Salvia miltiorrhiza suppresses cardiomyocyte ferroptosis after myocardial infarction by activating Nrf2 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Ferroptosis, a recently identified non-apoptotic form of cell death reliant on iron, is distinguished by an escalation in lipid reactive oxygen species (ROS) that are iron-dependent. This phenomenon has a strong correlation with irregularities in iron metabolism and lipid peroxidation. Salvia miltiorrhiza Bunge (DS), a medicinal herb frequently utilized in China, is highly esteemed for its therapeutic effectiveness in enhancing blood circulation and ameliorating blood stasis, particularly during the treatment of cardiovascular diseases (CVDs). Numerous pharmacological studies have identified that DS manifests antioxidative stress effects as well as inhibits lipid peroxidation. However, ambiguity persists regarding the potential of DS to impede ferroptosis in cardiomyocytes and subsequently improve myocardial damage post-myocardial infarction (MI). AIM OF THE STUDY: The present work focused on investigating whether DS could be used to prevent the ferroptosis of cardiomyocytes and improve post-MI myocardial damage. MATERIALS AND METHODS: In vivo experiments: Through ligation of the left anterior descending coronary artery, we constructed both a wild-type (WT) and NF-E2 p45-related factor 2 knockout (Nrf2-/-) mouse model of MI. Effects of DS and ferrostatin-1 (Fer-1) on post-MI cardiomyocyte ferroptosis were examined through detecting ferroptosis and myocardial damage-related indicators as well as Nrf2 signaling-associated protein levels. In vitro experiments: Erastin was used for stimulating H9C2 cardiomyocytes to construct an in vitro ferroptosis cardiomyocyte model. Effects of DS and Fer-1 on cardiomyocyte ferroptosis were determined based on ferroptosis-related indicators and Nrf2 signaling-associated protein levels. Additionally, inhibitor and activator of Nrf2 were used for confirming the impact of Nrf2 signaling on DS's effect on cardiomyocyte ferroptosis. RESULTS: In vivo: In comparison to the model group, DS suppressed ferroptosis in cardiomyocytes post-MI and ameliorated myocardial damage by inducing Nrf2 signaling-related proteins (Nrf2, xCT, GPX4), diminishing tissue ferrous iron and malondialdehyde (MDA) content. Additionally, it enhanced glutathione (GSH) levels and total superoxide dismutase (SOD) activity, effects that are aligned with those of Fer-1. Moreover, the effect of DS on alleviating cardiomyocyte ferroptosis after MI could be partly inhibited through Nrf2 knockdown. In vitro: Compared with the erastin group, DS inhibited cardiomyocyte ferroptosis by promoting the expression of Nrf2 signaling-related proteins, reducing ferrous iron, ROS, and MDA levels, but increasing GSH content and SOD activity, consistent with the effect of Fer-1. Additionally, Nrf2 inhibition increased erastin-mediated ferroptosis of cardiomyocytes through decreasing Nrf2 signaling-related protein expressions. Co-treatment with DS and Nrf2 activator failed to further enhance the anti-ferroptosis effect of DS. CONCLUSION: MI is accompanied by cardiomyocyte ferroptosis, whose underlying mechanism is probably associated with Nrf2 signaling inhibition. DS possibly suppresses ferroptosis of cardiomyocytes and improves myocardial damage after MI through activating Nrf2 signaling.

Environmental Variables Including Heavy Metals Significantly Shape the Soil Bacterial Community Structure in the Tatun Volcano Group, Northern Taiwan.

Recent studies suggested the presence of magma chambers from the Tatun volcano group under northern Taiwan's surface, the result of episodic volcanism for 0.2-2.8 million years. However, the microbial community in volcanic soil has not yet been characterized. Therefore, the present study investigated the spatial distribution of microbial communities and their relationships with environmental variables, including heavy metals. Next-generation sequencing was used to analyze the microbial community structures in three areas with different land uses: Lengshuikeng (recreational area), Zhuzihu (agricultural area), and Huangzuishan (conservation area). High contents of environmental factors, such as nitrogen (0.46-1.14%) and phosphorus (2.01-13.88 ppm), were detected. Large concentrations of heavy metals, such as copper (55.90-127.60 ppm) and zinc (36.13-147.73 ppm), were found among the three sites, whereas those of lead (83.13 ppm) and chromium (48.33 ppm) were higher in the Zhuzihu area. The most prevalent phylum across all sites was Proteobacteria, followed by Actinobacteria, Acidobacteria, and Chloroflexi, while the most abundant bacterial species was Koribacteraceae: NA_01, followed by Cyanobacteria: NA. A network ana-lysis showed that Koribacteracea: NA_01 positively correlated with bacterial groups, including Flavisolibacter sp., Oxalobacteraceae: NA, and Actinomycetales: NA_01. Based on Shannon and Simpson's diversity indices, the diversity of bacteria was significantly less in the Huangzuishan area than in the Lengshuikeng and Zhuzihu areas. Bacterial assemblages also significantly differed (P<0.05) among the three sites. The present results provide clear evidence to show that environmental variables, including heavy metals, are key factors affecting the bacterial community structure in volcanic soil.

Herbal Formula SS-1 Increases Tear Secretion for Sjögren's Syndrome.

Background: Sjögren's syndrome (SS) is an autoimmune inflammatory disease that primarily affects the exocrine glands, leading to glandular dysfunction. The hallmark symptoms of SS are dry eyes and mouth, compromising the quality of life of patients and decreasing their capacity to perform their daily activities. Objective: This study aims to evaluate the efficacy of the herbal formula SS-1 for its potential therapeutic benefits for patients with Sjögren's syndrome. Materials and Methods: The bioactivity profile of SS-1 was determined using four different SS-1 concentrations across 12 human primary cell systems of the BioMAP profile. After that, a randomized, double-blind, crossover, placebo-controlled trial was performed including 57 patients treated with SS-1 for 28 weeks. Results: Biologically multiplexed activity profiling in cell-based models indicated that SS-1 exerted anti-proliferative activity in B cells and promoted anti-inflammatory and immunomodulatory activity. In the clinical trial, Schirmer's test results revealed significant improvements in both eyes, with increases of 3.42 mm (95% CI, 2.44-4.41 mm) and 3.45 mm (95% CI, 2.32-4.59 mm), respectively, and a significant reduction in artificial tear use, which was -1.38 times/day, 95% CI, -1.95 to -0.81 times/day. Moreover, the increases in B-cell activating factor (BAFF) and B-cell maturation antigen (BCMA) levels were dampened by 53.20% (295.29 versus 555.02 pg/ml) and 58.33% (99.16 versus 169.99 pg/ml), respectively. Conclusion: SS-1 treatment significantly inhibited B-cell maturation antigen. No serious drug-related adverse effects were observed. Oral SS-1 administration may be a complementary treatment for Sjögren's syndrome.

Deciphering Trifolium pratense L. holobiont reveals a microbiome resilient to future climate changes.

The plant microbiome supports plant growth, fitness, and resistance against climate change. Trifolium pratense (red clover), an important forage legume crop, positively contributes to ecosystem sustainability. However, T. pratense is known to have limited adaptive ability toward climate change. Here, the T. pratense microbiomes (including both bacteria and fungi) of the rhizosphere and the root, shoot, and flower endospheres were comparatively examined using metabarcoding in a field located in Central Germany that mimics the climate conditions projected for the next 50-70 years in comparison with the current climate conditions. Additionally, the ecological functions and metabolic genes of the microbial communities colonizing each plant compartment were predicted using FUNGuild, FAPROTAX, and Tax4Fun annotation tools. Our results showed that the individual plant compartments were colonized by specific microbes. The bacterial and fungal community compositions of the belowground plant compartments did not vary under future climate conditions. However, future climate conditions slightly altered the relative abundances of specific fungal classes of the aboveground compartments. We predicted several microbial functional genes of the T. pratense microbiome involved in plant growth processes, such as biofertilization (nitrogen fixation, phosphorus solubilization, and siderophore biosynthesis) and biostimulation (phytohormone and auxin production). Our findings indicated that T. pratense microbiomes show a degree of resilience to future climate changes. Additionally, microbes inhabiting T. pratense may not only contribute to plant growth promotion but also to ecosystem sustainability.

Dauricine inhibits proliferation and promotes death of melanoma cells via inhibition of Src/STAT3 signaling.

Melanoma is the most common type of skin cancer. Signal transducer and activator of transcription 3 (STAT3) signaling has been demonstrated to be a therapeutic target for melanoma. Dauricine (Dau), an alkaloid compound isolated from the root of Menispermum dauricum DC., has shown tumor-suppressing effects in multiple human cancers, but its potential in melanoma remains unexplored. In this study, we demonstrated that Dau significantly inhibited the viability and proliferation of A375 and A2058 melanoma cells. Death of melanoma cells was also markedly promoted by Dau. Moreover, Dau inhibited phosphorylation-mediated activation of STAT3 and Src in a dose-dependent manner. Notably, constitutive activation of Src partially abolished the antiproliferative and cytotoxic activities of Dau on melanoma cells. Molecular docking showed that Dau could dock on the kinase domain of Src with a binding energy of -10.42 kcal/mol. Molecular dynamics simulations showed that Src-Dau binding was stable. Surface plasmon resonance imaging analysis also showed that Dau has a strong binding affinity to Src. In addition, Dau suppressed the growth of melanoma cells and downregulated the activation of Src/STAT3 in a xenograft model in vivo. These data demonstrated that Dau inhibits proliferation and promotes cell death in melanoma cells by inhibiting the Src/STAT3 pathways.

Notopterygium incisum extract (NRE) rescues cognitive deficits in APP/PS1 Alzhneimer's disease mice by attenuating amyloid-beta, tau, and neuroinflammation pathology.

ETHNOPHARMACOLOGICAL RELEVANCE: Alzheimer's disease (AD) is a frequently occurring disease of the elderly, and "deficiency" is the root of AD. Most famous experts of traditional Chinese medicine believe that the disease is based on deficiency, and the deficiency of kidney essence is the basis. Notopterygium incisum (Qiang huo) is beneficial to bladder, liver, and kidneys. It is used to treat liver and kidney deficiency, language difficulties, and mental coma. Qiang huo yu feng tang has been used to treat liver and kidney deficiency, unclear language and mental paralysis in many traditional Chinese medicine books and records. In modern times, it has been used to treat AD and exhibited favourable efficacy. AIM OF THE STUDY: This study attempts to investigate the effects of furocoumarins from Notopterygium incisum (NRE) on the Aß cascade, tau pathology and inflammatory pathology of AD. MATERIALS AND METHODS: In this study, we reported a detailed protocol for stabilizing HEK APPswe293T cells with lentivirus for the first time. This cell line can secrete high concentration of Aß. In addition, we treated N2a cells with AKT/PKC specific inhibitors (wortmannin/GF-109203X) and established a tau pathological cell model (AKT/PKC N2a) by activating GSK3ß and triggering hyperphosphorylation of tau. The Aß levels and the expression of phosphorylated tau were detected by ELISA and Western blot. The cognitive ability of NRE on APP/PS1 mice was detected using a Morris water maze (MWM) assay and Aß contents were also evaluated. RESULTS: In HEK APPswe293T cells, NRE (10, 20, 40 µg/mL) significantly inhibited the secretion and production of Aß in dose dependent manner. In addition, NRE also suppressed the expression of phosphorylated tau in wortmannin/GF-109203X treated N2a cells. Furthermore, NRE ameliorated the cognitive impairment of APP/PS1 mice, and the contents of Aß, IL-1ß and TNF-α were significantly depressed in hippocampus and cortex. CONCLUSION: In conclusion, our results demonstrated that NRE has a potential anti-AD effect via the inhibition of the Aß cascade, tau pathology and neuroinflammation in vitro and in vivo.

Effects and mechanism of action of Huang-Lian-Jie-Du-Tang in atopic dermatitis-like skin dysfunction in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Atopic dermatitis (AD), a disorder prevalent during childhood and adulthood, seriously affects the patient's quality of life. Although Huang-Lian-Jie-Du-Tang (HLJDT) has shown anti-inflammatory effects in previous studies, its effects and mechanism of action underlying AD disorder are still largely unknown. OBJECTIVE: This study explored the anti-inflammatory and immunomodulatory effects of HLJDT on the AD-like dermal disorder, induced in vitro by lipopolysaccharide (LPS)-triggered inflammation, and in vivo by 2,4-dinitrochlorobenzene (DNCB). MATERIALS AND METHODS: In vivo HLJDT effects were investigated by determining the severity of dermatitis, which consisted of observing signs of skin lesions, visually and through haematoxylin and eosin (HE) staining, in mouse ears and dorsal skin, measuring serum levels of interleukin (IL)-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, interferon (IFN)-γ, the tumour necrosis factor (TNF)-α, and determining the splenic index, number of splenic CD4+/CD8+ T-lymphocytes, as well as the phosphorylation levels of mitogen-activated protein kinases (including MAPKs-p38, ERK, and JNK), IκB-α, and nuclear factor kappa B (NF-κB) (p65) within dermal lesions. Morphological changes in LPS-induced inflammation were observed under a microscope, and ELISA and qPCR assays were used to measure IL-1α, IL-1ß, IL-6, and TNF-α expression levels. The protein expression levels of P-ERK/ERK, P-p38/p38, P-JNK/JNK, P-IKß-α, and P-p65 were measured through western blotting. Additionally, p65 expression was assessed by immunofluorescence, and LPS binding to RAW264.7 cell membrane was studied with laser confocal microscopy. RESULTS: HLJDT could remarkably mitigate DNCB-induced AD-like lesion symptoms, alleviating inflammatory mediator infiltration in mouse ears and dorsal skin tissue, down-regulating serum expression levels of IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IFN-γ, and TNF-α, normalising the splenic CD4+/CD8+ T-lymphocyte ratio, and inactivating MAPKs (including p38, ERK, and JNK), IκB-α, and NF-κB (p65) in dorsal skin. Furthermore, HLJDT inhibited LPS-induced differentiation of RAW264.7 cells, as evidenced by the decreased protein and mRNA expression of IL-1α, IL-1ß, IL-6, and TNF-α. Additionally, it decreased ERK, p38, JNK, IKß-α, and p65 phosphorylation levels in the MAPKs/NF-κB pathway, inhibited p65 nuclear translocation, and reduced LPS binding to the RAW264.7 cell membrane. CONCLUSIONS: HLJDT significantly improved AD-like symptoms via inhibition of the MAPKs/NF-κB pathway. Therefore, administration of HLJDT might be a potential treatment for AD in the clinical setting.

Rheum palmatum extract exerts anti-hepatocellular carcinoma effects by inhibiting signal transducer and activator of transcription 3 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Hepatocellular carcinoma (HCC) is among the most common malignancies. Signal transducer and activator of transcription 3 (STAT3), with abnormal expression and constitutive activation, has been reported to promote proliferation, metastasis, survival and angiogenesis of HCC cells. Rheum palmatum (RP), a traditional Chinese medicinal herb, exhibited tumor-suppressing effects in multiple human cancers, but its potential functions in HCC remain unexplored. AIM OF THE STUDY: This study aimed to examine the involvement of STAT3 signaling in the anti-HCC effects of RP extract. MATERIALS AND METHODS: SMMC-7721 and HepG2 HCC cell lines were treated with RP extract for 24 h, and then viability, migration, and invasion of HCC cells and angiogenesis of human umbilical vein endothelial cells (HUVECs) were analyzed using MTS, wound-healing, Transwell invasion and tube formation assays, respectively. Western blotting and immunohistochemistry (IHC) were used to examine the activation of key molecules in STAT3 signaling, including STAT3, JAK2, and Src. Additionally, we explored the in vivo antitumor effects of RP extract in a xenograft tumor nude mouse model of HCC. RESULTS: The result showed that RP extract reduced viability, migration, and invasion of SMMC-7721 and HepG2 cells and angiogenesis of HUVECs. It suppressed the phosphorylation of STAT3 and its upstream kinases including JAK2 and Src. In addition, RP extract treatment downregulated STAT3 target genes, including survivin, Bcl-xL, Mcl-1, Bcl-2, MMP-2, MMP-9, Cyclin D1, CDK4, c-Myc, and VEGF-C. Furthermore, RP extract suppressed the xenograft tumor growth and activation of STAT3 in xenograft tumor mice. CONCLUSION: Collectively, the results showed that RP extract prevented HCC progression by inhibiting STAT3, and might be useful for the treatment of HCC.

ATOH1/RFX1/RFX3 transcription factors facilitate the differentiation and characterisation of inner ear hair cell-like cells from patient-specific induced pluripotent stem cells harbouring A8344G mutation of mitochondrial DNA.

Degeneration or loss of inner ear hair cells (HCs) is irreversible and results in sensorineural hearing loss (SHL). Human-induced pluripotent stem cells (hiPSCs) have been employed in disease modelling and cell therapy. Here, we propose a transcription factor (TF)-driven approach using ATOH1 and regulatory factor of x-box (RFX) genes to generate HC-like cells from hiPSCs. Our results suggest that ATOH1/RFX1/RFX3 could significantly increase the differentiation capacity of iPSCs into MYO7AmCherry-positive cells, upregulate the mRNA expression levels of HC-related genes and promote the differentiation of HCs with more mature stereociliary bundles. To model the molecular and stereociliary structural changes involved in HC dysfunction in SHL, we further used ATOH1/RFX1/RFX3 to differentiate HC-like cells from the iPSCs from patients with myoclonus epilepsy associated with ragged-red fibres (MERRF) syndrome, which is caused by A8344G mutation of mitochondrial DNA (mtDNA), and characterised by myoclonus epilepsy, ataxia and SHL. Compared with isogenic iPSCs, MERRF-iPSCs possessed ~42-44% mtDNA with A8344G mutation and exhibited significantly elevated reactive oxygen species (ROS) production and CAT gene expression. Furthermore, MERRF-iPSC-differentiated HC-like cells exhibited significantly elevated ROS levels and MnSOD and CAT gene expression. These MERRF-HCs that had more single cilia with a shorter length could be observed only by using a non-TF method, but those with fewer stereociliary bundle-like protrusions than isogenic iPSCs-differentiated-HC-like cells could be further observed using ATOH1/RFX1/RFX3 TFs. We further analysed and compared the whole transcriptome of M1ctrl-HCs and M1-HCs after treatment with ATOH1 or ATOH1/RFX1/RFX3. We revealed that the HC-related gene transcripts in M1ctrl-iPSCs had a significantly higher tendency to be activated by ATOH1/RFX1/RFX3 than M1-iPSCs. The ATOH1/RFX1/RFX3 TF-driven approach for the differentiation of HC-like cells from iPSCs is an efficient and promising strategy for the disease modelling of SHL and can be employed in future therapeutic strategies to treat SHL patients.

Anti-inflammatory and immune response regulation of Si-Ni-San in 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin dysfunction.

ETHNOPHARMACOLOGICAL RELEVANCE: Si-Ni-San (SNS) is a well-known decoction in traditional Chinese medicine. Although studies have indicated that the anti-inflammatory and anti-allergic properties of SNS and its components can account for their therapeutic effects, the role and mechanism of SNS in treating skin dysfunction remain unclear. AIM OF THE STUDY: Atopic dermatitis (AD), a disorder known for its prevalence in infants and adults, severely influences the quality of life of affected patients. In this study, we aimed to investigate the anti-inflammatory and immune response modulations of SNS in 2,4-dinitrochlorobenzene (DNCB)-induced AD-like skin dysfunction. MATERIALS AND METHODS: Dermatitis was induced in Kunming mice by the topical application of DNCB. SNS or dexamethasone (positive control) was topically applied every day over the course of the 21-day study. The following were assessed: dermatitis severity scores; ear and dorsal skin haematoxylin and eosin staining; interleukin (IL)- 1α, IL-1ß, IL-2, IL-4, IL-6, and tumour necrosis factor (TNF)-α cytokine levels in the serum; spleen index; spleen CD4 + /CD8 + T lymphocyte ratio; and phosphorylation levels of mitogen-activated protein kinases (MAPKs- p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK)), IκB-α, and nuclear factor (NF)-κB (p65) in skin lesions. RESULTS: SNS significantly alleviated the symptoms of AD-like lesions induced by DNCB, decreased the infiltration of inflammatory cells in the ear and dorsal tissues, suppressed the increased cytokine levels in the serum, reduced the CD4 + /CD8 +T lymphocyte ratio in the spleen, and downregulated the activation of MAPKs, IκB-α, and NF-κB (p65) in the dorsal skin. The effects were similar to those of dexamethasone. CONCLUSIONS: SNS alleviated the DNCB-induced AD-like skin dysfunction in mice through anti-inflammatory and immune system modulation, indicating that SNS shows potential for AD treatment in clinical settings.

Icariside II attenuates myocardial fibrosis by inhibiting nuclear factor-κB and the TGF-ß1/Smad2 signalling pathway in spontaneously hypertensive rats.

Studies have demonstrated that icariin plays important roles in preventing hypertension and improving myocardial hypertrophy, inflammatory and infiltration. Icariside (ICS II) is the main metabolite of icariin, which has anti-inflammatory and anti-oxidant activities and protects against ischaemic brain injury. Whether ICS II improves myocardial fibrosis in spontaneously hypertensive rats (SHRs) and the related mechanism remain unknown. Some studies have suggested that TGF-ß and the nuclear factor κB signalling pathway play a key role in the progression of myocardial fibrosis. Therefore, in the current study, we aimed to evaluate the effects of ICS II on induced myocardial fibrosis in SHRs and explore the mechanism underlying this activity. The SHRs were treated with ICS II (4, 8, and 16 mg/kg) via daily gavage for 12 weeks. Left ventricular function was detected using the Vevo2100 system, and the collagen area was measured by Masson staining. The results indicated that ICS II markedly improved left ventricular function and decreased the left ventricular myocardial collagen area compared with the SHR group. To further investigate the mechanism underlying this activity, we measured the protein expression of interleukin-1ß (IL-1ß), tumour necrosis factor-α (TNF-α), transforming growth factor-ß1 (TGF-ß1), Smad2, inhibitory κB (IκB), and nuclear factor κB (NF-κB) p65 by Western blot. The results showed that ICS II inhibited NF-κB p65 expression and the TGF-ß1/Smad2 signalling pathways. In conclusion, the present results suggest that ICS II suppresses myocardial fibrosis in SHRs, and this effect might be at least partially mediated through suppression of NF-kB signalling and the TGF-ß1/Smad2 signalling pathway.

Vasculoprotective Effects of Water Extracts of Black, Green and Dark Tea in Vitro.

To investigate the preventive effects of tea on hyperglycemia and vascular complications of diabetes, we report the extraction and composition as well as the vasculoprotective effects of black tea extract (BTE), green tea extract (GTE), and dark tea extract (DTE). High Performance Liquid Chromatography (HPLC) and colorimetric methods were conducted to analyze for tea catechins, caffeine, polyphenols, amino acids and polysaccharides of BTE, GTE and DTE. The inhibitory effects of α-glucosidase, aldose reductase (AR), advanced glycation end-products (AGEs) and glucose uptake promotion effect in BTE, GTE and DTE were explored in vitro. Contents of six major catechin forms and total catechin as well as polyphenols are higher in GTE and DTE than BTE. BTE, GTE, and DTE showed the inhibitory effects of a-glucosidase, AR, and AGEs, but only DTE exhibited the glucose uptake promotion effect in HepG2 cells. The results suggest that regular consumption of tea can help prevent the progression of hyperglycemia and the vascular complications of diabetes.