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Background Despite progress in developing wound care strategies, there is currently no treatment that promotes the self-tissue repair capabilities. H2 has been shown to effectively protect cells and tissues from oxidative and inflammatory damage. While comprehensive effects and how H2 functions in wound healing are still obscure. Methods Here, we established a cutaneous aseptic wound model and applied a high concentration of H2 (66% H2) in a treatment chamber. Molecular mechanisms and the effects of healing were evaluated by gene functional enrichment analysis, blood perfusion/oxygen detection assay, in vitro tube formation assay, enzyme-linked immunosorbent assay, immunofluorescent staining, non-targeted metabonomic analysis, flow cytometry, transmission electron microscope, and live-cell imaging. Results We revealed that a high concentration of H2 (66% H2) greatly increased the healing rate (3 times higher than the control group) on day 11 post wounding. The effect was not dependent on O2 or anti-reactive oxygen species functions. Histological and cellular experiments proved the fast re-epithelialization in the H2 group. Extracellular matrix components early (3 days post wounding) deposition were found in the H2 group of the proximal wound, especially for the dermal col-I, epidermal col-III, and dermis-epidermis-junction col-XVII. H2 accelerated early autologous epidermal stem cell proliferation (1–2 days in advance) and then differentiation into myoepithelial cells. Other beneficial outcomes include sustained moist healing, greater vascularization, less T-helper-1 and T-helper-17 cell-related systemic inflammation, and better tissue remodelling. Conclusion We have discovered a novel pattern of wound healing induced by molecular hydrogen treatment. These H2-induced multiple advantages in healing may be related to the enhancement of cell viability in various cells and the maintenance of mitochondrial functions at a basic level in the biological processes of life.

Proliferation is an important characteristic of life, and many signaling pathways participate in this complicated process. The MAPK/Erk pathway is a classic pathway in cell proliferation. In this study, expression levels of key factors in the MAPK/Erk pathway were measured to assess the proliferation level among normal skin, physiological scar, and keloid tissue. Thirty patients were selected randomly from the Department of Plastic Surgery at Peking Union Medical College Hospital from January 2019 to December 2020. Histological appearance and fiber tissue content were observed by Hematoxylin and eosin staining and Masson staining. Expression levels of key factors in the MAPK/Erk pathway (ATF2, c-Jun, c-Myc, p38 and STAT1) and relative proteins (HIF-1α and PCNA) in tissues were detected by immunohistochemistry and analyzed as the percentage of positively stained cells in both the tissue epidermis and dermis. Western blot was used for quantitative analysis of the above factors. In results, keloid tissue showed a significantly higher fiber and less cell content. In the immunohistochemical result, higher expression of key factors was observed in the epidermis than in the dermal layer, and the expression of all factors was increased remarkably in keloid tissue. In western blot analysis, all factors (except STAT1) showed higher expression in keloid tissue. In our former research, keloid showed similar apoptosis level as physiological scar and normal skin. On combining our former conclusion and results in this study, an imbalance condition between the high proliferation level and normal apoptosis level may lead to the growth characteristics of keloid.

The potential for preventive and therapeutic applications of H2 have now been confirmed in various disease. However, the effects of H2 on health status have not been fully elucidated. Our previous study reported changes in the body weight and 13 serum biochemical parameters during the six-month hydrogen intervention. To obtain a more comprehensive understanding of the effects of long-term hydrogen consumption, the plasma metabolome and gut microbiota were investigated in this study. Compared with the control group, 14 and 10 differential metabolites (DMs) were identified in hydrogen-rich water (HRW) and hydrogen inhalation (HI) group, respectively. Pathway enrichment analysis showed that HRW intake mainly affected starch and sucrose metabolism, and DMs in HI group were mainly enriched in arginine biosynthesis. 16S rRNA gene sequencing showed that HRW intake induced significant changes in the structure of gut microbiota, while no marked bacterial community differences was observed in HI group. HRW intake mainly induced significant increase in the abundance of Lactobacillus, Ruminococcus, Clostridium XI, and decrease in Bacteroides. HI mainly induced decreased abundances of Blautia and Paraprevotella. The results of this study provide basic data for further research on hydrogen medicine. Determination of the effects of hydrogen intervention on microbiota profiles could also shed light on identification of mechanism underlying the biological effects of molecular hydrogen.

The potential for preventive and therapeutic applications of H2 have now been confirmed in various disease. However, the effects of H2 on health status have not been fully elucidated. Our previous study reported changes in the body weight and 13 serum biochemical parameters during the six-month hydrogen intervention. To obtain a more comprehensive understanding of the effects of long-term hydrogen consumption, the plasma metabolome and gut microbiota were investigated in this study. Compared with the control group, 14 and 10 differential metabolites (DMs) were identified in hydrogen-rich water (HRW) and hydrogen inhalation (HI) group, respectively. Pathway enrichment analysis showed that HRW intake mainly affected starch and sucrose metabolism, and DMs in HI group were mainly enriched in arginine biosynthesis. 16S rRNA gene sequencing showed that HRW intake induced significant changes in the structure of gut microbiota, while no marked bacterial community differences was observed in HI group. HRW intake mainly induced significant increase in the abundance of Lactobacillus, Ruminococcus, Clostridium XI, and decrease in Bacteroides. HI mainly induced decreased abundances of Blautia and Paraprevotella. The metabolic function was determined by metabolic cage analysis and showed that HI decreased the voluntary intake and excretions of rats, while HRW intake did not. The results of this study provide basic data for further research on hydrogen medicine. Determination of the effects of hydrogen intervention on microbiota profiles could also shed light on identification of mechanism underlying the biological effects of molecular hydrogen.

To provide a high-throughput, efficient, and accurate method to monitor multiple-fungicide resistance of Botrytis cinerea in the field, we used the suspension array, sequencing, and mycelial growth assay in our research. Discriminating-dose bioassays for detecting carbendazim, diethofencarb, boscalid, and iprodione resistance (CarR, DieR, BosR, and IprR, respectively) were used to analyze 257 isolates collected from Hebei Province in China during 2016 and 2017. High resistance frequencies to carbendazim (100%), diethofencarb (92.08%), and iprodione (86.59%) were detected. BosR isolates accounted for 11.67% of the total. In addition, 103 isolates were randomly selected for phenotype and genotype detection. The high-throughput suspension array was utilized to detect eight genotypes simultaneously, including BenA-E198, BenA-198A, SdhB-H272, SdhB-272Y, BcOS1-I365, BcOS1-365S, erg27-F412, and erg27-412S, which were associated with resistance toward carbendazim or diethofencarb, boscalid, iprodione, and fenhexamid (FenR), respectively. Most of the benzimidazole-resistant isolates (81.55%) possessed the E198V mutation in the BenA gene. Ninety-three isolates with dual resistance to carbendazim and diethofencarb showed the E198V/K mutation. All BosR isolates carried the H272R mutation in the SdhB gene. The I365S and Q369P+N373S (66.99%) mutations in the BcOS1 gene were more frequently observed. No mutation was detected in the erg27 gene in Hebei isolates. There were 13 resistance profile phenotypes. Phenotypes with triple resistance were the most common (83.50%), and CarRDieRBosSIprRFenS was the major type. CarR isolates that carried E198V/K/A were all highly resistant (HR) and only one F200Y mutant was moderately resistant (MR) to carbendazim. Isolates that possessed E198V/K were MR or HR to diethofencarb. BosR isolates that possessed H272R mutation were lowly resistant (LR). IprR isolates were all LR or MR. The distribution of half maximal effective concentrations of CarR isolates with E198V/K mutations and IprR isolates with Q369P+N373S mutations significantly increased from 2016 to 2017. Combined with our observations, a combination method of the high-throughput suspension array and the mycelial growth assay was suggested to accurately monitor multiple resistance of B. cinerea in the field.

AIM: To reveal the cytokines involved in idiopathic orbital inflammatory disease (IOID) and the relationship between Th17 cells, IgE and IOID pathogenesis. METHODS: Whole blood samples were processed immediately after collection and serological IgG4, IgG, and IgE antibodies were tested using ELISA. IOID and orbital cavernous hemangioma (CH) tissue samples underwent Bio-Plex multiplex cytokine detection. Hematoxylin-Eosin (HE) staining of all paraffin samples suggested the histological features of IOIDs, and expressions of IgG4 and IL-17A in affected tissues were detected by immunohistochemistry. RESULTS: Among 40 IOID plasma samples, 52.5% (21/40) were positive for IgG4 and 25% (10/40) were positive for IgE. Overlapped IgG4 or IgE positive samples accounted for 22.5% (9/40). Therefore, IOID samples were separated into three groups. The IgE+/IgG4+ group had a relevantly lower level of pro-inflammatory cytokine expression. IL-4 (Th2 cell related), IL-10 and TGF-β1 (Treg cell immunity related) were elevated in all three groups. Some of the Th17 cell related cytokines (i.e. IL-17A/F, IL-25, IL-23, and IL-33) displayed higher expression levels in the IgE-/IgG4- group compared to the other two groups. CONCLUSION: We discovered an IgG4-IgE co-positive group as well as Th17 cell immune involvement in IgG4-IgE co-negative subgtroup in IOID for the first time. The pathogenesis of IOID could differ from different subgroups according to the IgG4 and IgE detection. Therefore, we recommend that, Treatment stratagy should be made according to the clinical assessment of IgG4-IgE and Th17 profile detection.

To provide a high-throughput, efficient, and accurate method to monitor multiple-fungicide resistance of

Apoptosis is a form of programmed cell death that occurs in multicellular organisms. Fibroblasts are the main cellular ingredients in keloid tissue, which has a relatively low apoptosis level. A natural metabolite of estradiol, 2-Methoxyestradiol (2ME2) exerts a pro-apoptotic effect on tumor cells. In this study, the expression levels of key factors in the apoptosis pathway and the expression level of the proliferating cell nuclear antigen (PCNA) were measured to assess the levels of apoptosis and proliferation in both normal skin fibroblasts and keloid fibroblasts. Twelve samples were obtained from 12 patients: 6 keloid patients and 6 non-keloid patients. All 12 of the patients were randomly selected from the Department of Plastic Surgery at Peking Union Medical College Hospital from June 2016 to December 2016. After cell culture, fibroblasts were divided into the following 6 groups: normal skin fibroblasts (S); keloid fibroblasts (K); keloid fibroblasts treated with 2ME2 (2ME2); keloid fibroblasts treated with DMSO (DMSO); keloid fibroblasts treated with the caspase inhibitor Ac-DEVD-CHO (IN); and keloid fibroblasts treated with both Ac-DEVD-CHO and 2ME2 (IN+2ME2). Fibroblasts at up to passage 3 were used for analysis. Cell activity was measured by the cell counting kit-8. TUNEL staining was used to observe the cell apoptotic morphology. The key apoptosis factors (caspase-3, caspase-8, caspase-9, Bcl-2, Bax, and cytochrome-c) and PCNA expression levels were detected by immunofluorescence analysis and Western blotting. A certain concentration of 2ME2 was also used in group S to evaluate the toxicity. Compared with that in the other groups, 2ME2 significantly inhibited cell activity and led to apoptotic appearance of fibroblasts. In protein analysis, 2ME2 remarkably increased the expression of apoptosis factors and decreased the PCNA expression. Apoptosis levels were reduced by both the caspase inhibitor and 2ME2; thus indicating that the pro-apoptosis effect of 2ME2 was achieved through a caspase-dependent mechanism in keloid fibroblasts. Toxicity assessment showed that 2ME2 had a very low influence on normal skin fibroblasts. 2ME2, considered to be a new promising type of chemotherapy drug, exerts a pro-apoptosis effect by regulating the caspase family and an anti-proliferation effect towards keloid fibroblasts, and it presents low toxicity towards normal fibroblasts

There is increasing evidence concerning the occurrence of malignant lymphoma among people suffering from Mikulicz disease, also termed benign lymphoepithelial lesion (BLEL) and immunoglobulin G4‑associated disease. However, the underlying molecular mechanism of the malignant transformation remains unclear. The present study aimed to investigate the gene expression profile between BLEL and malignant lymphoepithelial lesion (MLEL) conditions using tissue microarray analysis, to identify genes and pathways which may be associated with the risk of malignant transformation. Comparing gene expression profiles between BLEL tissues (n=13) and MLEL (n=14), a total of 1,002 differentially expressed genes (DEGs) were identified including 364 downregulated and 638 upregulated DEGs in BLEL. The downregulated DEGs in BLEL were frequently associated with immune‑based functions, immune cell differentiation, proliferation and survival, and metabolic functions, whereas the upregulated DEGs were primarily associated with organ, gland and tissue developmental processes. The B cell receptor signaling pathway, the transcription factor p65 signaling pathway, low affinity immunoglobulin γ Fc region receptor II‑mediated phagocytosis, the high affinity immunoglobulin ε receptor subunit γ signaling pathway and Epstein‑Barr virus infection, and pathways in cancer, were the pathways associated with the downregulated DEGs. The upregulated DEGs were associated with three pathways, including glutathione metabolism, salivary secretion and mineral absorption pathways. These results suggested that the identified signaling pathways and their associated genes may be crucial for understanding the molecular mechanisms underlying malignant transformation from BLEL, and they may be considered to be markers for predicting malignancy among the BLEL group.

Apoptosis is a process of programmed cell death that occurs in multicellular organisms. The mitochondrial pathway plays a paramount role in apoptosis. In this study, the expression levels of key factors in the mitochondrial pathway and the cell proliferation factor (PCNA) were measured to evaluate the level of apoptosis and proliferation in keloid scars, physiological scars and normal skin tissue. Thirty samples were taken from 30 patients: 10 keloid patients, 10 physiological scar patients and 10 patients without obvious scarring. All 30 patients were selected randomly from the Department of Plastic Surgery at Peking Union Medical College Hospital from June 2016 to December 2016. Hematoxylin and eosin staining and Masson staining were used to observe the differences in histology and fiber tissue content. Mitochondrial pathway factors (caspase-3, caspase-8, caspase-9, Bcl-2, Bax, cytochrome-c) and PCNA expression levels were detected by immunohistochemistry and were analyzed as the percentage of positively stained cells in the epidermis and dermis. Relative protein expression levels were measured by western blotting. Compared with physiological scars and normal skin tissue, keloid tissue had an increase in fiber number and decrease in cell content. In our immunohistochemical and western blot analyses, all tissue types showed similar expression levels of the mitochondrial pathway factors. However, the percentage of PCNA-positive cells and the relative protein expression level of PCNA were significantly higher in keloid tissue. Keloid has a similar apoptosis level as physiological scars and normal skin but has a higher expression of PCNA, indicating that keloid scars have high levels of proliferation and normal apoptosis.

Since its first description in 1966, macrophage migration inhibitory factor (MIF) was found to play a critical role in inflammatory and immune responses as well as in disease pathogenesis especially in tumor pathogenesis and cancer progression. MIF is expressed in different cell types and is associated with many disease severity and tumor pathogenesis. Here, we investigated the influence of TLR7 and TLR8 agonist resiquimod (R848), an immune response inducer used as a prophylactic agent for several infectious diseases as well as anticancer agents and vaccine adjuvant on MIF expression in cells and organs. Humans, mice and rats cell lines from different tissues (blood, retinal, nasopharynx, brain and liver) and C57BL/6J mice organs (brain, liver and spleen) were used for this investigation. In vitro, R848 induced MIF gene overexpression except in brain and liver cells. Furthermore, it enhanced cells ability to release soluble MIF and differently regulated mRNA expression of MIF-related receptors (CD74, CXCR4, CXCR2 and CD44). Its influence on MIF gene expression and MIF proteins release was more consistent in cancer cells. In vivo, a strong positive expression of MIF was observed in different regions in brain and spleen in response to R848 treatment; however in liver, increased MIF expression was observed in hepatocytes only. On the other hand, R848 treatment had induced a slight enhancement of MIF concentration in the plasma of C57BL/6J mice. Taken together, these data suggest that R848 differently regulates MIF mRNA expression depending on organ types and could influence MIF concentration in cellular microenvironment.