Hypocrellin A通过ROS/JNK通路促进瘢痕疙瘩成纤维细胞凋亡的作用及机制

目的:探索Hypocrellin A(HA)通过活性氧簇(ROS)/c-Jun氨基末端激酶(JNK)通路促进瘢痕疙瘩成纤维细胞凋亡的作用及机制。方法:原代培养KFs并进行传代,第3代KFs分为对照组、不同剂量HA组(0.125、0.25、0.5、1.0μmol/L HA处理)、HA+乙酰半胱氨酸(NAC)组(1.0μmol/L HA+1.0 mmol/L NAC处理)。检测细胞增殖的光密度OD450,凋亡率,胶原代谢指标I型胶原(Col-I)、α-平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(FN)、氧化应激指标丙二醛(MDA)、超氧化物歧化酶(SOD)、总抗氧化力(T-AOC)及ROS的含量,内质网应激标志物GRP78、p-IRE-1及ROS/JNK通路中p-JNK、cleaved caspase-3的表达水平。结果:不同剂量HA组的OD450、Col-I、α-SMA、FN、SOD、T-AOC的含量均低于对照组,凋亡率、MDA、ROS含量及GRP78、p-IRE-1、p-JNK、cleaved caspase-3的表达水平均高于对照组(P<0.05)且HA剂量越高,上述指标的变化越显著;HA+NAC组的OD450、Col-I、α-SMA、FN、SOD、T-AOC的含量均高于1.0μmol/L HA组,凋亡率、MDA、ROS含量及GRP78、p-IRE-1、p-JNK、cleaved caspase-3的表达水平均低于1.0μmol/L HA(P<0.05)。结论:HA通过促进KFs凋亡,这一作用与激活ROS/JNK通路介导的氧化应激和内质网应激有关。

Hypocrellin A与二苄胺和N-甲基苄胺的光化学反应

为了探讨HypocrellinA (HA)与胺基衍生物反应的自由基形成机制 ,我们采用ESR谱分别研究了HA与二苄胺 (DBA)和N 甲基苄胺 (NMBA)的光化学反应 .在含胺类物质和溶解氧的溶剂系统下 ,可见光照射HA激发成HA ,然后将溶解氧转变成1O2 ,1O2 诱导DBA或NMBA的氯仿溶液都能产生半醌自由基和氮氧自由基 .氮氧自由基的信号强度随光照时间的增加而减弱 ,而半醌自由基的信号强度随光照时间的增加而增强 ,氮氧自由基的含量与光照产生的半醌自由基成反比 .在除氧条件下 ,光照含HA和DBA的氯仿溶液体系时 ,半醌自由基是主要的自由基 .结果表明 ,在有氧的氯仿溶液中 。

Apoptotic Effects of Hypocrellin A on HeLa Cells

Hypocrellin A（ HA）, a photosensitive perylenequinone compound of Hypocrella bambusae, inhibited the proliferation of several tumor cell lines. Human cervical cancer cells, HeLa ceils, were used as a model to elucidate the molecular mechanisms of HA-induced tumor cell death. The results show that HA can induce the oligonucleosomal fragmentation of DNA in HeLa cells and also can increase the expression of apoptosis inducer Bax mRNA and that it decreases the expression of apoptosis suppressor, Bcl-2 mRNA, in mitochondria. It can be concluded from the data that HA-induced apoptosis is related to the balance between Bcl-2 and Bax gene expressions.

High-Throughput Drug Screening on Borrelia garinii and Borrelia afzelii Identified Hypocrellin A as an Active Drug Candidate Against Borrelia Species

Lyme disease(LD)is a tick-transmitted infection caused by Borrelia burgdorferi sensu lato species,which include B.burgdorferi,Borrelia afzelii and Borrelia garinii.The majority of patients with early LD can be cured by the standard treatment,yet some still suffer from posttreatment LD syndrome.The presence of Borrelia persisters has been proposed as a contributing factor,because they cannot be completely eradicated by the currently used antibiotics for LD.Finding new pharmaceuticals targeting Borrelia persisters is crucial for developing more effective treatments.Here,we first confirmed the existence of persisters in B.garinii and B.afzelii cultures and then conducted a high-throughput screening of a customdrug library against persister-rich stationary-phase B.garinii and B.afzelii cultures.Among 2427 compounds screened,hypocrellin A(HA),anthracycline class of drugs and topical antibiotics along with some other natural compounds were identified to have strong potential for killing persisters of B.garinii and B.afzelii.HA was the most active anti-Borrelia compound,capable of eradicating stationary-phase Borrelia persisters,in particular when combined with doxycycline and/or ceftriaxone.Liposoluble antioxidant vitamin E was found to antagonize the activity of HA,indicating HA’s target is the cell membrane where HA triggers the generation of reactive oxygen species in the presence of light.HA was found to have distinct bactericidal activity against Borrelia species but had poor or no activity against gram-positive and gram-negative bacteria.Identification of the abovementioned drug candidates may help develop more effective therapies for LD.

Hypocrellin A-based photodynamic action induces apoptosis in A549 cells through ROS-mediated mitochondrial signaling pathway

Over recent decades, many studies have reported that hypocrellin A(HA) can eliminate cancer cells with proper irradiation in several cancer cell lines. However, the precise molecular mechanism underlying its anticancer effect has not been fully defined. HA-mediated cytotoxicity and apoptosis in human lung adenocarcinoma A549 cells were evaluated after photodynamic therapy(PDT). A temporal quantitative proteomics approach by isobaric tag for relative and absolute quantitation(iTRAQ) 2 D liquid chromatography with tandem mass spectrometric(LC–MS/MS) was introduced to help clarify molecular cytotoxic mechanisms and identify candidate targets of HA-induced apoptotic cell death. Specific caspaseinhibitors were used to further elucidate the molecular pathway underlying apoptosis in PDT-treated A549 cells. Finally, down-stream apoptosis-related protein was evaluated. Apoptosis induced by HA was associated with cell shrinkage, externalization of cell membrane phosphatidylserine, DNA fragmentation,and mitochondrial disruption, which were preceded by increased intracellular reactive oxygen species(ROS) generations. Further studies showed that PDT treatment with 0.08 mmol/L HA resulted in mitochondrial disruption, pronounced release of cytochrome c, and activation of caspase-3,-9, and-7.Together, HA may be a possible therapeutic agent directed toward mitochondria and a promising photodynamic anticancer candidate for further evaluation.

HYPOCRELLIN A—A NEW DRUG FOR PHOTOCHEMOTHERAPY

We have isolated a photodynamic pigment from the natural sources of Hypocrella bambusae (B. et Br.) sacc. (Hypocreaceae, Ascomycetes). The red pigment is soluble in most of the common organic solvents. It is readily soluble in both chloroform and acetone, somewhat soluble in hot light petroleum, but insoluble in water. When the acetone solution of this compound was exposed to light, it would combine with two oxygen atoms and turn into a yellowish-red compound like cladochrome. The red pigment was recrystallized from acetone (m. p. 209—210℃). Both elemental analysis (C 65.81％,

Raman spectroscopic characteristics of microcosmic and photosensitive damage on space structure of liposomes sensitized by hypocrellin and its derivatives

Raman spectroscopic characteristics of microcosmic and photosensitive damage on space structure of DPPC liposomes sensitized by hypocrellin and its derivatives are obvious. The trans conformation decreased and the gauche conformation increased. The longitudinal order_parameter in chains and the lateral order_parameter between chains decreased in different degrees. The lateral packing became loose. Photodamage on the liposomes sensitized by 5 Br hypocrellin B was stronger than that by hypocrellins A and B.

Synthesis of a hypocrellin B-polyamide conjugate as DNA-targeting photosensitizer

A new hypocrellin B （HB） derivative bearing a bispyrrolecarboxamide-containing side chain was synthesized, which presents improved absorptivity in phototherapeufic window than HB and affinity feature towards dsDNA.

Lanthanum elicitation on hypocrellin A production in mycelium cultures of Shiraia bambusicola is mediated by ROS generation

Rare earth elements(REEs)have been widely applied as efficient elicitors to improve production of biologically important secondary metabolites in plants.However,their effects on microbial secondary metabolites were seldom reported.In this study,lanthanum(LaCl3)was used as an elicitor in mycelium cultures of Shiraia bambusicola to stimulate the production of hypocrellin A(HA),a promising photosensitizer for anticancer photodynamic therapy.La^3+at 1.0 g/L not only increases HA content by 2.50-fold in mycelia,but stimulates HA exudation to the medium with the highest production(225.05 mg/L)on day 6.Further analysis shows that La^3+can induce the intracellular ROS(reactive oxygen species)accumulation with 2.33-fold higher contents of H2O2 in mycelium.HA production induced by La^3+treatment can be suppressed by ROS scavengers.Further analysis reveals that the elicitation of La^3+on HA accumulation is mediated by ROS generation to enhance expression of HA biosynthetical genes.The expression of ATP-binding cassette transporter gene(SbABC)for HA exudation is also regulated partially via ROS generated by NADPH oxidase.These results indicate that the enhanced HA production by La^3+might be mediated by ROS signaling pathway.Our results provide a basis for understanding REE elicitation on fungal metabolites and a practical biotechnological process for enhanced production of HA.

Spectroscopic Studies of a Water-soluble Derivative of Hypocrellin A and Its One- and Two-Electron Reduction Products

In this study the spectroscopic characteristics of a water-soluble derivative of hypocrellin A (HA), 14-dehydroxy-15-deacetyl-hypocrellin A-13-sulfonate(13-SO3Na-DDHA),and its one- and two-electron reduction products have been investigated. From the changes in absorbance with pH it was observed that the two phenolic hydroxy groups at C-3 and C-10 positions of 13-SO3Na-DDHA or HA dissociated stepwise with increase of pH values. The pKa values for 13-SO3Na-DDHA and HA were determined using an effective method established in this study. Attempts were also made to use absorption and ESR spectroscopies to study the photoreduction of 13-SO3Na-DDHA. It was found that 13-SO3Na-DDHA was directly reduced to its two-electron reduction product in buffered aqueous solution (pH 7. 7). However, in DMF-buffer (1 :1/ v : v,pH 7. 7), it proceeded with one-electron reduction to generate its semiquinone radical anions. The semiquinone radical anions decayed according to second-order kinetics. indicating that the termination went through protonation and disproportionation of the radical anions. Despite of four pheholic hydroxy groups in the molecule of the two-electron reduction product only two hydroxy groups seem to dissociate with increase of pH. This reflects that strong intramolecular hydrogen bonds are probably formed in the mono- and dian-ionic forms of the two-electron reduction product.

PHOTOCHEMISTRY OF HYPOCRELLIN A (V)——PHOTOREACTION OF HYPOCRELLIN A AND MERCAPTOETHANOL

I.INTRODUCTION Hypocrellin A(abbreviated HA) from Hypocrellia bambusae (B. ct Br.)sacc. growing in Yunnan Province, China, is an efficient phototherapeutic agent. Recently HA irradiation with visible light has been successfully used for the treatment of white lesions of vulva and vitiligo. In molecular level, studies on phototherapeutic mechanism have been focused on the structure, photophysical and photochemical properties of HA and its derivatives. The photoreactions, which are closely related to phototherapeutic activities, have not been re-

Photochemical properties of hypocrellin A,B and their oxides

Hypocrellin A and B in methanol were autosensitized and photooxygenated to 1,4,6, 7 and 2,5,8,9 respectively.1 in methanol was photooxygenated to 6 and 7 under the sensitization by HB.1 in methanol was photooxygenated to 7 under the sensitization by Rose bengal.1 in benzene ir- radiated with medium-pressure-mercury lamp was photolyzed to 3 in the presence of iodine.

THE REDUCTION AND NUCLEOPHILIC ADDITION OF MERCAPTOETHANOL TO HYPOCRELLIN A

Ⅰ. INTRODUCTIONHypocrellin A （abbreviated as HA）is a perylenequinone derivative. The structure, ESR and absorption spectrum of the metastable product HAH2 produced by photoinduced net two-electron reduction of HA have been reported. The absorption spec-

THE PHOTOCHEMISTRY OF HYPOCRELLIN A (Ⅷ): THE PHOTOBONDING BETWEEN HYPOCRELLIN A AND CYSTEINE AND SENSITIZED OXIDATION OF CYSTEINE

Ⅰ. INTRODUCTION Hypocrellin A ( abbreviated as HA ) extracted from Hypocrella bambusae (B et Br.) Sacc. grows abundantly in Yunnan Province, China. HA irradiated with visible light has been used successfully in the treatment of some skin diseases. Recently it has been reported that HA could be selectively retained in tumor cells and could

Photodynamic action of hypocrellin A in liposomes

Hypocrellin A (HA), a perylenequinone derivative, is an efficient phototherapeutic agent. When HA is incorporated into small unilamellar liposomes of egg phosphatidylcholine, it generates 1O2 as demonstrated by 9,10-diphenylanthracene (9,10-DPA) photobleaching and detection of nitroxide radicals with ESR. The 1O2 quantum yield measured is 0.80±0.02. On irradiation of oxygen-saturated solution of HA-liposomes, hydroxyl radical OH is detected using DMPO as the ESR spin trapping agent. Hydroxyl radical is derived from superoxide radical anion O2-. The electron transfer reaction is also studied in deaerated solution. The results suggest that the photodynamic action of HA in lipid membranes proceeds via both Type I and Type II reactions.

Interaction of Hypocrellin B or Mono-cysteine Substituted Hypocrellin B with CT-DNA by Spectral Methods

The interaction of the anticancer drug hypocrellin B (HB) or the mono-cysteine substituted hypocrellin B (MCHB) and calf thymus deoxyribonucleic acid (CT-DNA) has been investigated using spectral methods. The results of UV-visible spectra show that the HB and MCHB could intercalate into the base-stacking domain of the CT-DNA double helix. The studies of fluorescence spectra and circular dichroism(CD) spectra also support the interacalation mechanism.

Study on the Singlet and Triplet StateElectron Transfer between Hypocrellin A and Aromatic Amines in Solvents of Various Polarity

In this paper, the singlet and triplet state electron transfer processes between hypocrellin A(HA) and aromatic amines in solvents of various polarity have been studied by fluorescence and time-resolved transient absorption spectra. The results show,in.polar solvent, the quenching of HA fluorescence by aromatic amines results in Stern-Volmer plot, the investigation of transient absorption indicated that triplet-state electron transfer process from 3HA to the ground-state of amines occured, and the wines of the singlet and triplet state electron transfer rate constant were calculated, however in weak polar solvent, only the triplet-triplet absorption of HA was observed.

Interaction between hypocrellin and aliphatic amines

The interaction of hypocrellin, including hypocrellin A (HA) and hypocrellin B (HB), with aliphatic amines in deaerated solutions has been studied by ESR and nanosecond transient absorption spectra. In polar solvents, the acid-base interaction between hypocrellin and amines was observed without irradiation. The signals of semiquinone radical anions of hypocrellm and the spin-trapping adduct of α-phenyl-N-tertbutyl-ratrone (PNB) with the aminoalkyl radicals have been detected in photoinduced ESR studies. The transient absorption of excited triplet state of HA and semiquinone radical anion of HA have been observed in laser flash photolysis studies.

Drug-delivery and multifunction possibilities of hypocrellin photosensitizers

Photodynamic therapy(PDT)has been a routine treatment of tumors and some microvascular diseases,but clinically available photosensitizers are still scarce.Among all kinds of photo-sensitizers,hypocellins possess the most characteristics of ideal photosensitizers,such as,high photo-activity but low dark toxicity,fast clearance from tssues.This review is focused on two main topics,drug-delivery problem of hypocrellins and how the environment-sensitive fluorescence of bypecrellins was used for recognition of various biomolecules.Drug-delivery of hypocrellins was mainly achieved in two strategies一preparing the drugdelivery vehicles and finding quantitatively amphiphilic derivatives.Hypocrellin fAuorescence originated from the intramolecular proton transfer is very distinct from other kinds of photosensitizers.Recently,it was proved that quan-titative hypocrellin ftuorescence could not only reoognize various biomolecules,including proteins,polysaocharides and lipids,but also distinguish the specific binding from nonspedifie binding with some kind of biomolecules.Meantime,hypocrellin fluorescence was pH sensitive.It is known that tumor cells or tses have the features of a large amount of lipid,neonatal collagen,over expression of polysaccharides,and lower pH values compared to normal tissues.According to the relative but not absolute specifcity,further studies on quantitative recognition of various biomolecules at a cellular level,may find a new clue to treat tumors by joint usage of photodynamic diagnosis(PDD)and PDT.

High Efficiency Hypocrellin Production by a Novel Mutant Isolated from Shiraia bambusicola

The fungus Shiraia bambusicola GZ19 M1 is biotechnologically important due to its ability to biosynthesis the pigment hypocrellins.Results showed that ethyl methane sulfonate( EMS) was effective mutagenic agent for strain and potentially produced large numbers of random mutations broadly and uniformly over the whole genome to generate unique strains. Wild-type cultures of S. bambusicola GZ19 were subjected to EMS( 80 mM) induction targeted at approximately 20% spores' survival. When surviving spores were selected in sufficient numbers and cultured on PDA medium for 7 d at 26℃,five novel mutagenized S. bambusicola strains were obtained. A mutant GZ19 M1 that exhibited an activity of more than two times over the wild strain was obtained. Also,batch experiments were carried out to achieve the suitable conditions for hypocrellin. Glucose and rice extract were the most favorable carbon and nitrogen sources for hypocrellin production by submerged culture of S. bambusicola GZ19 M1,and initial glucose and rice extract concentrations were at 35 and 250 g/L,respectively. The optimal surfactant were found to be 0. 008 V/V Tween 80,it was added into cultivation medium at 24 h. Hypocrellin concentration reached 498. 89 mg/L under optimal nutritional conditions,an increment of about 8. 70 times of hypocrellins production was observed compared with that of in non-optimized medium by the wild strain.