿病是導致腎功能衰竭最常見的原因。Diabetic nephropathy is a chronic kidney disease that causes renal fibrosis and eventually leads to diabetes mellitus (DM), which currently affects about 10% of the world's population. Such disease damage affects many organs, including the eyes, nerves, blood vessels, heart and kidneys. In the United States, diabetes is the most common cause of kidney failure.
腎纖維化來自腎臟長期炎症和組織損傷,是各種腎臟疾病進展到慢性腎衰竭的共同途徑和主要病理基礎,病理症狀包括蛋白尿,高血壓,酮尿,低血糖,通常會導致腎小球硬化、腎小管萎縮、細胞外基質異常增多和過度沉積等,最終要靠透析和腎移植來維持生命,而腎纖維化的病理反應主要是細胞外間質堆積、高血糖、高血脂和高血壓等。Renal fibrosis comes from long-term inflammation and tissue damage in the kidney. It is the common pathway and main pathological basis for various kidney diseases to progress to chronic renal failure. Pathological symptoms include proteinuria, hypertension, ketonuria, hypoglycemia, usually leading to glomerular sclerosis. Renal tubular atrophy, abnormal extracellular matrix and excessive deposition, etc., ultimately rely on dialysis and kidney transplantation to maintain life, and the pathological response of renal fibrosis is mainly extracellular matrix accumulation, hyperglycemia, hyperlipidemia and hypertension .
目前已有許多研究指出,第一型乙型轉型生長因子(transforming growth factor-β1,TGF-β1)是公認的腎纖維化指標。TGF-β1會誘導細胞外間質堆積,最後增加糖尿病腎病變的情況(Gunwar,S.,F. Ballester,et al.(1998). Glomerular basement membrane. J Biol Chem 273,8767-8775)。纖維蛋白為糖尿病腎病變最有相關一種目標蛋白質,是一種在早期糖尿病腎病變用以評估病理狀態的指標(Efstratiadis,G.,M. Divani,et al.(2009). Renal fibrosis. Hippokratia 13,224-229.)。Many studies have pointed out that the first type of transforming growth factor-β1 (TGF-β1) is a recognized index of renal fibrosis. TGF-β1 induces extracellular matrix accumulation and ultimately increases the condition of diabetic nephropathy (Gunwar, S., F. Ballester, et al. (1998). Glomerular basement membrane. J Biol Chem 273, 8767-8775). Fibrin is one of the most relevant target proteins in diabetic nephropathy and is an indicator for assessing pathological status in early diabetic nephropathy (Efstratiadis, G., M. Divani, et al. (2009). Renal fibrosis. Hippokratia 13, 224- 229.).
第一型乙型轉型生長因子(TGF-β1)是一種誘導細胞纖維化因子,與糖尿病腎病變有關(Shi,Y. and J. Massague(2003). Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113,685-700),雖然有衆多促纖維化因素參與了腎纖維化的進程,但TGF-β1以及其下游的Smad信號途徑發揮了重要作用。TGF-β1可以調控細胞增生、細胞分化、細胞移動及細胞纖維化等功能。Smad蛋白家族是細胞內負責將TGF-β1刺激所產生的訊息傳遞至細胞核的訊息傳遞者,當細胞膜上的TGF-β1接受器被TGF-β1刺激後,會將Smad蛋白加以磷酸化,而磷酸化後的Smad蛋白便將TGF-β1之訊息從細胞膜傳遞入細胞核內,進而引發下游基因之運作。Type I B transforming growth factor (TGF-β1) is an induced cell fibrosis factor associated with diabetic nephropathy (Shi, Y. and J. Massague (2003). Mechanisms of TGF-beta signaling from cell membrane to the Nucleus. Cell 113, 685-700), although many profibrotic factors are involved in the progression of renal fibrosis, TGF-β1 and its downstream Smad signaling pathway play an important role. TGF-β1 can regulate cell proliferation, cell differentiation, cell migration and cell fibrosis. The Smad protein family is a messenger responsible for the transmission of TGF-β1-stimulated messages to the nucleus. When the TGF-β1 receptor on the cell membrane is stimulated by TGF-β1, the Smad protein is phosphorylated. The resulting Smad protein transmits the TGF-β1 message from the cell membrane into the nucleus, which in turn triggers the operation of downstream genes.
Smad蛋白家族可分為三種:(一)receptor-activated Smads(包含Smad2、Smad3),命名R-Smads;(二)common mediator Smads(包含Smad4),命名Co-Smads;(三)inhibitory Smads(包含Smad7),命名I-Smad。TGF-β1之訊息傳遞,是利用被磷酸化之Smad2、Smad3與Smad4共同轉運至細胞核,控制TGF-β1敏感基因的轉錄,而刺激細胞纖維化,其中,Smad4因為可以和Smad2/3蛋白形成複合體,有效地達成傳遞任務,而居關鍵性地位,相反的,Smad7產生的抑制化效果,則會阻斷TGF-β1誘發的纖維化訊息傳遞。因此,可透過抑制或阻斷TGF-β1之訊息傳遞,應可有效阻止或減緩腎細胞纖維化。The Smad protein family can be divided into three types: (1) receptor-activated Smads (including Smad2, Smad3), named R-Smads; (2) common mediator Smads (including Smad4), named Co-Smads; (iii) inhibitory Smads (including Smad7), named I-Smad. TGF-β1 signaling is the use of phosphorylated Smad2, Smad3 and Smad4 to transport to the nucleus, control the transcription of TGF-β1 sensitive genes, and stimulate cell fibrosis, in which Smad4 can form a complex with Smad2/3 protein. The body, which effectively achieves the task of delivery, is in a critical position. On the contrary, the inhibitory effect produced by Smad7 blocks the TGF-β1-induced fibrotic message transmission. Therefore, it can effectively prevent or slow down renal cell fibrosis by inhibiting or blocking the signaling of TGF-β1.
在許多國家中,天然植物或有機植物產品通常被視為是一種科學藥物的替代方案,因為缺乏精確的科學研究支持,所以中草藥往往被列為是替代療法,而將它排除在主要治療方式之外。In many countries, natural or organic plant products are often considered as an alternative to scientific medicine. Because of the lack of precise scientific research support, Chinese herbal medicine is often classified as an alternative treatment, and it is excluded from the main treatment. outer.
王不留行(Vaccaria segetalis (Neck.) Grack),通常稱之為牛蛤,廣泛分佈於歐洲、美國、加拿大和亞洲的草原領域,是一種經常被用來活血和促進乳汁分泌、減緩紅斑、改善經期和治療乳腺感染的中草藥。在中國中藥和草藥的文獻中,王不留行水萃物可促進血液循環化瘀,可作為一種有效的生物反應調節劑,增強宿主體內循環平衡。 Vaccaria segetalis (Neck.) Grack, commonly known as burdock, is widely distributed in the grasslands of Europe, the United States, Canada, and Asia. It is often used to promote blood circulation and promote milk secretion, slowing erythema, Chinese herbal medicines that improve menstruation and treat breast infections. In the Chinese herbal medicine and herbal medicine literature, Wang Buliu's water extract can promote blood circulation and can be used as an effective biological response modifier to enhance the circulation balance in the host.
因此,本發明之目的,即在提供一種用於治療糖尿病腎病變引發之腎纖維化的藥學組成物,其包含有王不留行(Vaccaria segetalis (Neck.) Grack)之一經乙醇萃取的產物。Accordingly, it is an object of the present invention to provide a pharmaceutical composition for treating renal fibrosis caused by diabetic nephropathy comprising an ethanol-extracted product of one of Vaccaria segetalis (Neck.) Grack.
依據本發明的藥學組成物可利用熟習此藝者所詳知的技術而被製造成一適合於非經腸道地、局部地或口服地投藥的劑型,這包括,但不限於,注射品(injection)(例如,無菌的水溶液或分散體)、無菌的粉末、錠劑(tablet)、片劑(troche)、丸劑(pellet)、膠囊(capsule)以及類似之物。The pharmaceutical composition according to the present invention can be manufactured into a dosage form suitable for parenteral, topical or oral administration using techniques well known to those skilled in the art, including, but not limited to, injection products (injection). (eg, sterile aqueous solutions or dispersions), sterile powders, tablets, troches, pellets, capsules, and the like.
依據本發明的藥學組成物可以一選自於下列所構成的群組中的非經腸道途徑來投藥:腹膜內注射(intraperitoneal injection)、皮下注射(subcutaneous injection)、肌肉內注射(intramuscular injection)以及靜脈內注射(intravenous injection)。The pharmaceutical composition according to the present invention may be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intramuscular injection. And intravenous injection.
依據本發明的藥學組成物可進一步包含有一被廣泛地使用於藥物製造技術之藥學上可接受的載劑。例如,該藥學上可接受的載劑可包含一種或多種選自於下列的試劑:賦形劑、溶劑、乳化劑、懸浮劑、分解劑、黏結劑、安定劑、螯合劑、稀釋劑、膠凝劑、防腐劑、潤滑劑、吸收延遲劑、脂質體以及類似之物。The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier can comprise one or more agents selected from the group consisting of excipients, solvents, emulsifiers, suspending agents, decomposers, binders, stabilizers, chelating agents, diluents, gums Coagulants, preservatives, lubricants, absorption delaying agents, liposomes, and the like.
在本發明的一個較佳具體例中,該藥學組成物包含有一藥學上可接受的溶劑,而且該溶劑是下列的任一者:水、生理鹽水、磷酸鹽緩衝生理鹽水、含糖溶液以及含有醇的水性溶液。In a preferred embodiment of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable solvent, and the solvent is any one of the following: water, physiological saline, phosphate buffered physiological saline, sugar-containing solution, and An aqueous solution of alcohol.
本發明亦提供一種王不留行植物(Vaccaria segetalis (Neck.) Grack)之經乙醇萃取的產物供應用於製備一可治療糖尿病腎病變引發之腎纖維化的醫藥品的用途。The present invention also provides the use of an ethanol-extracted product of Vaccaria segetalis (Neck. Grack) for the preparation of a medicament for treating renal fibrosis caused by diabetic nephropathy.
依據本發明,王不留行(Vaccaria segetalis (Neck.) Grack)之經乙醇萃取的產物之投藥劑量與投藥次數會視下列因素而變化:要被治療的疾病之嚴重性,投藥途徑,以及要被治療的個體之體重、年齡、身體狀況與反應。According to the present invention, the dosage of the ethanol-extracted product of Vaccaria segetalis (Neck.) Grack and the number of administrations vary depending on factors such as the severity of the disease to be treated, the route of administration, and Body weight, age, physical condition and response of the individual being treated.
有關本發明之前述及其他技術內容、特點與功效,在以下配合參考圖式之一個較佳實施例的詳細說明中,將可清楚的呈現。The above and other technical contents, features and advantages of the present invention will be apparent from the following detailed description of the preferred embodiments.
要被瞭解的是:除非另外有所定義,在本文中所使用的所有技術性與科學術語具有熟悉本發明所屬技藝的人士所共同瞭解的意義。It is to be understood that all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of the invention, unless otherwise defined.
在研究開發治療糖尿病腎病變引發之腎纖維化的中草藥物時,發明人發現王不留行(Vaccaria segetalis (Neck.) Grack)在抑制腎細胞纖維化方面具有很好的效果,但到目前為止,都還未有關於王不留行乙醇萃取產物具有治療糖尿病腎病變之腎纖維化功效的相關研究報導被提出。In research and development of a Chinese herbal medicine for the treatment of renal fibrosis caused by diabetic nephropathy, the inventors found that Vaccaria segetalis (Neck.) Grack has a good effect in inhibiting renal cell fibrosis, but so far There have been no reports on the efficacy of Wang Buliu's ethanol extraction products for the treatment of renal fibrosis in diabetic nephropathy.
透過王不留行乙醇萃取產物可抑制細胞內TGF-βRI、pSmad2/3,Smad2/3和Smad4表現量,並相對誘發Smad7表現量的作用機制,可大幅降低NRK-49F細胞之細胞內與細胞外纖維連接蛋白表現,而確實可阻斷或延緩NRK-49F細胞之細胞纖維化訊息傳遞,所以王不留行乙醇萃取產物對於腎纖維化病症之治療理應具備療效,確實可進一步應用於製成治療腎纖維化的藥物。The effect of inhibiting the expression of TGF-βRI, pSmad2/3, Smad2/3 and Smad4 in cells by inhibiting the expression of Smad7 in cells can greatly reduce the intracellular and cellular cells of NRK-49F cells. The expression of fibronectin can actually block or delay the cell fibrosis signal transmission of NRK-49F cells, so Wang does not leave the ethanol extract product for the treatment of renal fibrosis, and it can be further applied. A drug for the treatment of renal fibrosis.
本發明王不留行乙醇萃取產物用於製備治療糖尿病腎病變引發之腎纖維化的藥物的用途之較佳實施例如下說明,但應瞭解的是,該實施例僅是供例示說明用,而不應被解釋為本發明的實施上的限制。A preferred embodiment of the use of the present invention for the preparation of a medicament for the treatment of renal fibrosis caused by diabetic nephropathy is described below, but it should be understood that this embodiment is for illustrative purposes only, and It should not be construed as limiting the implementation of the invention.
【實施例】[Examples]
A. 生物材料A. Biological materials
採用之細胞株為正常的大鼠腎臟細胞(Normal Rat Kidney cells,NRK-49F),NRK-49F細胞是一種纖維母細胞(fibroblasts),購自於美國類型培養物收集中心(American Type Culture Collection,ATCC),寄存編號(accession number)為CRL-1570。NRK-49F細胞是培養在25T培養瓶(25T flask)中,使用之培養液為添加有10%小牛血清(Bovine Calf Serum,BCS)、100 U/mL盤尼西林G(penicillin G)、100 μg/mL鏈黴素(streptomycin),及5.5 mM葡萄糖的DMEM(Dulbecco modified Eagle medium,購自GIBCO),並培養在37℃與含5% CO2 的培養箱中,每隔3天更換新鮮的培養液。The cell line used was normal rat kidney cells (NRK-49F), a fibroblasts purchased from the American Type Culture Collection (American Type Culture Collection, ATCC), the accession number is CRL-1570. NRK-49F cells were cultured in 25T flasks (25T flask) using 10% calf serum (BCS), 100 U/mL penicillin G, 100 μg/ mL streptomycin, and 5.5 mM glucose in DMEM (Dulbecco modified Eagle medium, purchased from GIBCO), and cultured in 37 ° C and 5% CO 2 incubator, every 3 days to replace the fresh medium .
B. 王不留行乙醇萃取產物B. Wang does not leave the ethanol extraction product
王不留行的莖段是於2009年9月購自台灣的中藥店。王不留行萃取產物之萃取方法如圖1所示,將10 g乾燥的王不留行莖段浸泡於100 mL的60%乙醇後,將此乙醇溶液加熱至95℃,並持續加熱6小時。然後,降溫至65℃,並以超音波震盪90分鐘,製成萃取混合液。接著,以孔隙0.22 μm的濾紙濾除此萃取混合液中之王不留行殘渣,而獲得所需之乙醇萃取液,再將乙醇萃取液加熱乾燥(65℃)成粉狀萃取物,並量測所得王不留行粉狀萃取物重量。然後,再將所得王不留行粉狀萃取產物重新分散於Milli-Q水中,調製成濃度為10 mg/mL的王不留行萃取液(Vaccaria segetalis (Neck.) Garcke extract,VSE)備用。The stem segment that Wang did not stay in was purchased from Taiwan in September 2009. The extraction method of Wang Wuliuxing extract product is shown in Figure 1. After soaking 10 g of dried Wang Buliu stem segments in 100 mL of 60% ethanol, the ethanol solution is heated to 95 ° C and heated for 6 hours. . Then, the temperature was lowered to 65 ° C, and ultrasonic waves were shaken for 90 minutes to prepare an extraction mixture. Then, the residue in the extraction mixture is filtered out with a filter paper having a pore size of 0.22 μm to obtain a desired ethanol extract, and the ethanol extract is heated and dried (65 ° C) to form a powdery extract. The measured king did not leave the weight of the powdery extract. Then, the obtained king powder-free extract product was redispersed in Milli-Q water to prepare a Vaccaria segetalis (Neck. Garcke extract, VSE) at a concentration of 10 mg/mL.
C. 細胞存活率試驗C. Cell viability test
測試不同劑量的VSE對TGF-β1誘導之NRK-49F細胞生長的影響。先將NRK-49F細胞接種於25 cm2 的培養瓶中24小時,細胞量約為2.7x105 個,培養液為含有5%小牛血清(BCS)、100 U/mL盤尼西林(青黴素)G、100 μg/mL鏈黴素的DMEM(Dulbecco modified Eagle medium,DMEM)。接著,改以含少量血清(0.5%)的培養液繼續培養,讓NRK-49F細胞飢餓24小時,然後更換培養液,培養液為含有0.1% BCS、100 U/mL盤尼西林G與100 μg/mL鏈黴素的DMEM,並分別添加TGF-β1(購自R&D公司,美國)與VSE。其中,控制組未添加VSE與TGF-β1,實驗組同時添加TGF-β1(5 ng/mL)與VSE,VSE濃度分別為0、25,50和100 μg/mL,然後繼續培養24小時後,以100倍顯微鏡觀察細胞形態。The effect of different doses of VSE on TGF-β1-induced NRK-49F cell growth was tested. The NRK-49F cells were first inoculated into a 25 cm 2 culture flask for 24 hours, and the cell volume was about 2.7× 10 5 . The culture medium contained 5% calf serum (BCS), 100 U/mL penicillin (penicillin) G, 100 μg/mL streptomycin in DMEM (Dulbecco modified Eagle medium, DMEM). Next, continue to culture with a small amount of serum (0.5%), let NRK-49F cells starve for 24 hours, and then change the culture medium containing 0.1% BCS, 100 U/mL penicillin G and 100 μg/mL. Streptomycin in DMEM and TGF-β1 (purchased from R&D, USA) and VSE, respectively. Among them, VSE and TGF-β1 were not added to the control group, and TGF-β1 (5 ng/mL) and VSE were added to the experimental group. The VSE concentrations were 0, 25, 50 and 100 μg/mL, respectively, and then continued to culture for 24 hours. The cell morphology was observed with a 100-fold microscope.
上述各組經24小時培養後,移除培養瓶中的培養液,以PBS沖洗兩次,然後加入1 mL trypsin-EDTA,待細胞脫落後,加入2 mL新鮮培養液中和trypsin-EDTA活性後,將混合液取至15 mL離心管進行離心作業(1200 rpm,5分鐘),移除上清液,再加入適量新鮮培養液,並充分打散細胞團塊,製成細胞懸浮液。取出50 μL細胞懸浮液與等體積的trypan blue充分混合形成一混合液,接著將10 μL的前述混合液置於一血球計數盤(Hemocytometer)上,進行存活細胞與死亡細胞的計數。After the above groups were cultured for 24 hours, the culture medium in the flask was removed, washed twice with PBS, and then 1 mL of trypsin-EDTA was added. After the cells were detached, 2 mL of fresh medium was added and trypsin-EDTA activity was added. The mixture was taken to a 15 mL centrifuge tube for centrifugation (1200 rpm, 5 minutes), the supernatant was removed, an appropriate amount of fresh medium was added, and the cell pellet was sufficiently broken to prepare a cell suspension. 50 μL of the cell suspension was thoroughly mixed with an equal volume of trypan blue to form a mixed solution, and then 10 μL of the aforementioned mixture was placed on a Hemocytometer, and counts of viable cells and dead cells were counted.
細胞存活率(cell viability)(%)=(a/b)×100%Cell viability (%) = (a / b) × 100%
a:未被trypan blue染色的細胞數(存活細胞數)a: Number of cells not stained by trypan blue (number of viable cells)
b:總細胞數b: total number of cells
D. TGF-β1誘發活體外腎臟細胞纖維化試驗D. TGF-β1 induces in vitro renal cell fibrosis test
以上述控制組,及VSE劑量為100 μg/mL之實驗組進行活體外腎細胞纖維化試驗,上述控制組與實驗組配製完成後,繼續培養24小時,並於此培養過程之第8小時、16小時、20小時與22小時的時間點,分別在其中一實驗組中加入50 μg/mL環己醯亞胺(cycloheximide,CHX),抑制蛋白質生成,也就是說,各實驗組之環己醯亞胺分別作用歷時16、8、4、2小時。經上述24小時培養後,分別收取各實驗組之細胞培養液進行離心處理(2000 rpm,5分鐘),收集離心上清液來進行細胞外的纖維連接蛋白(fibronectin)表現量的測定。The in vitro renal cell fibrosis test was carried out in the above control group and the experimental group with a VSE dose of 100 μg/mL. After the preparation of the above control group and the experimental group, the culture was continued for 24 hours, and at the 8th hour of the culture process, At 16 hours, 20 hours and 22 hours, 50 μg/mL cycloheximide (CHX) was added to one of the experimental groups to inhibit protein production, that is, each experimental group. The imines lasted for 16, 8, 4, and 2 hours, respectively. After the above-mentioned 24 hours of culture, the cell culture solution of each experimental group was separately centrifuged (2000 rpm, 5 minutes), and the supernatant was collected to measure the expression of extracellular fibronectin.
細胞外的纖維連接蛋白表現量的測定,是使用human Fibronectin EIA KIT來進行分析。取上述離心上清液(100 μL)加入至一個96井的培養盤中,各井表面已被預先塗覆小鼠單株抗-人類纖維連接蛋白抗體(mouse monoclonal anti-human Fibronectin antibodies),混合均勻後,於37℃下反應歷時1小時。然後,移除各井中的液體,並以400 μL洗滌緩衝液(含有0.1% Tween 20的PBS)沖洗三次,然後將該培養盤中的液體排空。The amount of extracellular fibronectin expressed was determined using human Fibronectin EIA KIT. The above supernatant (100 μL) was added to a 96-well culture dish, and the surface of each well was pre-coated with mouse monoclonal anti-human fibronectin antibodies. After homogenization, the reaction was carried out at 37 ° C for 1 hour. Then, the liquid in each well was removed and washed three times with 400 μL of washing buffer (PBS containing 0.1% Tween 20), and then the liquid in the plate was emptied.
接著,在每一井中加入100 μL標有peroxidase(POD)的抗人類細胞外纖維母蛋白的二級抗體,並予以混合均勻後,於37℃下反應歷時1小時。接著,移除各井中的液體,並以400 μL的洗滌緩衝液反復沖洗四次,繼而排空各井中的液體。然後,在每一井中加入100 μL的受質溶液(Substrate Solution),並於室溫下反應歷時15分鐘呈色後,繼而將100 μL的終止溶液(Stop Solution)(1N H2 SO4 )加入至各井中以終止反應。最後,於加完終止溶液後一小時內,以ELISA讀取機(ELISA reader,Bio-Rad,Model 1550)在波長450 nm下來測量各井的吸光值(OD450)。Next, 100 μL of secondary antibody against peroxidase (POD) anti-human extracellular fibrin was added to each well and mixed well, and the reaction was carried out at 37 ° C for 1 hour. Next, the liquid in each well was removed and washed four times with 400 μL of wash buffer, which in turn drained the liquid in each well. Then, 100 μL of Substrate Solution was added to each well, and the reaction was carried out at room temperature for 15 minutes, followed by 100 μL of Stop Solution (1N H 2 SO 4 ). Go to each well to stop the reaction. Finally, the absorbance (OD450) of each well was measured by an ELISA reader (ELISA reader, Bio-Rad, Model 1550) at a wavelength of 450 nm within one hour after the addition of the stop solution.
此外,以纖維連接蛋白標準溶液(濃度分別為400 ng/mL、200 ng/mL、100 ng/mL、50 ng/mL、25 ng/mL以及12.5 ng/mL)的吸光值(OD450)作為對照組,繪製標準曲線(standard curve)。In addition, the absorbance (OD450) of fibronectin standard solutions (concentrations of 400 ng/mL, 200 ng/mL, 100 ng/mL, 50 ng/mL, 25 ng/mL, and 12.5 ng/mL) was used as a control. Group, draw a standard curve.
E. 西方墨點分析(Western blotting analysis)E. Western blotting analysis
(1)蛋白質樣品的製備與蛋白質濃度量測:(1) Preparation of protein samples and measurement of protein concentration:
首先,控制組與實驗組經24小時培養後,移除培養盤中的培養液,並以PBS清洗細胞兩次,將培養盤內的殘餘液體吸乾後,加入適量的trypsin-EDTA並於37℃下作用歷時5分鐘,待細胞自培養皿的底部脫離後,加入新鮮的DMEM(含有10% BCS、100 U/mL盤尼西林G與100 μg/mL鏈黴素)中和trypsin-EDTA活性,並對所形成的混合物進行離心處理(4℃,1,200 rpm,5分鐘),移除上清液,並加入400 μL的細胞溶解緩衝液(cell lysis buffer)混合均勻後,在冰浴下以超音波處理器(ultrasonicator)震碎細胞,操作條件為震盪歷時5秒,停止歷時1秒,重複10個循環。接著,於4℃下進行離心(13,000 rpm,10分鐘),並收集上清液,且藉由Bio-Rad DC蛋白質分析法(Bio-Rad DC Protein Assay)測定此上清液在595 nm的吸光值,測定蛋白質的濃度。First, after the control group and the experimental group were cultured for 24 hours, the culture solution in the culture plate was removed, and the cells were washed twice with PBS, and the residual liquid in the culture plate was blotted dry, and then an appropriate amount of trypsin-EDTA was added and 37 The action was carried out at °C for 5 minutes. After the cells were detached from the bottom of the dish, fresh DMEM (containing 10% BCS, 100 U/mL penicillin G and 100 μg/mL streptomycin) was added to neutralize trypsin-EDTA activity, and The resulting mixture was centrifuged (4 ° C, 1,200 rpm, 5 minutes), the supernatant was removed, and 400 μL of cell lysis buffer was added and mixed, and ultrasonic waves were taken in an ice bath. The ultrashonicator shatters the cells under an operating condition of 5 seconds for the oscillation, 1 second for the stop, and 10 cycles. Subsequently, centrifugation (13,000 rpm, 10 minutes) was carried out at 4 ° C, and the supernatant was collected, and the supernatant was measured for absorbance at 595 nm by Bio-Rad DC Protein Assay. Value, the concentration of the protein is determined.
此外,部分的上清液會被用於以下之SDS-聚丙烯醯胺凝膠電泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)分析。In addition, a portion of the supernatant was used for the following SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.
(2) 西方墨點分析(Western blotting Analysis):(2) Western blotting analysis:
將350 μL之上述『(1)蛋白質樣品的製備與蛋白質濃度量測』收集之上清液,及350 μL的2X樣品緩衝液(2X sample buffer)(含有0.125 M Tris-HCl、4% SDS、20%甘油與10%巰基乙醇(β-mercaptoethanol),pH 6.8)混合均勻後,置於100℃的水浴中加熱歷時5分鐘。接著,進行離心處理(4℃,13,000 rpm,10分鐘)後,收集上清液備用,並以此上清液作為蛋白質樣品。Collect 350 μL of the above (1) Preparation of Protein Samples and Protein Concentration Measurements to collect the supernatant, and 350 μL of 2X sample buffer (containing 0.125 M Tris-HCl, 4% SDS, After 20% glycerol was uniformly mixed with 10% mercaptoethanol (pH 6.8), it was heated in a water bath at 100 ° C for 5 minutes. Next, after centrifugation (4 ° C, 13,000 rpm, 10 minutes), the supernatant was collected for use, and the supernatant was used as a protein sample.
取15 μg的蛋白質樣品進行SDS-聚丙烯醯胺凝膠電泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)。電泳結束後,將凝膠片上被分離的蛋白質轉印至聚二氟乙烯膜(polyvinylidene difluoride membrane,PVDF membrane)。接著,將轉印後的PVDF膜取出並於室溫下以10%脫脂乳(配於TBST,Tris-Buffered Saline/Tween-20)進行封阻處理(blocking),繼而以TBST予以洗滌10分鐘。15 μg of protein sample was taken for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After the electrophoresis is completed, the separated protein on the gel sheet is transferred to a polyvinylidene difluoride membrane (PVDF membrane). Next, the transferred PVDF membrane was taken out and blocked with 10% skim milk (with TBST, Tris-Buffered Saline/Tween-20) at room temperature, followed by washing with TBST for 10 minutes.
然後,再加入含5%低脂牛奶的TBST稀釋之初級抗體(primary antibody),所使用之一級抗體有兔子抗Smad7、抗Smad4、抗fibronectin、抗TGF-βRI、抗Smad2/3和抗pSmad2/3等多株抗體(rabbit anti Smad7 polyclonal antibody ,Santa Cruz Inc.,USA),於室溫下均勻作用1小時。初級抗體反應完後,以TBST洗滌6次,每次10分鐘。接著,加入含5%低脂牛奶的TBST稀釋之二級抗體(secondary antibody),所用二級抗體為山羊抗兔子IgG抗體(goat anti rabbit IgG antibody,CALBIOCHEM,Cat. No. 401315),於室溫下作用歷時1小時後,以TBST予以洗滌6次,每次10分鐘。Then, a TBST-diluted primary antibody containing 5% low-fat milk was added, and the primary antibodies used were rabbit anti-Smad7, anti-Smad4, anti-fibronectin, anti-TGF-βRI, anti-Smad2/3, and anti-pSmad2/ A multi-strain antibody (rabbit anti Smad7 polyclonal antibody, Santa Cruz Inc., USA) was allowed to act uniformly for 1 hour at room temperature. After the primary antibody was reacted, it was washed 6 times with TBST for 10 minutes each time. Next, a TBST diluted secondary antibody containing 5% low fat milk was added, and the secondary antibody used was goat anti rabbit IgG antibody (CALBIOCHEM, Cat. No. 401315) at room temperature. After 1 hour, the lower action was washed 6 times with TBST for 10 minutes each time.
另外,以β-actin(β-肌動蛋白)作為內部對照組(internal control),並且使用兔子抗β-肌動蛋白抗體(rabbit anti β-actin antibody,Sigma,USA)作為一級抗體,以山羊抗兔子IgG抗體(CALBIOCHEM,Cat. No. 401315)作為二級抗體(secondary antibody),來進行相同的實驗。In addition, β-actin (β-actin) was used as an internal control, and rabbit anti-β-actin antibody (Sigma, USA) was used as a primary antibody to goats. The same experiment was carried out as an anti-rabbit IgG antibody (CALBIOCHEM, Cat. No. 401315) as a secondary antibody.
接著,以化學發光ECL偵測系統(Chemilucent ECL Detection System)(Cat. No. 2600,Millipore,USA)來處理PVDF膜,並進行Smad7、Smad4、fibronectin、TGF-β RI、Smad2/3和pSmad2/3之蛋白質表現量分析。Next, the PVDF membrane was treated with Chemilucent ECL Detection System (Cat. No. 2600, Millipore, USA) and subjected to Smad7, Smad4, fibronectin, TGF-β RI, Smad2/3 and pSmad2/ 3 protein expression analysis.
在本實施例中,各組的Smad7蛋白質表現量是以β-肌動蛋白蛋白質表現量來予以標準化(normalized),然後將控制組經標準化的Smad7蛋白質表現量當作100%,而其他各組則相對於正常對照組的Smad7蛋白質表現量予以標準化,而Smad4、fibronectin、TGF-β RI、Smad2/3和pSmad2/3之蛋白質表現量,皆用此方法。In the present example, the amount of Smad7 protein expression of each group was normalized by the amount of β-actin protein expression, and then the normalized Smad7 protein expression amount of the control group was regarded as 100%, and the other groups were The Smad7 protein expression was normalized to the normal control group, and the protein expression levels of Smad4, fibronectin, TGF-β RI, Smad2/3, and pSmad2/3 were all used.
F. 免疫螢光染色(Immunofluorescence)F. Immunofluorescence staining (Immunofluorescence)
透過免疫螢光分析觀察細胞內纖維連接蛋白的表現,NRK-49F細胞以4×103 cell/well種於chamber slid之8孔盤中,於37℃培養箱培養24小時後,更換新鮮的DMEM培養液(含5%的BCS)培養24小時,之後換新鮮的DMEM培養液(含0.1%的BCS),同時分組加入預定濃度之TGF-β1(5 ng/ml)與VSE(50、100 μg/mL),並繼續培養24小時。The expression of fibronectin in the cells was observed by immunofluorescence analysis. NRK-49F cells were seeded in an 8-well plate of chamber slid at 4 × 10 3 cells/well, and cultured in a 37 ° C incubator for 24 hours, and then replaced with fresh DMEM. The culture medium (5% BCS) was cultured for 24 hours, then replaced with fresh DMEM medium (containing 0.1% BCS), and a predetermined concentration of TGF-β1 (5 ng/ml) and VSE (50, 100 μg) were added in groups. /mL) and continue to culture for 24 hours.
接著,進行細胞免疫螢光染色。將舊的培養液去除,以PBS清洗2次,然後以4% paraformaldehyde固定30分鐘,再以PBS清洗4次後,加入0.1% Triton X-100/in PBS作用5分鐘,使NRK-49F細胞膜產生孔隙得以讓染劑進入,再以PBS清洗4次。然後,在室溫下使用1% BSA(bovine serum albumin)/PBS作用60分鐘,以兔子抗人類纖維連接蛋白抗體(rabbit antihuman fibronectin antibody)於4℃環境作用隔夜,再以PBS清洗5次,接著以FITC-conjugated goat anti-mouse IgG antibody於室溫下作用60分鐘,再以PBS清洗5次。最後,加入封固劑(Mounting Medium)進行封片,使用螢光顯微鏡照相記錄。Next, cell immunofluorescence staining was performed. The old culture solution was removed, washed twice with PBS, then fixed with 4% paraformaldehyde for 30 minutes, washed 4 times with PBS, and then added with 0.1% Triton X-100/in PBS for 5 minutes to produce NRK-49F cell membrane. The pores allowed the dye to enter and were washed 4 times with PBS. Then, using 1% BSA (bovine serum albumin)/PBS for 60 minutes at room temperature, rabbit anti-human fibronectin antibody was applied overnight at 4 ° C, and then washed 5 times with PBS, followed by 5 times. The cells were treated with FITC-conjugated goat anti-mouse IgG antibody for 60 minutes at room temperature and then washed 5 times with PBS. Finally, the mounting was carried out by adding Mounting Medium and photographing using a fluorescent microscope.
G. 統計學分析(Statistical Analysis)G. Statistical Analysis
在上述實施例中,各組的實驗被重複3次,而實驗數據是以平均值標準偏差(standard deviation,S.D)來表示。兩組實驗數據之間的差異是藉由unpaired Student’s t-test進行評估,而三組以上實驗數據間的差異,是藉由單因子變異數分析(one-way ANOVA)以及unpaired Student’s t-test進行評估。若所得到的統計比對結果是p<0.05,代表有統計學顯著性(statistical significance)。In the above embodiment, the experiments of each group were repeated 3 times, and the experimental data was expressed by the standard deviation (S.D.). The difference between the two sets of experimental data was assessed by unpaired Student's t-test, and the difference between the three sets of experimental data was analyzed by one-way ANOVA and unpaired Student's t-test. Evaluation. If the obtained statistical alignment result is p < 0.05, it represents statistical significance.
【試驗結果】【test results】
A. VSE對TGF-β1誘導之NRK-49F細胞的存活率影響A. Effect of VSE on survival rate of TGF-β1-induced NRK-49F cells
如圖2~4所示,在TGF-β1(5 ng/mL)和VSE(25,50與100 μg/mL)共同刺激下,NRK-49F細胞仍具有很高之存活率表現,顯示在TGF-β1和VSE共同刺激下,即便VSE濃度提高到100 μg/mL,也不會導致NRK-49F細胞死亡或促進NRK-49F細胞生長。As shown in Figures 2 to 4, NRK-49F cells still have a high survival rate under the stimulation of TGF-β1 (5 ng/mL) and VSE (25, 50 and 100 μg/mL), which is shown in TGF. Under the co-stimulation of β1 and VSE, even if the VSE concentration is increased to 100 μg/mL, it will not cause NRK-49F cell death or promote NRK-49F cell growth.
B. VSE對TGF-β1刺激之NRK-49F細胞的影響B. Effect of VSE on TGF-β1-stimulated NRK-49F cells
以酵素連結免疫吸附分析(ELISA)分析VSE拮抗TGF-β1誘導NRK-49F細胞之纖維蛋白表現量,藉以證實VSE在抗纖維化中所扮演的角色。如圖5所示,在僅有TGF-β1(5 ng/mL)刺激下,NRK-49F細胞之細胞外纖維連接蛋白表現量會顯著增加,但是當同時以VSE處理時,細胞外纖維連接蛋白表現量則會顯著下降,當VSE濃度提高至50 μg/mL以上時,纖維連接蛋白表現量便已遠低於控制組(未經TGF-β1與VSE處理),證實VSE確實能夠顯著抑制TGF-β1誘導的纖維連接蛋白分泌表現。The enzyme-linked immunosorbent assay (ELISA) was used to analyze the fibrin expression of NRK-49F cells induced by VSE antagonizing TGF-β1, thereby confirming the role of VSE in anti-fibrosis. As shown in Figure 5, the expression of extracellular fibronectin in NRK-49F cells was significantly increased under stimulation with only TGF-β1 (5 ng/mL), but when treated with VSE at the same time, extracellular fibronectin The amount of performance decreased significantly. When the VSE concentration increased above 50 μg/mL, the expression of fibronectin was much lower than that of the control group (without TGF-β1 and VSE treatment), confirming that VSE can significantly inhibit TGF- 11 induced fibronectin secretion performance.
C. VSE對於TGF-β1刺激之NRK-49F細胞的TGF-βRI(TGF-β1 receptor protein)與Smad表現的影響Effects of VSE on TGF-β1 (TGF-β1 receptor protein) and Smad expression in TGF-β1-stimulated NRK-49F cells
如圖6所示,在TGF-β1誘導下,會顯著增加NRK-49F細胞內的TGF-βRI表現,但是當同時以VSE對NRK-49F細胞進行處理時,發現VSE能顯著減少TGF-βRI蛋白質和纖維連接蛋白的表現。As shown in Figure 6, TGF-β1 induced a significant increase in TGF-βRI expression in NRK-49F cells, but when NSE-49F cells were simultaneously treated with VSE, VSE was found to significantly reduce TGF-βRI protein. And the performance of fibronectin.
如圖7、8所示,而在TGF-β1與VSE對NRK-49F細胞之Smad蛋白的訊息分子表現之影響方面,在僅有TGF-β1(5 ng/mL)刺激下,pSmad2/3、Smad2/3和Smad4的表現會顯著增加,但在同時以VSE進行處理時,pSmad2/3,Smad2/3和Smad4表現量會大幅下降,這結果顯示VSE可能是經由抑制TGF-β1下游的訊息,而逆轉TGF-β1誘導的細胞纖維化。As shown in Figures 7 and 8, the effect of TGF-β1 and VSE on the signal molecule expression of the Smad protein of NRK-49F cells was stimulated by TGF-β1 (5 ng/mL) alone, pSmad2/3, The performance of Smad2/3 and Smad4 was significantly increased, but the expression of pSmad2/3, Smad2/3 and Smad4 decreased significantly when treated with VSE at the same time. This result indicates that VSE may be through the inhibition of TGF-β1 downstream information. The TGF-β1-induced cell fibrosis was reversed.
如圖9所示,另一方面,在僅有TGF-β1(5 ng/mL)刺激時,抑制性Smad蛋白(Smad7)的表現量相當不明顯,但在VSE之同時作用下,Smad7表現量即大幅提高,且在VSE添加劑量僅為25 μg/mL情況下,便足以誘使Smad7表現量增加數十倍,逆轉了TGF-β1誘導下所減少的Smad7蛋白質表現。As shown in Figure 9, on the other hand, when only TGF-β1 (5 ng/mL) was stimulated, the expression of inhibitory Smad protein (Smad7) was rather insignificant, but at the same time as VSE, Smad7 expression That is, it is greatly improved, and when the VSE additive amount is only 25 μg/mL, it is enough to induce the Smad7 expression to increase by several tens of times, which reverses the reduced Smad7 protein expression induced by TGF-β1.
因此,根據上述結果,顯示VSE確實可透過提高Smad7表現量,同時抑制TGF-βRI、pSmad2/3、Smad2/3與Smad4表現量的方式,來達到延緩或阻斷TGF-β1誘導的細胞內細胞纖維化訊息傳遞,換句話說,VSE應可用以改善腎細胞纖維化症狀。Therefore, based on the above results, it was shown that VSE can actually delay or block TGF-β1-induced intracellular cells by increasing the amount of Smad7 expression while inhibiting the expression of TGF-βRI, pSmad2/3, Smad2/3 and Smad4. Fibrosis message delivery, in other words, VSE should be used to improve renal cell fibrosis symptoms.
D. 環己醯亞胺與VSE對於TGF-βRI降解的影響D. Effect of cycloheximide and VSE on the degradation of TGF-βRI
圖10顯示NRK-49F細胞分別在TGF-β1(5 ng/mL)單獨刺激下,及在TGF-β1(5 ng/mL)與VSE同時刺激下,於不同的時間點(8、16、20和22小時)加入環己醯亞胺(50 μg/mL)的TGF-βRI表現,結果顯示VSE確實會降低NRK-49F細胞之TGF-βRI蛋白表現。Figure 10 shows that NRK-49F cells were stimulated with TGF-β1 (5 ng/mL) alone, and stimulated simultaneously with TGF-β1 (5 ng/mL) and VSE at different time points (8, 16, 20). TGF-βRI expression of cycloheximide (50 μg/mL) was added to 22 hours), and it was shown that VSE did reduce the TGF-βRI protein expression of NRK-49F cells.
E. 免疫螢光分析纖維連接蛋白的表現E. Immunofluorescence analysis of fibronectin expression
TGF-β1會誘導腎小球腎炎與糖尿病腎病變的患者產生腎纖維化,TGF-β1誘導腎臟細胞外和細胞內纖維連接蛋白生成的表現如圖11、12所示。NRK-49F細胞經飢餓24小時後,分別以TGF-β1(5 ng/mL)單獨刺激,及以TGF-β1(5 ng/mL)與VSE(50、100 μg/mL)共同刺激。結果顯示,TGF-β1確實會大量增加NRK-49F細胞之細胞內與細胞外纖維連接蛋白的表現,但在同時有VSE刺激下,不僅細胞內纖維連接蛋白表現量降低,細胞外纖維連接蛋白表現也同時降低,證實VSE會顯著抑制NRK-49F細胞在TGF-β1誘導的纖維連接蛋白表現。TGF-β1 induces renal fibrosis in patients with glomerulonephritis and diabetic nephropathy, and TGF-β1 induces extracellular and intracellular fibronectin production in the kidney as shown in Figs. NRK-49F cells were stimulated with TGF-β1 (5 ng/mL) alone after 24 hours of starvation, and co-stimulated with VSE (50, 100 μg/mL) with TGF-β1 (5 ng/mL). The results showed that TGF-β1 did increase the expression of intracellular and extracellular fibronectin in NRK-49F cells, but under the stimulation of VSE, not only the expression of fibronectin in cells decreased, but also the expression of extracellular fibronectin. At the same time, it was confirmed that VSE significantly inhibited TGF-β1-induced fibronectin expression in NRK-49F cells.
綜上所述,透過上述各項分析結果顯示,VSE(王不留行萃取液)(100 μg/mL)並不會對NRK-49F細胞產生危害,且證實VSE對同時以TGF-β1刺激之NRK-49F細胞,可藉由抑制細胞內TGF-βRI、pSmad2/3,Smad2/3和Smad4表現量,並相對誘發Smad7表現量的作用機制,大幅降低NRK-49F細胞之細胞內與細胞外纖維連接蛋白表現,而確實可阻斷或延緩NRK-49F細胞之細胞纖維化訊息傳遞,因此,本發明王不留行萃取液(VSE)對於糖尿病腎病變引發之腎纖維化病症之治療理應具備療效,確實可進一步應用於製成治療糖尿病腎病變引發之腎纖維化的藥物。因此,確實可達到本發明之目的。In summary, the results of the above analysis showed that VSE (100 μg/mL) did not cause harm to NRK-49F cells, and it was confirmed that VSE was stimulated by TGF-β1 at the same time. NRK-49F cells can significantly reduce the intracellular and extracellular fibers of NRK-49F cells by inhibiting the expression of TGF-βRI, pSmad2/3, Smad2/3 and Smad4 in cells and relatively inducing the expression of Smad7. Connexin expression can indeed block or delay the cell fibrosis signal transmission of NRK-49F cells. Therefore, the present invention does not retain the extract (VSE) for the treatment of renal fibrosis caused by diabetic nephropathy. It can be further applied to the preparation of a drug for treating renal fibrosis caused by diabetic nephropathy. Therefore, the object of the present invention can be achieved.
惟以上所述者,僅為本發明之較佳實施例而已,當不能以此限定本發明實施之範圍,即大凡依本發明申請專利範圍及發明說明內容所作之簡單的等效變化與修飾,皆仍屬本發明專利涵蓋之範圍內。The above is only the preferred embodiment of the present invention, and the scope of the invention is not limited thereto, that is, the simple equivalent changes and modifications made by the scope of the invention and the description of the invention are All remain within the scope of the invention patent.
圖1是本發明王不留行萃取液之萃取製造流程圖;圖2是顯示經由TGF-β1與VSE刺激後之NRK-49F細胞形態;圖3顯示TGF-β1與VSE刺激對NRK-49F細胞存活率的影響;圖4顯示TGF-β1與VSE刺激對NRK-49F細胞之細胞數的影響;圖5(a)顯示纖維連接蛋白標準曲線;圖5(b)顯示VSE對TGF-β1刺激之NRK-49F細胞的纖維連接蛋白表現量的影響;圖6(a)顯示NRK-49F細胞經TGF-β1與VSE處理後,經Western blot analysis之TGF-βRI區帶影像;圖6(b)顯示VSE會抑制TGF-β1刺激之NRK-49F細胞的TGF-βRI表現量;圖7(a)顯示NRK-49F細胞經TGF-β1與VSE處理後,經Western blot analysis之pSmad2/3與Smad2/3區帶影像;圖7(b)顯示VSE會抑制TGF-β1刺激之NRK-49F細胞的pSmad2/3與Smad2/3表現量;圖8(a)顯示NRK-49F細胞經TGF-β1與VSE處理後,經Western blot analysis之Smad4區帶影像;圖8(b)顯示VSE會抑制TGF-β1刺激之NRK-49F細胞的Smad4與纖維連接蛋白表現量;圖9(a)顯示NRK-49F細胞經TGF-β1與VSE處理後,經Western blot analysis之Smad7區帶影像;圖9(b)顯示VSE 會誘發TGF-β1刺激之NRK-49F細胞的Smad7表現量;圖10(a)顯示NRK-49F細胞經TGF-β1與VSE處理後,經Western blot analysis之TGF-βRI區帶影像;圖10(b)顯示VSE對降解TGF-βRI的影響;圖11顯示VSE對NRK-49F細胞之細胞內纖維連接蛋白表現的影響;及圖12顯示VSE對NRK-49F細胞之細胞外纖維連接蛋白表現的影響。BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart showing the extraction and preparation of the extract of the king of the present invention; Fig. 2 is a view showing the morphology of NRK-49F cells stimulated by TGF-β1 and VSE; and Fig. 3 shows that TGF-β1 and VSE are stimulated to NRK-49F cells. Effect of Survival; Figure 4 shows the effect of TGF-β1 and VSE stimulation on the number of cells in NRK-49F cells; Figure 5 (a) shows the fibronectin standard curve; Figure 5 (b) shows VSE stimulation of TGF-β1 The effect of fibronectin expression on NRK-49F cells; Figure 6(a) shows the TGF-βRI band image of NRK-49F cells treated with TGF-β1 and VSE after Western blot analysis; Figure 6(b) shows VSE inhibited TGF-β1-stimulated TGF-βRI expression in TRK-β1 cells; Figure 7(a) shows that NRK-49F cells were treated with TGF-β1 and VSE, and Western blot analysis of pSmad2/3 and Smad2/3 The image of the zone; Figure 7(b) shows that VSE inhibits the expression of pSmad2/3 and Smad2/3 in TRK-β1-stimulated NRK-49F cells; Figure 8(a) shows that NRK-49F cells are treated with TGF-β1 and VSE After that, the Smad4 region was imaged by Western blot analysis; Figure 8(b) showed that VSE inhibited the expression of Smad4 and fibronectin in TGF-β1-stimulated NRK-49F cells; Figure 9(a) shows NRK-49F TGF-β1 cells after treatment with the VSE, Smad7 by Western blot analysis of the region with an image; FIG. 9 (b) show VSE The Smad7 expression of TRK-β1-stimulated NRK-49F cells was induced; Figure 10 (a) shows the TGF-βRI band image of NRK-49F cells treated with TGF-β1 and VSE after Western blot analysis; Figure 10 ( b) shows the effect of VSE on the degradation of TGF-βRI; Figure 11 shows the effect of VSE on the expression of intracellular fibronectin in NRK-49F cells; and Figure 12 shows the effect of VSE on the expression of extracellular fibronectin in NRK-49F cells. .
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張翠、董佳濤、王海英, 通腑洩濁湯對腎間質纖維化大鼠alpha-SMA、LN、Col-III表達的影響, 中成藥 2008年第30卷第11期, 1584-1589 李帆、梁敬鈺, 王不留行的研究進展, 海峽藥學 2007年第19卷第3期, 1-5 * |
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