Mechanism of action of cordycepin in the treatment of hepatocellular carcinoma via regulation of the Hippo signaling pathway

Hepatocellular carcinoma(HCC)is one of the common most malignant tumors.This study aimed to determine the in vitro and in vivo anticancer activity of cordycepin and elucidate its mechanism of action.The results of in vitro and in vivo studies revealed that cordycepin inhibited proliferation and migration in HepG-2 cells and inhibited the growth of HepG-2 xenograft-bearing nude mice by inducing apoptosis.Transcriptome sequencing analysis revealed a total of 403 differential genes,which revealed that cordycepin may play an anti-HCC role by regulating Hippo signaling pathway.The regulatory effects of cordycepin on the Hippo signaling pathway was further investigated using a YAP1 inhibitor.The results demonstrated that cordycepin upregulated the expression of MST1 and LAST1,and subsequently inhibited YAP1,which activated the Hippo signaling pathway.This in turn downregulated the expression of GBP3 and ETV5,and subsequently inhibited cell proliferation and migration.Additionally,YAP1 regulated the expression of Bax and Bcl-2,regulated the mitochondrial apoptotic pathway,and induced apoptosis by upregulating the expression of the caspase-3 protein.In summary,this study reveals that cordycepin exerts its anti-hepatocarcinoma effect through regulating Hippo signaling pathway,and GBP3 and ETV5 may be potential therapeutic targets for hepatocarcinoma.

国产虫草素(cordycepin)抗小鼠迟发型超敏反应的实验研究

目的:研究国产虫草素抗小鼠迟发型超敏反应的作用及其免疫机理。方法:用2,4-二硝基氯苯(2,4-DNCB)对昆明种小鼠皮肤致敏和诱发,制成迟发型超敏反应模型。以国产虫草素溶液作为实验组,分为两个剂量组(实验组1:12mg/kg;实验组2:16mg/kg),以生理盐水作为阴性对照组,所有溶液均为间隔24小时(最后一次间隔为4小时)重复腹腔注射给药。计算激发后各组小鼠左右耳耳廓肿胀厚度差、耳廓增重值及肿胀度,进行统计分析,并经过HE染色观察虫草素对小鼠致炎耳炎症病理变化及脾脏病理变化的影响。结果:两种给药剂量的国产虫草素溶液对模型鼠红斑,水肿,渗出的接触性皮炎损害有明显抑制作用,可减轻由于充血、水肿等炎症反应导致的局部皮损增厚及重量的增加。与生理盐水对照组相比,两实验组虫草素溶液的抗炎作用均有显著性差异(厚度差:P<0·0001;肿胀度:P<0·05);两实验组间的抗炎作用也有显著性差异(厚度差及肿胀度均为P<0·05),且与给药剂量相关。三组小鼠脾指数未见明显差异(P>0·05),脾脏组织病理变化未见明显差异。结论:虫草素以24小时间隔(最后一次给药间隔为4小时)经腹腔注射后,可能通过其免疫调节作用对迟发型超敏反应引起的小鼠接触性皮炎发挥明显的抑制效应,该效应与给药剂量相关,同时对脾脏组织未见明显毒性作用。

虫草素及其代谢物3′-脱氧肌苷在大鼠体内的药动学

目的建立血浆虫草素及代谢物3’-脱氧肌苷(3′-Deo)的液相色谱-串联质谱(LC-MS/MS)测定方法,研究其在大鼠体内的药动学。方法以2-氯腺苷(2-Chl)为内标,甲醇沉淀蛋白,色谱柱为Kinetex C18(3 mm×100 mm,2.6μm),流动相为水(5 mmol·L^(-1)乙酸铵)-甲醇溶液,梯度洗脱。电喷雾离子源,正离子多反应监测。研究大鼠灌胃虫草素(10 mg·kg^(-1))后血浆虫草素及3′-Deo药动学。结果虫草素和3′-Deo分别在0.5~100和1~200 ng·mL^(-1)范围内线性关系良好,定量下限分别为0.5和1 ng·mL^(-1)。大鼠灌胃虫草素后,血浆虫草素浓度较低,主要转化为3′-Deo。虫草素和3′-Deo的峰浓度(C_(max))分别为(5.4±3.4)和(142.0±50.0)ng·mL^(-1),血药浓度-时间曲线下面积(AUC_(0-360 min))分别为(658.4±459.3)和(18034.9±4981.1)ng·min·mL^(-1)。结论该方法简单、灵敏、准确,适用于虫草素与3′-Deo血药浓度测定及药动学研究。

北虫草饮品配方优化及其理化特性

以北虫草粉为主要原料,添加麦芽糖醇、羧甲基纤维素钠、柠檬酸和D-异抗坏血酸钠制作北虫草饮品。通过单因素试验和响应面试验对北虫草饮品的配方进行优化,测定酸价、过氧化值等指标。最佳工艺条件结果表明:羧甲基纤维素钠和六偏磷酸钠添加量为0.04%,北虫草粉添加量为9.5%,麦芽糖醇和木糖醇添加量为6.4%,柠檬酸和D-异抗坏血酸钠添加量为0.25%,该条件下,各项理化性质均符合饮品的质量和卫生指标,北虫草饮品的差异性化合物主要有虫草素、苏氨酸、甲硫氨酸。

蛹虫草缓解新型冠状病毒感染研究进展

严重急性呼吸综合征冠状病毒2(SARS-CoV-2)引起的COVID-19在全球范围内大流行,危害了人类健康和公共安全,随着对SARS-CoV-2的结构、功能和致病过程的了解,越来越多的潜在药物被开发。蛹虫草(Cordyceps militaris)是我国传统的药用真菌,具有显著的抗病毒作用,虫草素作为蛹虫草的主要活性成分能够与SARS-CoV-2刺突蛋白、主蛋白酶(Mpro)相结合,抑制病毒RNA依赖的RNA聚合酶(RDRP)活性,阻断SARS-CoV-2在机体内复制。蛹虫草还具有提升机体免疫力、修复受损组织的作用。本文概述虫草素抗SARS-CoV-2的机制和蛹虫草其他相关药理作用,以期为蛹虫草用于新型冠状病毒感染的辅助治疗提供参考。

虫草素对穿支皮瓣存活的影响及其机制

目的:探讨虫草素对于穿支皮瓣存活的影响及可能机制。方法:将20只雄性SD大鼠随机分为对照组和虫草素组,每组10只。构建背部穿支皮瓣模型,虫草素组给予虫草素20mg/kg,对照组给予0.9%氯化钠溶液,连续7d。观察各组术后7d皮瓣远端坏死情况以及血流灌注情况,同时取各组皮瓣组织检测皮下水肿率。通过病理学染色、氧化应激相关试剂盒检测、Tunel染色、Westernblot等方法检测虫草素对于穿支皮瓣存活的作用以及相应组织内氧化应激、细胞凋亡和自噬指标的变化。结果:术后7d,各组穿支皮瓣远端均出现不同程度的坏死。虫草素组相较于对照组,皮瓣远端坏死率以及皮下水肿率更低,有更佳的血流灌注(P<0.05)。HE染色及Masson染色结果显示虫草素组皮瓣内微小血管更多,皮下结构和胶原排列更加有序。氧化应激相关指标检测结果显示,虫草素组的丙二醛含量下调(P<0.05)、超氧化物歧化酶和谷胱甘肽含量上调(P<0.05)。Tunel荧光染色结果显示,虫草素组凋亡细胞更少(P<0.05)。Westernblot结果显示,虫草素可以调控皮瓣组织内氧化应激相关蛋白核因子E2相关因子2、血红素加氧酶-1水平升高(P<0.05),凋亡相关蛋白Bcl-2水平升高、cleaved-caspase3和Bax水平下降(P<0.05),自噬相关蛋白p-AMPK和微管相关蛋白轻链3II水平升高、p62水平下降(P<0.05)。结论:虫草素能促进穿支皮瓣远端存活,其机制可能是通过抑制氧化应激、抑制细胞凋亡和调控自噬水平起作用。

虫草素及其在生猪养殖中的应用

“禁抗令”出台后,“替抗”已成为畜牧业的热门话题。虫草素是蛹虫草的核心活性成分,具有广谱抗菌、抗氧化和调节免疫等功能,且取材和化学成分天然、不易产生抗药性、在体内无有害残留等突出优点,将有可能为“替抗”做出贡献。本文主要对虫草素的来源、合成方式、药理作用进行综述,并重点探讨虫草素在生猪养殖业中的应用进展,旨在为开发虫草素产品并应用于生猪养殖提供一定的参考信息。

米曲霉异源表达合成虫草素

【目的】通过米曲霉中异源表达虫草素合成关键基因,构建米曲霉工程菌株合成虫草素。【方法】以米曲霉尿苷/尿嘧啶和组氨酸双营养缺陷型菌株AoΔpyrGΔHisB为背景菌株,利用农杆菌介导的转化方法对虫草素合成关键基因CmCns1-CmCns3进行过表达;通过荧光显微镜观察CmCns1和CmCns2在米曲霉中的亚细胞定位;利用高效液相色谱法(HPLC)测定转基因米曲霉虫草素含量,同时对米曲霉发酵液添加合成虫草素的前体甘氨酸和腺嘌呤,探究其在米曲霉中对合成虫草素的影响。【结果】蛹虫草CmCns1和CmCns2在米曲霉中定位于脂滴;并且米曲霉中单独过表达CmCns1、共表达CmCns1和CmCns2以及共表达CmCns1-CmCns3均能合成虫草素,发酵48 h虫草素胞外产量最高达到37.74 μg/mL;向米曲霉发酵液添加甘氨酸和腺嘌呤,不能有效提升虫草素的含量。【结论】成功在米曲霉异源表达合成虫草素。

不同种类氨基酸对蛹虫草发酵液中虫草素含量的影响

[目的]研究液体培养基中添加氨基酸对蛹虫草发酵液中虫草素含量的影响,并确定8种氨基酸最佳添加浓度。[方法]考察24种氨基酸(添加浓度为1 g/L)对虫草素含量的影响,采用DPS V18.10软件进行差异显著性分析,并对促进虫草素合成作用显著的8种氨基酸添加浓度进行研究。[结果]L-赖氨酸、L-半胱氨酸、L-精氨酸、L-天门冬酰胺、L-甘氨酸、L-丙氨酸、L-丝氨酸、L-苏氨酸、L-组氨酸、肌氨酸对蛹虫草发酵液中虫草素含量具有不同程度的提高作用;4 g/L的L-甘氨酸对虫草素的合成促进作用最强,发酵液中虫草素含量为1022.25 mg/L。[结论]该研究为蛹虫草液体发酵生产虫草素提供参考依据。

Anti-Cancer Effects of Cordycepin on Oral Squamous Cell Carcinoma Proliferation and Apoptosis in Vitro

Cordycepin is an active component of parasitic fungus, Cordyceps militaris, and investigated for its pharmacologic efficacy. Increasing evidence supports the anti-tumoral effects of Cordycepin in various types of human solid tumors. We sought to determine the effects of Cordycepin on oral squamous cell carcinoma in vitro and in vivo. Two oral squamous cell carcinoma cell lines, KB and HSC3, were used in this study. Cells were treated with Cordycepin or diluent, followed by determinations of proliferation by sulforhodamine method and apoptosis by TUNEL assay in vitro. For in vivo experiments, tumor cells were transplanted into nude mice, followed by treatment with Cordycepin or control diluent. In addition, cells were examined for expression of adenosine receptor isotypes, and tested whether cordycepin-induced effects were mediated through adenosine receptors by combinatorial treatment of cordycepin and antagonists specific to each isotype of adenosine receptors. Two cell lines expressed protein of all types of adenosine receptors stronger than normal oral keratinocytes. Cordycepin showed anti-proliferating effect and apoptotic effect on both cell lines in vitro in a dose dependent manner. However, any adenosine receptors did not reverse the effect of cordycepin. In our in vivo experiments, cordycepin failed to decrease the tumor volume significantly, and failed to induce more apoptosis of tumor cells. Cordycepin has anti-proliferating effect and induces apoptosis not mediated by adenosine receptor on oral squamous cell carcinoma cells in vitro. However, in vivo results suggest that cordycepin in itself has a limited value as a novel chemotherapeutic agent for oral squamous cell carcinoma.

Determination of Cordycepin in Cordyceps kyushuensis by Capillary Electrophoresis and its Antitumour Activity

A simple, rapid and low-cost method of determination for cordycepin in Cordyceps kyushuensis by capillary zone electrophoresis (CZE) was developed. Based on the finding that there is a high concentration of cordycepin in both natural and cultured Cordyceps kyushuensis, the in vitro antitumor activity of cordycepin and the water extracts of Cordyceps kyushuensis has been investigated. This is the first report about the antitumor effect of Cordyceps kyushuensis.

Layered Double Hydroxide as Cordycepin Delivery Nanocarrier

Layered double hydroxide was investigated as cordycepin delivery nanocarrier for the first time in this study. Negatively charged biomolecule-cordycepin was intercalated in the gallery spaces of [Mg-Al-NO3], which was corff＇trmed by the results of X-ray diffraction and electrophoretic mobility. Cell experiment suggested that the new bio-LDH nanohybrid could prevent cordycepin decomposition by adenosine deaminase. This new formulation could possibly be used as a novel form cordycepin intravenous injection.

Study on Intercalative Nanohybrid of Cordycepin in Layered Double Hydroxide

A novel negatively charged biomolecule-cordycepin has been intercalated within the gallery spaces of [Mg-Al-NO3]. Results of TEM, PXRD and FT-IR spectroscopy confirmed that cordycepin could be intercalated into [Mg-Al-NO3] interlayers as the charge-compensating species. Initial studies suggest that the new bioinorganic nanocomposite may be used as a novel inorganic reservoir or carrier of pharmaceutically active compounds.

Preliminary Study on Cordycepin-DNA Interaction by Raman Spectroscopy

The interaction of cordycepin with calf thymus DNA was investigated at physiological pH with drug/DNA molar ratio of 8. The Raman spectroscopy results indicated that the intercalation of high concentration cordycepin and the interaction of cordycepin with PO2 group led to a major reduction of B-form DNA structure in favor of A-form DNA.

Matrix-assisted laser desorption ionization mass spectrometry based quantitative analysis of cordycepin from Cordyceps militaris

Cordycepin,which has great immunomodulatory activities such as anticancer,antifungal,antivirus,antileukemia and lipid-lowering ones,is the secondary metabolite of Cordyceps militaris(C.militaris).Liquid submerged fermentation is the common cultivation process to produce cordycepin.To optimize the fermentation process and improve production,monitoring the cordycepin secretion in the fermentation is essential.The measurement based on chromatography-mass spectrometry methods is generally involved in the complex sample pretreatments and time-consuming separation,so more rapid and convenient methods are required.Matrix-assisted laser desorption ionization mass spectrometry(MALDI-MS)is more attractive for faster and direct detection.Therefore,MALDI-MS detection combined with isotope-labeled internal standard was applied to the measurement of cordycepin content in the fermentation broth and mycelium.This method made accurate quantification of cordycepin in the range of 5-400μg/mL with a relative standard deviation of 5.6%.The recovery rates of fermentation samples after the 1,13,and 25 days were 90.15%,94.27%,and 95.06%,respectively.The contents of cordycepin in the mycelium and fermentation broth were 136 mg/g and 148.39 mg/mL on the 20 th culture day,respectively.The cordycepin secretion curve of the liquid fermentation of C.militaris was real-time traced over 25 days.

虫草素对脓毒症幼鼠急性肝损伤的改善作用

目的 探讨虫草素(Cor)对脓毒症幼鼠急性肝损伤的改善及对肝细胞自噬的影响。方法 将65只SD幼鼠随机分为假手术组、模型组、Cor低剂量组和Cor高剂量组,Cor低剂量、Cor高剂量组分别灌胃给予10、20 mg·kg^(-1)虫草素,1日1次,连续7 d;假手术组、模型组灌胃等量生理盐水,除假手术组外,其余各组复制脓毒症幼鼠模型。检测血清中丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)和乳酸脱氢酶(LDH)水平;ELISA检测血清中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)和IL-6含量;计算肝脏指数;HE染色观察幼鼠肝组织病理变化;TUNEL法检测幼鼠肝组织细胞凋亡;Western blot检测自噬相关蛋白的表达。结果 与假手术组比较,模型组、Cor低剂量、Cor高剂量组ALT、AST和LDH水平升高,血清TNF-α、IL-1β和IL-6含量增加,细胞凋亡率升高(P<0.05);与假手术组比较,模型组、Cor低剂量组肝脏指数升高(P<0.05),模型组微管相关蛋白1轻链3Ⅱ(LC3-Ⅱ)、Beclin1相对表达量降低,p62相对表达量升高(P<0.05),Cor高剂量组LC3-Ⅱ、Beclin1相对表达量升高,p62相对表达量降低(P<0.05);与模型组比较,Cor低剂量、高剂量组ALT、AST、LDH水平、肝脏指数和细胞凋亡率降低,TNF-α、IL-1β和IL-6含量减少,LC3-Ⅱ、自噬基因Beclin1相对表达量升高,p62相对表达量降低(P<0.05);与Cor低剂量组比较,Cor高剂量组ALT、AST、LDH水平、肝脏指数和细胞凋亡率降低,TNF-α、IL-1β和IL-6含量减少,LC3-Ⅱ、Beclin1相对表达量升高,p62相对表达量降低(P<0.05);HE染色显示,假手术组肝细胞细胞核圆且大,细胞排列整齐、分布均匀,肝小叶欠清晰,炎症细胞浸润较少;模型组肝细胞排列紊乱、肿胀甚至出现坏死,肝小叶结构破坏,出现灶性松散的炎症细胞浸润;Cor低剂量、高剂量组肝细胞排列相对整齐,炎症细胞浸润减轻,肝小叶结构相对清晰,Cor高剂量组肝细胞结构、排列更清晰,炎症细胞浸润更少。结论 Cor可以显著改善脓毒症幼鼠的急性肝损伤症状并诱导肝细胞自噬。

Cordycepin regulates the malignant biological behaviors of lung cancer cell lines A549

Objective:To study the effect of cordycepin on proliferation, apoptosis and invasion-related molecule expression in lung cancer cell lines A549. Methods:Lung cancer cell lines A549 were cultured and treated with different doses of cordycepin (0, 0.25, 0.5, 1.0, 2.0 and 4.0 ng/mL) for 24 h, and then the proliferation, apoptosis and invasion-related molecule mRNA expression in cells were detected. Results:After 0.25, 0.5, 1.0, 2.0 and 4.0 ng/mL cordycepin treatment, Caspase-3, Caspase-8, NOX1 and LATS1 mRNA expression were significantly higher than those after 0 ng/mL cordycepin treatment (P<0.05) while CyclinD1, Bcl-2, c-Myc, c-FLIP, TRAF6, N-cadherin and Vimentin mRNA expression were significantly lower than those after 0 ng/mL cordycepin treatment (P<0.05). The greater the cordycepin dosage, the higher the Caspase-3, Caspase-8, NOX1 and LATS1 mRNA expression, and the lower the CyclinD1, Bcl-2, c-Myc, c-FLIP, TRAF6, N-cadherin and Vimentin mRNA expression. Conclusions: Cordycepin can promote pro-apoptosis gene expression and inhibit pro-proliferation and pro-invasion gene expression in lung cancer cell lines A549.

Effect of cordycepin on non-small cell lung cancer cell line H358 proliferation and related gene expression

Objective: To study the effect of cordycepin on non-small cell lung cancer cell line H358 proliferation and related gene expression. Methods: C57BL/6 mice were selected as experimental animals, and the mouse model with transplanted tumor was established after non-small cell lung cancer cell line H358 was cultured;the mice with transplanted tumor were divided into the intervention group who received Corbrin Capsule therapy and the model group who received no drug therapy. 30 d after treatment, the expression of proliferation activity indexes, proliferation genes and invasion genes in transplanted tumor tissue were determined. Results: 30 d after treatment, the mRNA expression and protein expression of PCNA and Ki-67 in transplanted tumor lesion of intervention group were significantly lower than those of model group, and Notch-1, Bmi-1, MIF, Rap2a, NGAL and SIRT1 mRNA expression in transplanted tumor lesion were significantly lower than those of model group while PAQR3, LATS1, E-cadherin, ZO-1 and TES mRNA expression were significantly higher than those of model group. Conclusion: Cordycepin can inhibit the proliferation and invasive growth of non-small cell lung cancer cell line H358 in transplanted tumor tissue.

基于蛹虫草转录物组学的虫草素生物合成相关基因的表达和功能分析

蛹虫草Cordyceps militaris是我国著名的中药,而虫草素是蛹虫草重要的生物活性成分之一。通过转录物组分析野生型C.militaris、虫草素高产菌株C.militaris GYS60和低产菌株C.militaris GYS80虫草素生物合成相关基因的表达和功能变化。利用Illumina NovaSeq 6000测序获得3株菌株的转录物组数据,以鉴定差异表达基因。与C.militaris相比,在GYS60和GYS80中,分别有145和470个上调基因及96和594个下调基因;GYS60与GYS80相比,GYS60有306个上调基因和207个下调基因。这些基因经过GO和KEGG分析,与C.militaris相比,GYS60和GYS80分别有10和8个与嘌呤途径相关的基因差异表达;与GYS80相比,GYS60有12个基因差异表达。定量聚合酶链式反应验证8个关键酶的基因表达与转录物组分析一致。在GYS60中,参与虫草素生物合成的嘌呤核苷酸代谢中的氧化还原酶(CCM_07507,cmORE)、3′,5′-环AMP磷酸二酯酶(CCM_02777,cmAPE)、转移酶(CCM_04722,cmTRF)和腺苷酸环化酶(CCM_06928,cmADC)表达量显著上调。这些发现提高了我们对虫草素生物合成相关基因的理解和对其他次级代谢产物代谢通路的启示。