Potential Anti-Inflammatory Constituents from Aesculus wilsonii Seeds.

A chemical study of Aesculus wilsonii Rehd. (also called Suo Luo Zi) and the in vitro anti-inflammatory effects of the obtained compounds was conducted. Retrieving results through SciFinder showed that there were four unreported compounds, aeswilosides I-IV (1-4), along with fourteen known isolates (5-18). Their structures were elucidated by extensive spectroscopic methods such as UV, IR, NMR, [α]D, and MS spectra, as well as acid hydrolysis. Among the known ones, compounds 5, 6, 8-10, and 12-16 were obtained from the Aesculus genus for the first time; compounds 7, 11, 17, and 18 were first identified from this plant. The NMR data of 5 and 18 were reported first. The effects of 1-18 on the release of nitric oxide (NO) from lipopolysaccharide (LPS)-induced RAW264.7 cells were determined. The results showed that at concentrations of 10, 25, and 50 µM, the novel compounds, aeswilosides I (1) and IV (4), along with the known ones, 1-(2-methylbutyryl)phloroglucinyl-glucopyranoside (10) and pisuminic acid (15), displayed significant inhibitory effects on NO production in a concentration-dependent manner. It is worth mentioning that compound 10 showed the best NO inhibitory effect with a relative NO production of 88.1%, which was close to that of the positive drug dexamethasone. The Elisa experiment suggested that compounds 1, 4, 10, and 15 suppressed the release of TNF-α and IL-1ß as well. In conclusion, this study enriches the spectra of compounds with potential anti-inflammatory effects in A. wilsonii and provides new references for the discovery of anti-inflammatory lead compounds, but further mechanistic research is still needed.

Antihyperglycemic effects of triterpenoid saponins from the seeds of Aesculus chinensis Bge.

Six undescribed triterpenoid saponins, namely aescuchinosides A-F, along with seven known triterpenoid saponins, were isolated from the seeds of Aesculus chinensis. Barrigenol-like triterpenoids (BATs) constitute these saponins. Protoaescigenin serves as their aglycone, with various oxygen-containing groups, including acetyl, isobutyryl, tigloyl, and angeloyl groups situated at C-21, C-22, and C-28. Various techniques, including 1D and 2D-NMR spectroscopy, high-resolution mass spectrometry, and acid hydrolysis, were employed to determine the structures of these compounds. The antihyperglycemic effects of the isolated compounds were examined in insulin -resistant HepG2 cells induced by palmitic acid treatment. At a concentration of 6 µM, aesculinoside F exhibited a significant increase in glucose consumption. In addition, aesculinoside F demonstrated the potential to improve insulin resistant by upregulating the PI3K/AKT pathway. These results indicate that the seeds of A.chinensis hold promising potential for preventing insulin resistant related disease.

Aesculus hippocastanum extract and the main bioactive constituent ß-escin as antivirals agents against coronaviruses, including SARS-CoV-2.

Respiratory viruses can cause life-threatening illnesses. The focus of treatment is on supportive therapies and direct antivirals. However, antivirals may cause resistance by exerting selective pressure. Modulating the host response has emerged as a viable therapeutic approach for treating respiratory infections. Additionally, considering the probable future respiratory virus outbreaks emphasizes the need for broad-spectrum therapies to be prepared for the next pandemics. One of the principal bioactive constituents found in the seed extract of Aesculus hippocastanum L. (AH) is ß-escin. The clinical therapeutic role of ß-escin and AH has been associated with their anti-inflammatory effects. Regarding their mechanism of action, we and others have shown that ß-escin and AH affect NF-κB signaling. Furthermore, we have reported the virucidal and broad-spectrum antiviral properties of ß-escin and AH against enveloped viruses such as RSV, in vitro and in vivo. In this study, we demonstrate that ß-escin and AH have antiviral and virucidal activities against SARS-CoV-2 and CCoV, revealing broad-spectrum antiviral activity against coronaviruses. Likewise, they exhibited NF-κB and cytokine modulating activities in epithelial and macrophage cell lines infected with coronaviruses in vitro. Hence, ß-escin and AH are promising broad-spectrum antiviral, immunomodulatory, and virucidal drugs against coronaviruses and respiratory viruses, including SARS-CoV-2.

Nitrogenous compounds from Aesculus wilsonii seeds.

Eight nitrogenous compounds including five undescribed ones, aeswilnitrousol A (1), aeswilnitrousosides BD (2-4), and 6-(2-hydroxy-3-methylbutylamino)-8-oxoadenine (5) were isolated from the seeds of Aesculus wilsonii. Their structures and absolute configurations were established based on spectroscopic determination, calculated electronic circular dichroism (ECD) analysis, as well as chemical reaction methods. Among the three known compounds, 7 and 8 were obtained from the Aesculus genus for the first time, and 6 was gained from this plant initially. The 13C NMR data of 7 and 8 were reported for the first time. Moreover, the inhibitory effect of all the isolates against LPS-induced nitric oxide production in RAW264.7 macrophages was evaluated. As a result, compounds 2 and 8 exhibited anti-inflammatory activity in a concentration-dependent manner at 10, 25, and 50 µM.

Characterization of the horse chestnut genome reveals the evolution of aescin and aesculin biosynthesis.

Horse chestnut (Aesculus chinensis) is an important medicinal tree that contains various bioactive compounds, such as aescin, barrigenol-type triterpenoid saponins (BAT), and aesculin, a glycosylated coumarin. Herein, we report a 470.02 Mb genome assembly and characterize an Aesculus-specific whole-genome duplication event, which leads to the formation and duplication of two triterpenoid biosynthesis-related gene clusters (BGCs). We also show that AcOCS6, AcCYP716A278, AcCYP716A275, and AcCSL1 genes within these two BGCs along with a seed-specific expressed AcBAHD6 are responsible for the formation of aescin. Furthermore, we identify seven Aesculus-originated coumarin glycoside biosynthetic genes and achieve the de novo synthesis of aesculin in E. coli. Collinearity analysis shows that the collinear BGC segments can be traced back to early-diverging angiosperms, and the essential gene-encoding enzymes necessary for BAT biosynthesis are recruited before the splitting of Aesculus, Acer, and Xanthoceras. These findings provide insight on the evolution of gene clusters associated with medicinal tree metabolites.

Phenolic-Based Discrimination between Non-Symptomatic and Symptomatic Leaves of Aesculus hippocastanum Infested by Cameraria ohridella and Erysiphe flexuosa.

The herbivore Cameraria ohridella (kingdom Animalia) and the pathogen Erysiphe flexuosa (kingdom Fungi) are considered pests and biotic stressors of Aesculus hippocastanum (chestnut trees). The impact of both pests on the accumulation of secondary metabolites in chestnut leaves was investigated. Specifically, the interactive effect of both pests on metabolite accumulation and their potential role in enhancing the resistance of chestnut trees to biological stress was the focus of this study. Aesculus hippocastanum leaves with varying degrees of Cameraria ohridella infestation and Erysiphe flexuosa infection were used in this research. Leaf samples were collected during the plant vegetative growth phase and evaluated for pest infection and secondary metabolite content. Eight main polyphenols were identified in the leaves: (1) neochlorogenic acid, (2) (-)-epicatechin, (3) procyanidin trimer A-type, (4) procyanidin tetramer A-type, (5) quercetin-3-O-arabinoside, (6) quercetin-3-O-rhamnoside, (7) kaempferol-3-O-arabinoside, and (8) kaempferol-3-O-rhamnoside. It was found that the accumulation of metabolites, primarily those derived from epicatechin and quercetin, during the initial vegetation phase (up to 11.05 or 09.05), strongly depended on the later degree of pest infection. The differences observed in the metabolite dynamics in the chestnut leaves, depending on the extent of infection, indicate the development of a metabolic response mechanism in chestnut trees to biological stress.

Impact analysis of different applications of cyantraniliprole on control of horse chestnut leaf miner (Cameraria ohridella) larvae supported by biophoton emission.

Cameraria ohridella is one of the most invasive pests of horse chestnut. Cyantraniliprole is one of the most perspectively active insecticides, which can transport within the plant in several ways, and its efficacy against this pest has not yet been tested. All three modes of application were effective against the target pest, but there was a difference in the time of action between them. However, no demonstrable difference in the speed of action was detected between the doses used. A more intense rate of acropetal translocation was confirmed compared to basipetal translocation. A trend-like effect between the applied concentration of cyantraniliprole and the photon emission intensity per unit area of plant tissue was observed in the translaminar and acropetal treatment settings. In both cases, a clear increase in photon emission was observed, indicating metabolic upregulation. Therefore, we can conclude that biophoton emission measurements can be utilized to conduct efficient pesticide translocation investigations.

Identification and functional characterization of AcMYB113 in anthocyanin metabolism of Aesculus chinensis Bunge var. chinensis leaves.

Anthocyanins can be induced by environmental factors such as low-temperature and play essential roles in plant color formation. In this study, leaves of Aesculus chinensis Bunge var. chinensis with different colors under natural low-temperature in autumn were collected and grouped into green leaf (GL) and red leaf (RL). To reveal the underlying mechanism of color formation in RL, a combined analysis of the metabolome and transcriptome was conducted with GL and RL. Metabolic analyses revealed that total anthocyanin content and primary anthocyanin components were increased RL relative to GL and cyanidin was the main anthocyanin compound in RL. Transcriptome analysis provided a total of 18720 differentially expressed genes (DEGs), of which 9150 DEGs were upregulated and 9570 DEGs were downregulated in RL relative to GL. KEGG analysis showed that DEGs were mainly enriched in flavonoid biosynthesis, phenylalanine metabolism, and phenylpropanoid biosynthesis. Furthermore, co-expression network analysis indicated that 56 AcMYB transcription factors were highly expressed in RL compared with GL, among which AcMYB113 (an R2R3-MYB TF) had a strong correlation with anthocyanins. Overexpression of AcMYB113 in apple resulted in dark-purple transgenic calluses. In addition, the transient expression experiment showed that AcMYB113 enhanced anthocyanin synthesis by activating pathways of anthocyanin biosynthesis in leaves of Aesculus chinensis Bunge var. chinensis. Taken together, our findings reveal new insights into the molecular mechanism of anthocyanin accumulation in RL and provide candidate genes for the breeding of anthocyanin-rich cultivars.

Phytochemical Estimation and Therapeutic Amelioration of Aesculus hippocastanum L. Seeds Ethanolic Extract in Gastric Ulcer in Rats Possibly by Inhibiting Prostaglandin Synthesis.

OBJECTIVE: To quantify phytochemicals using liquid chromatography and mass spectroscopy (LCMS) analysis and explore the therapeutic effect of Aesculus hippocastanum L. (AH) seeds ethanolic extract against gastric ulcers in rats. METHODS: Preliminary phytochemical testing and LCMS analysis were performed according to standard methods. For treatment, the animals were divided into 7 groups including normal control, ulcer control, self-healing, AH seeds low and high doses, ranitidine and per se groups. Rats were orally administered with 10 mg/kg of indomethacin, excluding the normal control group (which received 1% carboxy methyl cellulose) and the per se group (received 200 mg/kg AH seeds extract). The test group rats were then given 2 doses of AH seeds extract (100 and 200 mg/kg, respectively), while the standard group was given ranitidine (50 mg/kg). On the 11th day, rats in all groups were sacrificed, and their stomach was isolated to calculate the ulcer index, and other parameters such as blood prostaglandin (PGE2), tissue superoxide dismutase (SOD), catalase (CAT), malonyldialdehyde (MDA), and glutathione (GSH). All isolated stomach tissues were analyzed for histopathological findings. RESULTS: The phytochemical examination shows that the AH seeds contain alkaloids, flavonoids, saponins, phenolic components, and glycosides. LCMS analysis confirms the presence of quercetin and rutin. The AH seeds extract showed significant improvement in gastric mucosa conditions after indomethacin-induced gastric lesions (P<0.01). Further marked improvement in blood PGE2 and antioxidant enzymes, SOD, CAT, MDA and GSH, were observed compared with self-healing and untreated ulcer-induced groups (P<0.01). Histopathology results confirmed that AH seeds extract improved the mucosal layer and gastric epithelial membrane in treated groups compared to untreated ulcer-induced groups. CONCLUSIONS: LCMS report confirms the presence of quercetin and rutin in AH seeds ethanolic extract. The therapeutic effect of AH seeds extract against indomethacin-induced ulcer in rat model indicated the regenerated membrane integrity, with improved cellular functions and mucus thickness. Further, improved antioxidant enzyme level would help to reduce PGE2 biosynthesis.

Horse Chestnut Saponins-Escins, Isoescins, Transescins, and Desacylescins.

Escins constitute an abundant family of saponins (saponosides) and are the most active components in Aesculum hippocastanum (horse chestnut-HC) seeds. They are of great pharmaceutical interest as a short-term treatment for venous insufficiency. Numerous escin congeners (slightly different compositions), as well as numerous regio-and stereo-isomers, are extractable from HC seeds, making quality control trials mandatory, especially since the structure-activity relationship (SAR) of the escin molecules remains poorly described. In the present study, mass spectrometry, microwave activation, and hemolytic activity assays were used to characterize escin extracts (including a complete quantitative description of the escin congeners and isomers), modify the natural saponins (hydrolysis and transesterification) and measure their cytotoxicity (natural vs. modified escins). The aglycone ester groups characterizing the escin isomers were targeted. A complete quantitative analysis, isomer per isomer, of the weight content in the saponin extracts as well as in the seed dry powder is reported for the first time. An impressive 13% in weight of escins in the dry seeds was measured, confirming that the HC escins must be absolutely considered for high-added value applications, provided that their SAR is established. One of the objectives of this study was to contribute to this development by demonstrating that the aglycone ester functions are mandatory for the toxicity of the escin derivative, and that the cytotoxicity also depends on the relative position of the ester functions on the aglycone.

Comparison of Semen Aesculi species from different geographical origins and horse chestnut based on metabolomics and chemometrics analysis.

BACKGROUND: Semen Aesculi, a traditional Chinese herbal medicine, has a long history of use for treating chest and abdominal pain with distension. In addition, the horse chestnut (Aesculus hippocastanum L.) is another species of Aesculus in Europe and has notable clinical significance in alleviating chronic venous insufficiency, hemorrhoids, and postoperative edema. Thus, highlighting the comparative study of Semen Aesculi and horse chestnut may broaden clinical applications. OBJECTIVES: To conduct a comprehensive comparative analysis on the chemical profiling of these two varieties and determine whether they have equivalent clinical efficacy by integrating plant metabolomics and multivariate statistical methods. METHODS: Initially, a comprehensive characterisation was performed using ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) platform, and in total 44 active ingredients were identified. Then, untargeted metabolomics combined with principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) was applied for the discrimination of a German species and three official Chinese species. Next, 24 marker compounds responsible for the discrimination of different species were screened out and used to predict the species of unknown samples by genetic algorithm-optimised support vector machine (GA-SVM) with a high prediction accuracy. Finally, a heatmap visualisation was employed for clarifying the distribution of the identified active ingredients. RESULTS: The three species of Chinese Semen Aesculi showed distinct separation from each other, while European horse chestnut and Aesculus chinensis Bunge were similar in chemical composition. CONCLUSIONS: This work provided experimental evidence for further expanding the clinical application of Chinese Semen Aesculi and promoted the species identification and quality control of Semen Aesculi.

NO Release Inhibitory Activity of Flavonoids from Aesculus Wilsonii Seeds through MAPK (P38), NF-κB, and STAT3 Cross-Talk Signaling Pathways.

The flavonoid constituents of Aesculus wilsonii, a source of the Chinese medicinal drug Suo Luo Zi, and their in vitro anti-inflammatory effects were investigated. Fifteen flavonoids, including aeswilflavonosides IA-IC (1:  - 3: ) and aeswilflavonosides IIA-IIE (4:  - 8: ), along with seven known derivatives were isolated from a seed extract. Their structures were elucidated by extensive spectroscopic methods, acid and alkaline hydrolysis, and calculated electronic circular dichroism spectra. Among them, compounds 3: and 7: possess a 5-[2-(carboxymethyl)-5-oxocyclopent-yl]pent-3-enylate or oleuropeoylate substituent, respectively, which are rarely reported in flavonoids. Compounds 2, 3, 7: , and 12:  - 15: were found to inhibit lipopolysaccharide-induced nitric oxide production in RAW 264.7 cell lines. In a mechanistic assay, the flavonoid glycosides 2, 3: , and 7: reduced the expressions of interleukin-6 and tumor necrosis factor-alpha induced by lipopolysaccharide. Further investigations suggest that 2: and 3: downregulated the protein expression of tumor necrosis factor-alpha and interleukin-6 by inhibiting the phosphorylation of p38. Compound 7: was found to reduce the production of inducible nitric oxide synthase, and the secretion of tumor necrosis factor-alpha and interleukin-6 through inhibiting nuclear factor kappa-light-chain-enhancer of activated B signaling pathway. Compounds 2, 3: , and 7: possessed moderate inhibitory activity on the expression of signal transducer and activator of transcription-3. Taken together, the data indicate that the flavonoid glycosides of A. wilsonii seeds exhibit nitric oxide release inhibitory activity through mitogen-activated protein kinase (p38), nuclear factor kappa-light-chain-enhancer of activated B, and signal transducer and activator of transcription-3 cross-talk signaling pathways.

New HPLC pattern-oriented approach for quality control of enteric coated tablets containing aescin from Aesculus hippocastanum.

From the seed of Aesculus hippocastanum L., a complex mixture of triterpene saponin glycosides known as aescin was isolated, purified and characterized to be an active pharmaceutical ingredient for preparing of drug products. A reversed phase high performance liquid chromatographic method with UV detection was developed for the determination of total triterpene glycosides in enteric coated tablets containing aescin in a pattern-oriented manner. The mobile phase consisted of a mixture of acetonitrile and 0.1 % phosphoric acid solution (40: 60). UV detection was performed at 220 nm. The chromatographic column was Gemini C18 column, 5 µm 250 mm × 4.6 mm, which was maintained at 25 ◦C, connected with a precolumn (C18, 4.0 × 3.0 mm, 5 µm). The linear range was from 53.4 to 160.1 µg·mL-1. The RSD of intra-and inter-day precision variations were less than 1 % and the mean recovery was (100.66 ± 0.49) % (RSD = 0.64 %). The method showed its suitability, simplicity, rapidity and precision. Accordingly, it was proven adequate for quality control of this herbal drug product either for registration with the regulartory authority or routine analysis.

The Secondary Metabolites Profile in Horse Chestnut Leaves Infested with Horse-Chestnut Leaf Miner.

Natural defensive substances synthesized by plants that could replace synthetic pesticides in the protection of plants against insect invasions are constantly being sought. The study assessed changes in the qualitative and quantitative composition of secondary metabolites in horse chestnut leaves collected in different locations and differing in the sensitivity of the plant to the invasion by the horse-chestnut leaf miner. An attempt was made to identify compounds that are most responsible for the increased plant resistance to this threat. Additionally, changes in the anatomy of chestnut leaves affected by the pest were presented. It was noticed that the trees differed in the composition of secondary metabolites already in the initial growing season, which should be related to the influence of habitat conditions. The analysis of the profile of the compounds in non-infested and infested horse chestnut leaves revealed a clear response of the plant to the stress factor, i.e., the foraging of the horse-chestnut leaf miner. Catechins seem to be compounds involved in plant resistance. The leaf anatomy showed enhanced accumulation of phenolic compounds at the pest foraging sites. Hypertrophy and thickened and cracked cell walls of the spongy parenchyma were visible in the vicinity of the mines.

Assessment of Antimicrobial Activity of Ethanolic and Aqueous Extracts of Aesculus hippocastanum L. (Horse Chestnut) Bark against Bacteria Isolated from Urine of Patients Diagnosed Positive to Urinary Tract Infections.

The search for new antimicrobials is essential to address the worldwide issue of antibiotic resistance. The present work aimed at assessing the antimicrobial activity of Aesculus hippocastanum L. (horse chestnut) bark against bacteria involved in urinary tract infections (UTIs). Bioactive compounds were extracted from A. hippocastanum bark using water and ethanol as solvents. The extracts were tested against 10 clinical uropathogenic strains including five Gram-positive and five Gram-negative bacteria. Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 25922 were used as reference bacteria. The susceptibility to antibiotics was assessed using the Kirby Bauer disc diffusion method and the antibacterial activity of the extracts was evaluated using the well diffusion method. The Minimum inhibitory concentration (MIC) and the minimum bactericidal concentrations (MBC) were asseded by the microdilution method. A. hippocastanum bark possessed a dry matter content of 65.73%. The aqueous extract (AE) and ethanolic extract (EE) showed a volume yield of 77.77% and 74.07% (v/v), and a mass yields of 13.4% and 24.3% (w/w) respectively. All the bacteria were susceptible to amoxiclav, imipenem and ceftriaxone but the clinical strains were resistant to at least one antibiotic. Kocuria rizophilia 1542 and Corynebacterium spp 1638 were the most resistant bacteria both with multidrug resistance index of 0.45. Except AE on Proteus Mirabilis 1543 and Enterococcus faecalis 5960 (0 mm), both AE and EE were active against all the microorganisms tested with inhibition diameters (mm) which ranged from 5.5-10.0 for AE and 8.0-14.5 for EE. The MICs of EEs varied from 1-4 mg/mL while those of AEs varied from 4-16 mg/mL. The ethanolic extracts (EE) were overall more active than the aqueous ones. The A. hippocastanum bark extracts had overall weak antibacterial activity (MIC ≥0.625 mg/mL) and bacteriostatic potential (MBC/MIC ≥16) on both Gram-positive and Gram-negative bacteria.

Red Horse Chestnut and Horse Chestnut Flowers and Leaves: A Potential and Powerful Source of Polyphenols with High Antioxidant Capacity.

Aesculus flowers and leaves are an excellent source of bioactive compounds, including flavanols, phenolic acids, and anthocyanins, and the leaves also contain antioxidant carotenoids and chlorophylls. The aim of this study was to analyse and compare the amounts of bioactive compounds present in Aesculus hippocastanum and Aesculus × carnea flowers and leaves over two years. These two species from six independent locations (parks and green areas) located in Warsaw were assessed in this study. The dry matter by the scale method and polyphenol, carotenoid, and chlorophyll content by the HPLC method of the flowers and leaves was evaluated. Red horse chestnut flowers contained significantly more total carotenoids (40.6 µg/g FW) and chlorophylls (36.9 µg/g FW) than horse chestnut flowers, and red horse chestnut flowers contained higher levels of anthocyanins (5.41 µg/g FW) than other species. We observed that horse chestnut flowers were characterized by a higher total polyphenols concentration (9.45 µg/g FW) compared to red horse chestnut flowers. In addition, the analysis of leaves showed that all quality parameters were higher in red horse chestnut species. Five individual anthocyanins were identified in both species' flowers, but a higher concentration was found in red horse chestnut flowers, and pelargonidin-3-O-glucoside was the predominant form among a pool of total anthocyanins. In both experimental years, leaves (109.25 mMol/100 g FW and 112.0 mMol/100 g FW) were characterized by a higher antioxidant activity than flowers (27.0 mMol/100 g FW and 27.5 mMol/100 g FW).

Multiyear Time-Shift Study of Bacteria and Phage Dynamics in the Phyllosphere.

AbstractCoevolution shapes diversity within and among populations but is difficult to study directly. Time-shift experiments, where individuals from one point in time are experimentally challenged against individuals from past, contemporary, and/or future time points, are a powerful tool to measure coevolution. This approach has proven useful both in directly measuring coevolutionary change and in distinguishing among coevolutionary models. However, these data are only as informative as the time window over which they were collected, and inference from shorter coevolutionary windows might conflict with those from longer time periods. Previous time-shift experiments from natural microbial communities of horse chestnut tree leaves uncovered an apparent asymmetry, whereby bacterial hosts were more resistant to bacteriophages from all earlier points in the growing season, while phages were most infective to hosts from only the recent past. Here, we extend the time window over which these infectivity and resistance ranges are observed across years and confirm that the previously observed asymmetry holds over longer timescales. These data suggest that existing coevolutionary theory should be revised to include the possibility of differing models for hosts and their parasites and examined for how such asymmetries might reshape the predicted outcomes of coevolution.

Aesculus hippocastanum L. extract differently modulates normal human dermal fibroblasts and cancer-associated fibroblasts from basal/squamous cell carcinoma.

Fibroblasts are actively involved in the formation of granulation tissue and/or tumor stroma. These cells possess the potential to differentiate into myofibroblasts acquiring a highly contractile phenotype characterized by the expression of α-smooth muscle actin (SMA). Considering TGF-ß1 as the main inducer of myofibroblast differentiation and horse chestnut extract (HCE) as an effective modulator of the wound healing, we have new evidence to demonstrate canonical TGF-ß1/SMAD and non-canonical/non-SMAD signaling in normal fibroblasts, isolated from healthy human skin (human dermal fibroblasts - HDFs), and their malignant counterparts (CAFs) isolated from basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) using western blot and immunofluorescence. Our study revealed that HCE stimulated the deposition of fibronectin by BCC fibroblasts (BCCFs), an effect not seen in other studied fibroblasts. Moreover, HCE in combination with TGF-ß1 showed a synergic effect on the presence of polymerized SMA-stress fibers, particularly visible in CAFs. Interestingly, the TGF-ß1 exposure led to activation of the canonical SMAD signaling in HDFs and BCCFs, whereas treatment of SCC fibroblasts (SCCFs) resulted in activation of the non-canonical AKT and/or ERK1/2 signaling. In conclusion, we observed specific differences in signaling between HDFs and CAFs that should be considered when developing new therapeutic approaches targeting wound/tumor microenvironments.

Chestnut polysaccharides restore impaired spermatogenesis by adjusting gut microbiota and the intestinal structure.

Our previous study confirmed the beneficial effects of chestnut polysaccharides (CPs) on the spermatogenesis process, but the exact mechanism is not clear. Several studies have demonstrated the importance of balanced gut microbiota in maintaining normal reproductive function. In this study, we investigated the biological functions of CPs from the perspective of gut microbiota function, expecting to find out the specific mechanism of CPs in restoring impaired spermatogenesis. Compared with the control group, the mice treated with busulfan showed a reduced number of germ cells, structural changes in the small intestine and composition alteration in the gut microbiota at several levels, including the phylum and genus. In contrast, the number of germ cells in seminiferous tubules was significantly increased, and the structure of the small intestine and the composition of the gut microbiota were altered in the busulfan-treated mice after the CPs treatment. The 16s rRNA analysis results showed that the Firmicutes was the predominant phylum in all groups followed by Proteobacteria, Bacteroidetes, Actinobacteria, Tenericutes, Cyanobacteria and unidentified bacteria. Interestingly, the subsequent functional analysis implied that the steroid hormone biosynthesis process is the major metabolic pathway in the CPs-mediated restoration process and the experimental results confirmed this speculation. In conclusion, this study confirmed that CPs can restore the impaired spermatogenesis process by adjusting the gut microbiota and intestinal structure, which will also provide technical support and a theoretical basis for the subsequent treatment of male infertility.

Interaction between hypoviral-regulated fungal virulence factor laccase3 and small heat shock protein Hsp24 from the chestnut blight fungus Cryphonectria parasitica.

Laccase3 is an important virulence factor of the fungus Cryphonectria parasitica. Laccase3 gene (lac3) transcription is induced by tannic acid, a group of phenolic compounds found in chestnut trees, and its induction is regulated by the hypovirus CHV1 infection. CpHsp24, a small heat shock protein gene of C. parasitica, plays a determinative role in stress adaptation and pathogen virulence. Having uncovered in our previous study that transcriptional regulation of the CpHsp24 gene in response to tannic acid supplementation and CHV1 infection was similar to that of the lac3, and that conserved phenotypic changes of reduced virulence were observed in mutants of both genes, we inferred that both genes were implicated in a common pathway. Building on this finding, in this paper we examined whether the CpHsp24 protein (CpHSP24) was a molecular chaperone for the lac3 protein (LAC3). Our pull-down experiment indicated that the protein products of the two genes directly interacted with each other. Heterologous co-expression of CpHsp24 and lac3 genes using Saccharomyces cerevisiae resulted in more laccase activity in the cotransformant than in a parental lac3-expresssing yeast strain. These findings suggest that CpHSP24 is, in fact, a molecular chaperone for the LAC3, which is critical component of fungal pathogenesis.