除非另作定義,否則本文中所使用之所有技術及科學術語具有與一般熟習本發明所從屬之技術者通常所瞭解之含義相同的含義。儘管可在本發明之實踐或試驗中使用與本文中所描述之彼等方法與材料相似或等效的任何方法與材料,但現在描述較佳的方法與材料。本文所提及之所有公開案均以引用的方式併入。 在本申請案中,除非另外特定陳述,否則單數之使用包括複數,介詞「一(a)」或「一(an)」意謂「至少一個」,且「或」之使用意謂「及/或」。 在本申請案中,字「包含(comprise)」或諸如「包含(comprises)」或「包含(comprising)」之變體表示包括任何所述整數或整數群但不排除指定方法、結構或組成中之任何其他整數或整數群。 如本文中所使用,「抑制(inhibit/inhibiting)」、「預防(prevent/preventing)」或「減少(reduce/reducing)」在本文中可互換地使用。此等術語係指相較於未經治療之細胞(組織或個體),經治療細胞(組織或個體)中之所量測參數之減小。比較亦可由治療前與治療後之間的相同細胞或組織或個體作出。減小為可足以檢測的。 如本文中所使用,「治療(treatment)」或「治療(treating)」或「改善(ameliorating)」在本文中可互換地使用。此等術語係指獲得有利或所要結果之方法,其包括但不限於治療益處及/或預防益處。治療益處係關於根除或改善所治療之潛在病症。此外,藉由根除或改善與潛在病症相關之一或多種生理症狀以使得在患者中觀測到改善來實現治療益處,但該病患仍可罹患潛在病症。需要治療之彼等者包括已患有病狀或病症以及易於患有病狀或病症或體內病狀或病症待預防之彼等者。 如本文中所使用,「治療有效量」意謂足以治療罹患疾病之個體或減輕與疾病相關之症狀或併發症的量。 如本文中所使用,「個體」係指人類或非人類動物。 在一個態樣中,本發明提供一種預防或治療個體中之纖維化的方法,其包含向個體投與有效量的源自靈芝、松杉靈芝、小孢子靈芝或紫芝的免疫調節蛋白,由此預防或治療纖維化。 在一個實施例中,纖維化選自心臟纖維化、硬皮病、骨胳肌纖維化、肝纖維化、腎臟纖維化、肺纖維化、糖尿病纖維化或OSF。在另一態樣中,纖維化為OSF。 在一些實施例中,免疫調節蛋白為源自靈芝之LZ-8、源自松杉靈芝之FIP-gts、源自小孢子靈芝之GMI或源自紫芝之FIP-gja。在另外實施例中,免疫調節蛋白具有如SEQ ID NO: 1或SEQ ID NO: 2中所展示之胺基酸序列。 SEQ ID NO: 1 (亦即,本文中所描述之GMI)及SEQ ID NO: 2之序列如下所列。在一個實施例中,該方法藉由抑制膠原蛋白凝膠收縮、遷移、侵襲及創傷癒合能力而降低肌纖維母細胞活性。在另一實施例中,該方法減少介白素(IL)-6及IL-8之表現量。 因此,本發明提供一種降低細胞或個體中之肌纖維母細胞活性的方法,其包含使源自靈芝、松杉靈芝、小孢子靈芝或紫芝之免疫調節蛋白與該細胞接觸或向該個體投與該免疫調節蛋白。在一些實施例中,免疫調節蛋白為源自靈芝之LZ-8、源自松杉靈芝之FIP-gts、源自小孢子靈芝之GMI或源自紫芝之FIP-gja。在另外實施例中,免疫調節蛋白具有如SEQ ID NO: 1或SEQ ID NO: 2中所展示之胺基酸序列。 在一個實施例中,肌纖維母細胞活性藉由抑制膠原蛋白凝膠收縮、遷移、侵襲及創傷癒合能力而降低。在另一實施例中,該方法減少介白素(IL)-6及IL-8之表現量。 在一個實施例中,用於本發明之方法中之有效量範圍介於約25 mg/kg至約55 mg/kg體重免疫調節蛋白。在一些實施例中,有效量範圍介於約25 mg/kg至約50 mg/kg、約25 mg/kg至約45 mg/kg、約25 mg/kg至約40 mg/kg、約25 mg/kg至約35 mg/kg、約25 mg/kg至約30 mg/kg、約30 mg/kg至約55 mg/kg、約32 mg/kg至約55 mg/kg、約34 mg/kg至約55 mg/kg、約36 mg/kg至約55 mg/kg、約38 mg/kg至約55 mg/kg、約40 mg/kg至約55 mg/kg、約30 mg/kg至約50 mg/kg、約32 mg/kg至約50 mg/kg、約34 mg/kg至約50 mg/kg、約36 mg/kg至約50 mg/kg、約38 mg/kg至約50 mg/kg、約40 mg/kg至約50 mg/kg、約30 mg/kg至約45 mg/kg、約32 mg/kg至約45 mg/kg、約34 mg/kg至約45 mg/kg、約36 mg/kg至約45 mg/kg、約38 mg/kg至約45 mg/kg、約40 mg/kg至約45 mg/kg、約30 mg/kg至約40 mg/kg、約32 mg/kg至約40 mg/kg、約34 mg/kg至約40 mg/kg或約36 mg/kg至約40 mg/kg體重。在一另外實施例中,有效量範圍介於約36 mg/kg至約40 mg/kg體重。 可向患者單獨投與或呈醫藥組合物(其中其與適合載劑及賦形劑混合)形式投與本發明之免疫調節蛋白或其重組合。本發明之免疫調節蛋白或其重組合或組合物可不經腸投與,諸如藉由靜脈注射或輸注、腹膜內注射、皮下注射或肌內注射。免疫調節蛋白或其重組合或組合物可經口投與或經由與載劑及/或賦形劑合適調配以形成錠劑、丸劑、膠囊、液體、凝膠、糖漿、漿液、懸浮液及類似物來投與。免疫調節蛋白或其重組合或組合物可局部投與,諸如藉由皮膚貼片。可將免疫調節蛋白或其重組合或組合物調配為適合於局部施用至黏膜表面之局部乳膏、皮膚貼片或黏膜貼片、液體或凝膠。 適用於根據本發明之用途的免疫調節蛋白或其重組合或組合物之劑量可基於本文中之揭露內容藉由熟習此項技術者來測定。藥劑將含有有效劑量(視活性劑之投與途徑及藥物動力學而定)的免疫調節蛋白或其重組合或組合物。適合的醫藥學上之載劑及賦形劑適用於調配物之特定投與途徑(亦即,口服、不經腸、局部或藉由吸入)。藉助於混合、溶解、粒化、糖衣錠製造、乳化、囊封、包覆或凍乾製程將免疫調節蛋白或其重組合混合至醫藥組合物中。用於不經腸投與之醫藥組合物包括呈水可溶形式的免疫調節蛋白或其重組合之水性溶液。另外,可將免疫調節蛋白或其重組合之懸浮液製備為油性注射懸浮液。適合親脂性溶劑或媒劑包括脂肪油,諸如芝麻油;或合成脂肪酸酯,諸如油酸乙酯或三酸甘油酯;或脂質體。水性注射懸浮液可含有增加懸浮液黏度之物質,諸如羧甲基纖維素鈉、山梨糖醇或聚葡萄糖。懸浮液可視情況含有穩定劑或試劑以增加複合物或組合之溶解度以允許更濃縮之溶液。 無需進一步詳細描述,咸信熟習此項技術者可基於先前描述最大程度地利用本發明。因此,以下實例應理解為僅為說明性的且無論如何不限制本發明範疇。實例 GMI 蛋白
藉由Mycomagic Biotechnology Co., Ltd. (Taipei,Taiwan)製造之GMI由小孢子靈芝產生及改善。根據描述於US 7,601,808中之方法製備GMI。OSF 組織獲得及細胞培養
在知情同意及協議經Institutional Review Board of Chung Shan Medical University Hospital批准之情況下自Oral Medicine Center (Chung Shan Medical University Hospital,Taichung,Taiwan)招募之OSF患者分離纖維化BMF (fBMF)。根據此項技術中已知之準則及方法培養源自正常頰黏膜(BMF)及fBMF之纖維母細胞。第三代與第八代之間的細胞培養用於此研究。MTT 檢定
針對fBMF及BMF中之GMI之細胞毒性效應的測定,在37℃下在不同濃度之GMI或媒劑的存在下,以1×105
細胞/500 gl/孔將細胞接種於24孔培養盤中48小時,隨後進行MTT (溴化3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑鹽)處理。將活細胞之藍甲䐶晶體溶解於DMSO中,且接著在570 nm下以分光光度法評估。將DMSO處理組設置為100%,且資料呈現為DMSO對照之百分比。藉由GraFit軟體(Erithacus Software Ltd.,West Sussex,UK)計算IC50值。膠原蛋白凝膠收縮 檢定
藉由膠原蛋白收縮檢定(Sigma-Aldrich Chemical Co.,St. Louis,MO,USA)執行肌纖維母細胞功能之生物活性。將2×105
個細胞懸浮於冷膠原蛋白溶液(2 mg/ml)中且接著以0.5 ml/孔將細胞-膠原蛋白混合物負載至24孔培養盤且在膠原蛋白聚合之後用1 ml培養介質覆蓋。為引起收縮,用無菌刮刀自培養皿之側面輕緩地解離膠原蛋白凝膠。在不同時間處記錄膠原蛋白凝膠大小(收縮指標)之直徑變化且藉由ImageJ軟體將其定量。創傷癒合 檢定
將細胞接種於6孔培養皿中。用無菌200 pL塑膠滴管尖端將創傷引入至細胞之匯合單層以創造經剝蝕區域。在顯微鏡下攝影0及48小時處之朝向創傷區域之中心移動的細胞。定量即時 PCR (qRT-PCR)
根據製造商的協議(Invitrogen Life Technologies,Carlsbad,CA,USA)使用Trizol試劑自細胞提取總RNA。使用用於RT-PCR之Superscript III第一鏈合成系統(Invitrogen Life Technologies,Carlsbad,CA,USA)反轉錄mRNA之qRT-PCR。在ABI StepOneTM即時PCR系統(應用生物系統(Applied Biosystem))上執行所得cDNA上之qRT-PCR反應。西方墨點分析
如先前所描述進行西方墨點分析(Yang PY等人,2017)。針對COL1A1、α-SMA、FN1及波形蛋白之一級抗體購自Santa Cruz Biotechnology, Inc. (Santa Cruz,CA,USA)。在一級抗體之後,膜與對應二級抗體一起培育。免疫反應性波段使用ECL增強版化學發光基板(Perkin-Elmer,Waltham,MA,USA)顯影且由LAS-1000增強版發光影像分析器捕獲(GE Healthcare,Piscataway,NJ,USA)。酶聯 免疫吸附分析 (ELISA)
針對IL-6及IL-8之檢測,在6孔培養盤中將fBMF與不同濃度之GMI一起培養48小時。收集細胞上清液且經離心以移除死亡的細胞。接著根據製造商的協議使用來自eBioscience之IL-6或IL-8特定套組藉由ELISA分析上清液。統計分析
SPSS統計版本13.0用於統計分析。司徒頓t試驗(Student's t test)或ANOVA分析用於測定實驗組之間的差異的統計顯著性;p值少於0.05視為在統計學上顯著。實 例 1 GMI 降低 fBMF 之膠原蛋白收縮及創傷癒合能力
肌纖維母細胞為特定收縮細胞且引起創傷閉合。吾等發現經GMI處理之fBMF在10 μg/ml及20 μg/ml下顯示細胞收縮能力之顯著降低(圖1A)。此外,創傷癒合能力在不經GMI處理之情況下與fBMF細胞相比在兩種濃度下明顯地受抑制(圖1B),從而指示GMI具有阻礙肌纖維母細胞之細胞遷移的潛能。實例 2 GMI 下調 fBMF 之遷移及 侵襲能力
為進一步評估GMI對經活化肌纖維母細胞特性之影響,用不同濃度之GMI處理fBMF且使用傳斯維爾系統(transwell system)檢測細胞遷移及侵襲能力。如圖2A中所展示,吾等觀測到GMI對遷移能力之劑量依賴性效果。此外,吾人之結果證實肌纖維母細胞之侵襲在20 μg/ml GMI之存在下減少了70% (圖2B)。當與來自遷移檢定之結果相關聯時,吾等展示GMI在肌纖維母細胞中展現顯著的抗侵襲能力。實例 3 GMI 抑止經檳榔鹼刺激之 BMF 中之肌纖維母細胞活性
吾人之先前研究已證實檳榔鹼(檳榔子中發現之生物鹼)可誘導BMF之肌纖維母細胞轉分化,因此,吾等試圖在以下試驗中使用經檳榔鹼處理之BMF驗證吾人之發現。如所預期,檳榔鹼處理誘導BMF之膠原蛋白收縮能力,而GMI之施加顯著地逆轉此現象(圖3A)。同樣,GMI以劑量依賴性形式降低藉由檳榔鹼誘導之創傷癒合能力(圖4B)。另外,增加的遷移能力顯著地藉由GMI處理阻斷(圖4A)。且檳榔鹼刺激之後的BMF之侵襲性表現型亦藉由GMI抑制(圖4B)。實 例 4 GMI 減少 fBMF 中之肌纖維母細胞標 記物表現及促炎性細胞介素
為調查下伏於GMI之抗纖維化效果的可能機制,吾等評估fBMF中之纖維發生標記物之表現。如圖5A中所展示,GMI以劑量依賴性方式顯著地下調此等fBMF中之I型膠原蛋白(COL1A1)、α-SMA、纖維結合蛋白(FN)及波形蛋白之mRNA表現。當符合基因表現時,COL1A1、α-SMA、FN及波形蛋白之蛋白量亦以劑量依賴性方式在fBMF中下調(圖5B)。另外,吾等檢測到促炎性細胞介素在此等fBMF中之表現,此係因為發現GMI展現抗發炎效果。且吾人之結果展示,在暴露GMI之後存在IL-6或IL-8之劑量依賴性抑止(圖5C及圖5D)。Unless otherwise defined, all technical and scientific terms used herein have the same meaning meaning meaning Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference. In the present application, the use of the singular "a" or "an" means "at least one", and the use of "or" means "and /". or". In the present application, the word "comprise" or variant such as "comprises" or "comprising" includes any such integer or integer group but does not exclude the specified method, structure or composition. Any other integer or integer group. As used herein, "inhibit/inhibiting", "preventing/preventing" or "reduce/reducing" are used interchangeably herein. These terms refer to the reduction in measured parameters in treated cells (tissue or individual) compared to untreated cells (tissue or individual). Comparisons can also be made from the same cells or tissues or individuals between pre-treatment and post-treatment. Decrease to be sufficient for detection. As used herein, "treatment" or "treating" or "ameliorating" is used interchangeably herein. These terms refer to methods of obtaining beneficial or desired results including, but not limited to, therapeutic benefits and/or prophylactic benefits. Therapeutic benefits are related to eradicating or ameliorating the underlying condition being treated. In addition, the therapeutic benefit is achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying condition such that an improvement is observed in the patient, but the patient may still be suffering from a potential condition. Those in need of treatment include those already suffering from a condition or disorder as well as those prone to have the condition or disorder or the condition or disorder in the body to be prevented. As used herein, "therapeutically effective amount" means an amount sufficient to treat an individual suffering from a disease or to alleviate a symptom or complication associated with the disease. As used herein, "individual" refers to a human or non-human animal. In one aspect, the invention provides a method of preventing or treating fibrosis in an individual comprising administering to the individual an effective amount of an immunomodulatory protein derived from Ganoderma lucidum, Ganoderma lucidum, Glomus lucidum or Ganoderma lucidum, thereby Prevent or treat fibrosis. In one embodiment, the fibrosis is selected from the group consisting of cardiac fibrosis, scleroderma, skeletal muscle fibrosis, liver fibrosis, renal fibrosis, pulmonary fibrosis, diabetic fibrosis, or OSF. In another aspect, the fibrosis is OSF. In some embodiments, the immunomodulatory protein is LZ-8 derived from Ganoderma lucidum, FIP-gts derived from Ganoderma lucidum, GMI derived from microspores Ganoderma lucidum or FIP-gja derived from Ganoderma lucidum. In additional embodiments, the immunomodulatory protein has the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2. The sequences of SEQ ID NO: 1 (i.e., GMI described herein) and SEQ ID NO: 2 are listed below. In one embodiment, the method reduces myofibroblastic activity by inhibiting collagen gel contraction, migration, invasion, and wound healing capabilities. In another embodiment, the method reduces the amount of interleukin (IL)-6 and IL-8 expression. Accordingly, the present invention provides a method of reducing myofibroblast activity in a cell or an individual comprising contacting or administering to the individual an immunomodulatory protein derived from Ganoderma lucidum, Ganoderma lucidum, Glomus lucidum or Ganoderma lucidum Immunomodulatory protein. In some embodiments, the immunomodulatory protein is LZ-8 derived from Ganoderma lucidum, FIP-gts derived from Ganoderma lucidum, GMI derived from microspores Ganoderma lucidum or FIP-gja derived from Ganoderma lucidum. In additional embodiments, the immunomodulatory protein has the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, myofibroblastic activity is reduced by inhibiting collagen gel contraction, migration, invasion, and wound healing capabilities. In another embodiment, the method reduces the amount of interleukin (IL)-6 and IL-8 expression. In one embodiment, an effective amount for use in the methods of the invention ranges from about 25 mg/kg to about 55 mg/kg body weight of immunomodulatory protein. In some embodiments, the effective amount ranges from about 25 mg/kg to about 50 mg/kg, from about 25 mg/kg to about 45 mg/kg, from about 25 mg/kg to about 40 mg/kg, about 25 mg. /kg to about 35 mg/kg, from about 25 mg/kg to about 30 mg/kg, from about 30 mg/kg to about 55 mg/kg, from about 32 mg/kg to about 55 mg/kg, about 34 mg/kg To about 55 mg/kg, from about 36 mg/kg to about 55 mg/kg, from about 38 mg/kg to about 55 mg/kg, from about 40 mg/kg to about 55 mg/kg, from about 30 mg/kg to about 50 mg/kg, from about 32 mg/kg to about 50 mg/kg, from about 34 mg/kg to about 50 mg/kg, from about 36 mg/kg to about 50 mg/kg, from about 38 mg/kg to about 50 mg /kg, from about 40 mg/kg to about 50 mg/kg, from about 30 mg/kg to about 45 mg/kg, from about 32 mg/kg to about 45 mg/kg, from about 34 mg/kg to about 45 mg/kg From about 36 mg/kg to about 45 mg/kg, from about 38 mg/kg to about 45 mg/kg, from about 40 mg/kg to about 45 mg/kg, from about 30 mg/kg to about 40 mg/kg, about From 32 mg/kg to about 40 mg/kg, from about 34 mg/kg to about 40 mg/kg or from about 36 mg/kg to about 40 mg/kg body weight. In an additional embodiment, the effective amount ranges from about 36 mg/kg to about 40 mg/kg body weight. The immunomodulatory proteins of the invention, or recombination thereof, can be administered to a patient separately or in the form of a pharmaceutical composition wherein it is mixed with a suitable carrier and excipient. The immunomodulatory proteins of the invention, or a recombination or composition thereof, may be administered parenterally, such as by intravenous or infusion, intraperitoneal injection, subcutaneous injection or intramuscular injection. The immunomodulatory protein or a recombination or composition thereof can be administered orally or via a suitable formulation with carriers and/or excipients to form lozenges, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like. Things to vote for. The immunomodulatory protein or a recombination or composition thereof can be administered topically, such as by dermal patching. The immunomodulatory protein or a recombination or composition thereof can be formulated as a topical cream, dermal patch or mucosal patch, liquid or gel suitable for topical application to the mucosal surface. Dosages of immunomodulatory proteins or combinations or compositions thereof suitable for use in accordance with the present invention can be determined by those skilled in the art based on the disclosure herein. The agent will contain an effective amount (depending on the route of administration of the active agent and pharmacokinetics) of the immunomodulatory protein or a recombination or combination thereof. Suitable pharmaceutical carriers and excipients are suitable for the particular route of administration of the formulation (i.e., orally, parenterally, topically or by inhalation). The immunomodulatory protein or a recombination thereof is mixed into the pharmaceutical composition by means of mixing, dissolving, granulating, dragee manufacturing, emulsifying, encapsulating, coating or lyophilizing processes. Pharmaceutical compositions for parenteral administration include aqueous solutions of immunomodulatory proteins in water soluble form or recombination thereof. Alternatively, the immunomodulatory protein or a reconstituted suspension thereof can be prepared as an oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil; or synthetic fatty acid esters such as ethyl oleate or triglycerides; or liposomes. Aqueous injection suspensions may contain materials which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or polydextrose. The suspension may optionally contain stabilizers or agents to increase the solubility of the complex or combination to allow for a more concentrated solution. Without further elaboration, it is apparent to those skilled in the art that the invention can Therefore, the following examples are to be construed as illustrative only and not limiting the scope of the invention in any way. Example GMI Protein GMI manufactured by Mycomagic Biotechnology Co., Ltd. (Taipei, Taiwan) was produced and improved by microspores. GMI was prepared according to the method described in US 7,601,808. OSF Tissue Acquisition and Cell Culture Isolation of Fibrotic BMF (fBMF) from OSF patients recruited from Oral Medicine Center (Chung Shan Medical University Hospital, Taichung, Taiwan) with informed consent and agreement approved by the Institutional Review Board of Chung Shan Medical University Hospital ). Fibroblasts derived from normal buccal mucosa (BMF) and fBMF are cultured according to criteria and methods known in the art. Cell culture between the third and eighth generations was used for this study. MTT assay For the determination of the cytotoxic effect of GMI in fBMF and BMF, cells were seeded in 24 well culture at 1×10 5 cells/500 gl/well in the presence of different concentrations of GMI or vehicle at 37 °C. After 48 hours in the pan, subsequent treatment with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was carried out. The blue cellulite crystals of living cells were dissolved in DMSO and then evaluated spectrophotometrically at 570 nm. The DMSO treatment group was set to 100% and the data presented as a percentage of the DMSO control. IC50 values were calculated by GraFit software (Erithacus Software Ltd., West Sussex, UK). Collagen gel shrinkage assay The biological activity of myofibroblast function was performed by collagen shrinkage assay (Sigma-Aldrich Chemical Co., St. Louis, MO, USA). 2×10 5 cells were suspended in cold collagen solution (2 mg/ml) and then the cell-collagen mixture was loaded to a 24-well culture plate at 0.5 ml/well and 1 ml culture medium was used after collagen polymerization. cover. To cause contraction, the collagen gel was gently dissociated from the side of the dish with a sterile spatula. Diameter changes in collagen gel size (contraction index) were recorded at different times and quantified by ImageJ software. Wound healing assay Cells were seeded in 6-well culture dishes. A wound is introduced into the confluent monolayer of cells using a sterile 200 pL plastic dropper tip to create a denuded area. Cells that moved toward the center of the wound area at 0 and 48 hours under the microscope. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from cells using Trizol reagent according to the manufacturer's protocol (Invitrogen Life Technologies, Carlsbad, CA, USA). qRT-PCR for reverse transcription of mRNA was performed using Superscript III First Strand Synthesis System (Invitrogen Life Technologies, Carlsbad, CA, USA) for RT-PCR. The qRT-PCR reaction on the resulting cDNA was performed on an ABI StepOneTM real-time PCR system (Applied Biosystem). Western blot analysis Western blot analysis was performed as previously described (Yang PY et al., 2017). One of the antibodies against COL1A1, α-SMA, FN1 and vimentin was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Following the primary antibody, the membrane is incubated with the corresponding secondary antibody. The immunoreactive bands were developed using an ECL enhanced version of a chemiluminescent substrate (Perkin-Elmer, Waltham, MA, USA) and captured by a LAS-1000 enhanced luminescence imager (GE Healthcare, Piscataway, NJ, USA). Enzyme-linked immunosorbent assay (ELISA) for the detection of IL-6 and IL-8, the 6-well plates in the fBMF incubated with varying concentrations of GMI 48 hours. The cell supernatant was collected and centrifuged to remove dead cells. The supernatant was then analyzed by ELISA using an IL-6 or IL-8 specific kit from eBioscience according to the manufacturer's protocol. Statistical analysis SPSS statistical version 13.0 was used for statistical analysis. The Student's t test or ANOVA analysis was used to determine the statistical significance of the differences between the experimental groups; a p value of less than 0.05 was considered statistically significant. Examples 1 GMI reduced collagen contraction and wound healing fBMF of myofibroblasts specific contractile cells and cause wound closure. We found that GMI-treated fBMF showed a significant decrease in cell shrinkage at 10 μg/ml and 20 μg/ml (Fig. 1A). Furthermore, wound healing ability was significantly inhibited at both concentrations compared to fBMF cells without GMI treatment (Fig. IB), indicating that GMI has the potential to block cell migration of myofibroblasts. Example 2 GMI Down-regulation of migration and invasion of fBMF To further assess the effect of GMI on the properties of activated myofibroblasts, fBMF was treated with different concentrations of GMI and cell migration and invasion abilities were measured using a transwell system. As shown in Figure 2A, we observed a dose-dependent effect of GMI on migration ability. Furthermore, our results confirmed that myofibroblastic invasion was reduced by 70% in the presence of 20 μg/ml GMI (Fig. 2B). When associated with results from migration assays, we demonstrate that GMI exhibits significant anti-invasive ability in myofibroblasts. Example 3 GMI inhibits myofibroblastic activity in benithine- stimulated BMF Previous studies have confirmed that arecoline (alkaloid found in betel nut) can induce BMF myofibroblast transdifferentiation, therefore, we tried to The BMF treated with arecoline was used to verify our findings. As expected, arecoline treatment induced collagen contractility of BMF, whereas application of GMI significantly reversed this phenomenon (Fig. 3A). Similarly, GMI reduced the ability of wound healing induced by arecoline in a dose-dependent manner (Fig. 4B). In addition, the increased migration capacity was significantly blocked by GMI processing (Fig. 4A). The invasive phenotype of BMF after arecoline stimulation was also inhibited by GMI (Fig. 4B). Examples of GMI. 4 fBMF reduction in the myofibroblast marker exhibits pro-inflammatory cytokine and of the possible mechanisms underlying the anti-fibrotic effect GMI under investigation, evaluation fBMF Wudeng fibrogenesis in the performance of the marker. As shown in Figure 5A, GMI significantly down-regulated the mRNA expression of type I collagen (COL1A1), α-SMA, fibronectin (FN), and vimentin in these fBMFs in a dose-dependent manner. The protein levels of COL1A1, α-SMA, FN, and vimentin were also down-regulated in fBMF in a dose-dependent manner when consistent with gene expression (Fig. 5B). In addition, we detected the expression of pro-inflammatory interleukins in these fBMFs because GV was found to exhibit anti-inflammatory effects. And our results show that there is a dose-dependent inhibition of IL-6 or IL-8 after exposure to GMI (Figure 5C and Figure 5D).