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Objective: To investigate the role of platelet-rich plasma (PRP) in inducing the M2 macrophage polarization via regulating AMPK singling pathway., Methods: The expressions of M1 marker CD11c and M2 marker CD206 in macrophages of blank control group, LPS group, LPS+PRP group, and LPS+PRP+Compound C group were detected by flow cytometry. Western blot was used to observe the effects of PRP on the expression of AMPK-mTOR signaling pathway-related proteins at different times (12 h, 18 h and 24 h) after LPS treatment. RNA interference technology was used to silence the expression of AMPK in macrophages, and the expression of TGF-β protein was subsequently examined by Western blot., Results: LPS significantly reduced the expression of CD206 and increased the expression of CD11c ( P <0.05). After the addition of PRP, the expression of CD206 was significantly increased ( P <0.05), while the expression of CD11c was significantly decreased ( P <0.05). Compared with LPS group, PRP treatment significantly increased the expressions of p-AMPK and p-ULK1 proteins at 12 h, 18 h and 24 h, while significantly decreased the expression of p-mTOR protein ( P <0.05). After the addition of AMPK inhibitor Compound C, the expression of CD206 was significantly reduced ( P <0.05) and the expression of CD11c was significantly increased compared with LPS+PRP group ( P <0.05). After silencing the expression of AMPK in macrophages, the promotion effect of PRP on TGF-β was significantly reduced ( P <0.05)., Conclusion: PRP can stimulate the transformation of macrophages to M2 type via AMPK signalling pathway.

Pyridinium aldoximes are best-known therapeutic antidotes used for clinical treatment of poisonings by organophosphorus nerve-agents and pesticides. Recently, we found that pralidoxime (2-PAM, a currently clinically used nerve-agent antidote) could also detoxify tetrachloro-1,4-benzoquinone (TCBQ), which is a carcinogenic quinoid metabolite of the widely used wood preservative pentachlorophenol under normal physiological conditions, via an unusually mild and facile Beckmann fragmentation mechanism accompanied by radical homolysis. However, it is not clear whether the less-chlorinated benzoquinones ( C n BQs, n ≤ 3) act similarly; if so, what is the structure-activity relationship? In this study, we found that (1) The stability of reaction intermediates produced by different C n BQs and 2-PAM was dependent not only on the position but also the degree of Cl-substitution on C n BQs, which can be divided into TCBQ- and DCBQ (dichloro-1,4-benzoquinone)-subgroup; (2) The p K a value of hydroxlated quinones ( C n -1 BQ-OHs, the hydrolysis products of C n BQs), determined the stability of corresponding intermediates, that is, the decomposition rate of the intermediates depended on the acidity of C n -1 BQ-OHs; (3) The p K a value of the corresponding C n -1 BQ-OHs could also determine the reaction ratio of Beckmann fragmentation to radical homolysis in C n BQs/2-PAM. These new findings on the structure-activity relationship of the halogenated quinoid carcinogens detoxified by pyridinium aldoxime therapeutic agents via Beckmann fragmentation and radical homolysis reaction may have broad implications on future biomedical and environmental research.

Discharges released from fluorochemical industrial plants lead to severe contamination of the environment with per- and polyfluoroalkyl substances (PFASs), which may pose risks to human health. In this study, 187 serum samples from teenagers (age = 14 years), 22 tap water samples and 40 soil samples were collected in areas within 0-11 km of a fluorochemical industrial plant in Huantai County, Shandong Province, and concentrations of 18 PFASs were quantified by UPLC-MS/MS. Perfluorooctanoic acid (PFOA) was found to be predominant, concentrations of which ranged from 40.4 to 845 ng/mL in serum, from 2.88 to 19.3 ng/L in tap water, from 4.40 to 189 ng/g in soil, and accounting for 84.1-98.6%, 15.9-79.8%, and 73.8-96.7% of the total PFASs, respectively. Statistical analysis demonstrated that concentrations of perfluorinated carboxylic acids (PFCAs) in soil (C5-C9) and serum (C8-C10) were associated with the industrial plant. And PFOA concentrations in tap water were not relevant to the industrial plant, which were comparable with the non-contaminated area and lower than the threshold value recommended by U.S. EPA (70 ng/mL), indicating that the contribution to the high concentration of serum PFOA of local teenagers by drinking water was limited. Moreover, PFCAs in soil only made a limited contribution to the serum PFCAs of local residents by direct inhalation and dermal exposure, but the potential health risk by the soil via food chain should be paid attention to. Furthermore, health risk assessment demonstrated that high concentrations of PFOA in serum could pose potential health risk to local teenagers. Therefore, effective measures should be taken to attenuate the health risks caused by the industrial plant to local residents, and further epidemiological studies should be carried out in the future., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We found recently that benzohydroxamic acid (BHA) could detoxify the chlorinated quinoid carcinogens via an unusual Lossen rearrangement reaction. However, it is not clear what would happen when the nitrogen hydrogen of BHA was substituted with methyl and other alkyl groups. Here we show that N-methyl benzohydroxamic acid (N-MeBHA, a simple model compound for the classic iron-chelator deferoxamine, which is a typical N-alkyl trihydroxamic acid) could react with 2,5-dichloro-1,4-benzoquinone (DCBQ) to form a relatively stable initial carbon-oxygen bonding conjugation intermediate CBQ-O-N-MeBHA. However, the major final product was identified, unexpectedly, as a carbon-nitrogen bonding conjugate CBQ(OH)-N(CH 3 )-COAr, which is the rearranged isomer of CBQ-O-N-MeBHA. Interestingly, a new 18-line nitrogen-centered radical and a carbon-centered quinone ketoxy radical were observed by the ESR spin-trapping method, which was further confirmed by HPLC-MS and 15 N-isotope labeling methods. We further found that both new DNA adducts and DNA strand breaks could be produced by the reactive nitrogen-centered radical. Taken together, we propose that the reaction between DCBQ and N-MeBHA was not via the Lossen rearrangement, but rather through a novel radical homolysis and recoupling pathway. Analogous results were observed for other chlorinated quinones and N-alkyl hydroxamic acids including the widely-used trihydroxamate iron-chelating drug deferoxamine. This represents the first report of unexpected radical pathway for the reaction between chlorinated quinones and N-alkyl hydroxamic acids under normal physiological conditions, which may have broad biological and environmental significance for future study of carcinogenic chloroquinones and hydroxamic acid drugs., (Copyright © 2019. Published by Elsevier Inc.)

Mannan-binding lectin (MBL), a circulating C-type lectin, is an important member of the defense collagen family. It exhibits a high potential for recognizing broad categories of pathogen-associated molecular patterns and initiating complement cascade responses. DCs are well-known specialist antigen-presenting cells that significantly trigger specific T cell-mediated immune responses. In our previous study, it was observed that high concentrations of MBL significantly attenuate LPS-induced maturation of monocyte-derived DCs (MoDCs). In the current study, it was postulated that MBL at similar supraphysiological concentrations would affect early differentiation of MoDCs in some way. CD14(+) monocytes from human peripheral blood mononuclear cells were cultured with granulocyte-macrophage colony-stimulating factor and IL-4 in the presence or absence of physiological (1 µg/mL) and supraphysiological concentrations (20 µg/mL) of MBL protein, respectively. Phenotypic analysis indicated that the differentiated DCs incubated with high concentrations of MBL expressed MHC class II and costimulatory molecules (e.g., CD80 and CD40) more weakly than did control groups. The secretion of IL-10 and IL-6 increased markedly, whereas their mixed lymphocyte reaction-stimulating capacity decreased. Members of the signal transducer and activator of transcription family were also found to be differentially regulated. Thus, beyond the role of MBL as an opsonin, our data reveal a possible inhibitory effect of MBL at high concentrations in monocyte-DC transition, which probably provides one way of regulating adaptive immune responses by strict regulation of DCs, making MBL a better prospect for controlling relevant pathological events such as autoimmune diseases., (© 2015 The Societies and Wiley Publishing Asia Pty Ltd.)

Halogenated quinones (XQ) are a class of carcinogenic intermediates and newly identified chlorination disinfection byproducts in drinking water. Organic hydroperoxides (ROOH) can be produced both by free radical reactions and enzymatic oxidation of polyunsaturated fatty acids. ROOH have been shown to decompose to alkoxyl radicals via catalysis by transition metal ions, which may initiate lipid peroxidation or transform further to the reactive aldehydes. However, it is not clear whether XQ react with ROOH in a similar manner to generate alkoxyl radicals metal-independently. By complementary applications of ESR spin-trapping, HPLC/high resolution mass spectrometric and other analytical methods, we found that 2,5-dichloro-1,4-benzoquinone (DCBQ) could significantly enhance the decomposition of a model ROOH tert-butylhydroperoxide, resulting in the formation of t-butoxyl radicals independent of transition metals. On the basis of the above findings, we detected and identified, for the first time, an unprecedented C-centered quinone ketoxy radical. Then, we extended our study to the more physiologically relevant endogenous ROOH 13-hydroperoxy-9,11-octadecadienoic acid and found that DCBQ could also markedly enhance its decomposition to generate the reactive lipid alkyl radicals and the genotoxic 4-hydroxy-2-nonenal (HNE). Similar results were observed with other XQ. In summary, these findings demonstrated that XQ can facilitate ROOH decomposition to produce reactive alkoxyl, quinone ketoxy, lipid alkyl radicals, and genotoxic HNE via a novel metal-independent mechanism, which may explain partly their potential genotoxicity and carcinogenicity.

The classic Lossen rearrangement is a well-known reaction describing the transformation of an O-activated hydroxamic acid into the corresponding isocyanate. In this study, we found that chlorinated benzoquinones (CnBQ) serve as a new class of agents for the activation of benzohydroxamic acid (BHA), leading to Lossen rearrangement. Compared to the classic one, this new kind of CnBQ-activated Lossen rearrangement has the following unique characteristics: (1) The stability of CnBQ-activated BHA intermediates was found to depend not only on the degree but also on the position of Cl-substitution on CnBQs, which can be divided into two subgroups. (2) It is the relative energy of the anionic CnBQ-BHA intermediates that determine the rate of this CnBQ-activated rearrangement, which is the rate-limiting step, and the Cl or H ortho to the reaction site at CnBQ is crucial for the stability of the anionic intermediates. (3) A pKa-activation energy correlation was observed, which can explain why the correlation exists between the rate of the rearrangement and the acidity of the conjugate acid of the anionic leaving group, the hydroxlated quinones. These findings may have broad implications for future research on halogenated quinoid carcinogens and hydroxamate biomedical agents.

Halogenated quinones are a class of carcinogenic intermediates and newly identified chlorination disinfection by-products in drinking water. 13-Hydroperoxy-9,11-octadecadienoic acid (13-HPODE) is the most extensively studied endogenous lipid hydroperoxide. Although it is well known that the decomposition of 13-HPODE can be catalyzed by transition metal ions, it is not clear whether halogenated quinones could enhance its decomposition independent of metal ions and, if so, what the unique characteristics and similarities are. Here we show that 2,5-dichloro-1,4-benzoquinone (DCBQ) could markedly enhance the decomposition of 13-HPODE and formation of reactive lipid alkyl radicals such as pentyl and 7-carboxyheptyl radicals, and the genotoxic 4-hydroxy-2-nonenal (HNE), through the complementary application of ESR spin trapping, HPLC-MS, and GC-MS methods. Interestingly, two chloroquinone-lipid alkoxyl conjugates were also detected and identified from the reaction between DCBQ and 13-HPODE. Analogous results were observed with other halogenated quinones. This represents the first report that halogenated quinoid carcinogens can enhance the decomposition of the endogenous lipid hydroperoxide 13-HPODE and formation of reactive lipid alkyl radicals and genotoxic HNE via a novel metal-independent nucleophilic substitution coupled with homolytic decomposition mechanism, which may partly explain their potential genotoxicity and carcinogenicity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Mannose-binding lectin (MBL), a plasma C-type lectin, plays an important role in innate immunity. However, the interaction, and the consequences of it, between MBL and the immune system remain ill defined. We have investigated the contributing mechanisms and effects of MBL on the proliferation of human monocytes. At lower concentrations (≤4 μg/ml) MBL was shown to partially enhance monocyte proliferation. By contrast, at higher concentrations (8-20 μg/ml) of MBL, cell proliferation was markedly attenuated. MBL-induced growth inhibition was associated with G0/G1 arrest, down-regulation of cyclin D1/D3, cyclin-dependent kinase (Cdk) 2/Cdk4 and up-regulation of the Cdk inhibitory protein Cip1/p21. Additionally, MBL induced apoptosis, and did so through caspase-3 activation and poly ADP-ribose polymerase (PARP) cleavage. Moreover, transforming growth factor (TGF)-β1 levels increased in the supernatants of MBL-stimulated monocyte cultures. We also found that MBL-dependent inhibition of monocyte proliferation could be reversed by the TGF-β receptor antagonist SB-431542, or by anti-TGF-β1 antibody, or by the mitogen-activated protein kinase (MAPK) inhibitors specific for p38 (SB203580), but not ERK (U0126) or JNK (SP600125). Thus, at high concentrations, MBL can affect the immune system by inhibiting monocyte proliferation, which suggests that MBL may exhibit anti-inflammatory effects.

We found, unexpectedly, that the radical form of the carbon-centered quinone ketoxy radical adduct with a recently developed spin-trapping agent BMPO can not only be directly detected and identified using HPLC/high resolution MS, but can also be isolated and purified using semi-preparative HPLC, enabling direct observation of its clean 6-line ESR signal.

The aim of this study was to evaluate the efficacy of autologous platelet-rich plasma (PRP) combined with erbium fractional laser therapy for facial acne or acne scars. PRP combined with erbium fractional laser therapy was used for the treatment of 22 patients, including 16 patients who suffered from facial acne scars and 6 patients who suffered from acne scars concomitant with acne. Whole blood (40 ml) was collected from each patient, and following differential centrifugation, PRP was harvested. After using an erbium fractional laser, we applied PRP to the entire face of every patient. Digital photos were taken before and after the treatment for evaluation by dermatologists and the patients rated the efficacy on a 5-point scale. The erythema was moderate or mild, while its total duration was <3 days; after receiving the treatment three times, 90.9% of the patients showed an improvement of >50%, and 91% of the patients were satisfied; no acne inflammation was observed after treatment. PRP combined with erbium fractional laser therapy is an effective and safe approach for treating acne scars or acne, with minimal side-effects, and it simultaneously enhanced the recovery of laser-damaged skin.

The treatment of chronic diabetic wounds remains complicated, despite new insight into the cellular and molecular basis of wound healing and cutaneous regeneration. A growing body of clinical trials has shown that platelet release has a notable effectiveness on refractory ulcer healing. However, patients with chronic diabetic ulcers usually have poor general health, and the large-volume blood absence required to produce autologous platelet-rich plasma often causes adverse effects. To overcome the limitation, the homologous platelet gel (PG) from healthy donor was used for the treatment of chronic diabetic lower extremity wound in the study. We show here that homologous derived platelets significantly enhanced EVC304 cell and HaCaT cell proliferation and homologous PG was capable of prompting cell migration. Twenty-one patients with refractory diabetic lower extremity ulcers, who had no response to conventional treatments, were treated in this study. Our data indicated that homologous PG was effective for the enhancement and acceleration of diabetic lower extremity wounds healing. We propose that homologous PG appeared to enhance vascularization and epithelialization, which might induce a quicker healing process and and encourage controlled studies in future.

Hydroxamic acids, which are best-known for their metal-chelating properties in biomedical research, have been found to effectively detoxify the carcinogenic polyhalogenated quinoid metabolites of pentachlorophenol and other persistent organic pollutants. However, the chemical mechanism underlying such detoxication is unclear. Here we show that benzohydroxamic acid (BHA) could dramatically accelerate the conversion of the highly toxic tetrachloro-1, 4-benzoquinone (p-chloranil) to the much less toxic 2,5-dichloro-3, 6-dihydroxy-1, 4-benzoquonine (chloranilic acid), with rate accelerations of up to 150,000-fold. In contrast, no enhancing effect was observed with O-methyl BHA. The major reaction product of BHA was isolated and identified as O-phenylcarbamyl benzohydroxamate. On the basis of these data and oxygen-18 isotope-labeling studies, we proposed that suicidal nucleophilic attack coupled with an unexpected double Lossen rearrangement reaction was responsible for this remarkable acceleration of the detoxication reaction. This is the first report of an unusually mild and facile Lossen-type rearrangement, which could take place under normal physiological conditions in two consecutive steps. Our findings may have broad biological and environmental implications for future research on hydroxamic acids and polyhalogenated quinoid carcinogens, which are two important classes of compounds of major biomedical and environmental interest.

We have shown recently that halogenated quinones could enhance the decomposition of hydroperoxides and formation of alkoxyl/hydroxyl radicals through a metal-independent mechanism. However, neither the proposed quinone enoxy radical intermediate, nor the major reaction products were unambiguously identified. In the present study, one of the major reaction products between 2,5-dichloro-1,4-benzoquinone (DCBQ) and t-butylhydroperoxide (t-BuOOH) was isolated and purified by semipreparative HPLC, and identified as 2-hydroxy-3-t-butoxy-5-chloro-1,4-benzoquinone [CBQ(OH)-O-t-Bu], which is the rearranged isomer of the postulated quinone-peroxide reaction intermediate. The formation of CBQ(OH)-O-t-Bu was found to be inhibited by the spin trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and concurrently, a new DMPO adduct with 1-chlorine isotope peak clusters at m/z 268 was observed. Further electron spin resonance (ESR) spin-trapping, (1)H-NMR and HPLC/Fourier transform ion cyclotron resonance (FTICR) mass spectrometric studies with oxygen-17-labeled and unlabeled hydrogen peroxide strongly suggest that the radical trapped by DMPO is a carbon-centered quinone ketoxy radical, which is the spin isomer of the proposed oxygen-centered quinone enoxy radical. Analogous results were observed when DCBQ was substituted by other halogenated quinones. This study represents the first detection and identification of an unusual carbon-centered quinone ketoxy radical, which provides direct experimental evidence to further support and expand our previously proposed mechanism for metal-independent decomposition of hydroperoxides by halogenated quinones.

The hydroxyl radical ((*)OH) has been considered to be one of the most reactive oxygen species produced in biological systems. It has been shown that (*)OH can cause DNA, protein, and lipid oxidation. One of the most widely accepted mechanisms for (*)OH production is through the transition metal-catalyzed Fenton reaction. Pentachlorophenol (PCP) was one of the most widely used biocides, primarily for wood preservation. PCP is now ubiquitously present in our environment and even found in people who are not occupationally exposed to it. PCP has been listed as a priority pollutant by the U.S. Environmental Protection Agency (EPA) and classified as a group 2B environmental carcinogen by the International Association for Research on Cancer (IARC). The genotoxicity of PCP has been attributed to its two major quinoid metabolites: tetrachlorohydroquinone and tetrachloro-1,4-benzoquinone (TCBQ). Although the redox cycling of PCP quinoid metabolites to generate reactive oxygen species is believed to play an important role, the exact molecular mechanism underlying PCP genotoxicity is not clear. Using the salicylate hydroxylation assay and electron spin resonance (ESR) secondary spin-trapping methods, we found that (*)OH can be produced by TCBQ and H(2)O(2) independent of transition metal ions. Further studies showed that TCBQ, but not its corresponding semiquinone radical, the tetrachlorosemiquinone radical (TCSQ(*)), is essential for (*)OH production. The major reaction product between TCBQ and H(2)O(2) was identified to be trichloro-hydroxy-1,4-benzoquinone (TrCBQ-OH), and H(2)O(2) was found to be the source and origin of the oxygen atom inserted into this reaction product. On the basis of these data, we propose that (*)OH production by TCBQ and H(2)O(2) is not through a semiquinone-dependent organic Fenton reaction but rather through the following novel mechanism: a nucleophilic attack of H(2)O(2) to TCBQ, leading to the formation of an unstable trichloro-hydroperoxyl-1,4-benzoquinone (TrCBQ-OOH) intermediate, which decomposes homolytically to produce (*)OH. These findings represent a novel mechanism of (*)OH formation not requiring the involvement of redox-active transition metal ions and may partly explain the potential carcinogenicity of the widely used biocides such as PCP and other polyhalogenated aromatic compounds.

The metal-independent decomposition of organic hydroperoxides and the formation of organic alkoxyl radicals in the absence or presence of halogenated quinones were studied with electron spin resonance (ESR) and the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO). We found that 2,5-dichloro-1,4-benzoquinone (DCBQ) markedly enhanced the decomposition of tert-butylhydroperoxide (t-BuOOH), leading to the formation of the DMPO adducts with t-butoxyl radicals (t-BuO* and methyl radicals *CH(3)). The formation of DMPO/t-BuO* and DMPO/*CH(3) was dose-dependent with respect to both DCBQ and t-BuOOH and was not affected by iron- or copper-specific metal chelators. Comparison of the data obtained with DCBQ and t-BuOOH with those obtained in a parallel study with ferrous iron and t-BuOOH strongly suggested that t-BuO* was produced by DCBQ and t-BuOOH through a metal-independent mechanism. Other halogenated quinones were also found to enhance the decomposition of t-BuOOH and other organic hydroperoxides such as cumene hydroperoxide, leading to the formation of the respective organic alkoxyl radicals in a metal-independent manner. Based on these data, we propose a mechanism for DCBQ-mediated t-BuOOH decomposition and formation of t-BuO*: a nucleophilic attack of t-BuOOH on DCBQ, forming a chloro-t-butylperoxyl-1,4-benzoquinone intermediate, which decomposes homolytically to produce t-BuO*. This represents a mechanism of organic alkoxyl radical formation not requiring the involvement of redox-active transition metal ions.

Background: To further investigate the relationship between inappropriate gonadotropin, hyperinsulinemia and androgen excess., Methods: We divided women with polycystic ovarian syndrome (PCOS) into two groups on the basis of LH/FSH (LH/FSH > or = 3, Group 1, n=30; LH/FSH<3, Group 2, n=25) and measured the responses of insulin, C-peptide (C-P), C-P/insulin (C-I) and testosterone concentrations to an oral glucose tolerance test (OGTT) in these patients and 15 control subjects., Results: Significant positive correlations were found between basal LH and T (r=O.58, p<0.05) in Group 1, and between fasting serum insulin and T (r=0.48, p<0.05) in Group 2. Although these patients had higher, delayed insulin and C-P responses to OGTT than the control group, much greater insulin and C-P levels and lower C/I values were found in Group 2. T concentrations decreased mildly during OGTT in Group 1 and the control, while they increased slightly in Group 2., Conclusion: Our data suggest that hyperinsulinemia in PCOS is due to a enhanced beta-cell secretion and an impaired hepatic clearance of this hormone, hyperandrogenism may be LH-dependent in Group 1 and insulin-dependent in Group 2.