Calycosin promotes axon growth by inhibiting PTPRS and alleviates spinal cord injury.

Our former studies have identified the alleviating effect of Calycosin (CA) on spinal cord injury (SCI). In this study, our purpose is to explore the influence of CA on SCI from the perspective of promoting axon growth. The SCI animal model was constructed by spinal cord compression, wherein rat primary cortex neuronal isolation was performed, and the axonal growth restriction cell model was established via chondroitin sulfate proteoglycan (CSPG) treatment. The expressions of axon regeneration markers were measured via immunofluorescent staining and western blot, and the direct target of CA was examined using silver staining. Finally, the expression of the protein tyrosine phosphatase receptor type S (PTPRS) was assessed using western blot. CA treatment increased neuronal process outgrowth and the expressions of axon regeneration markers, such as neurofilament H (NF-H), vesicular glutamate transporter 1 (vGlut1), and synaptophysin (Syn) in both SCI model rats and CSPG-treated primary cortical neurons, and PTPRS levels were elevated after SCI induction. In addition, PTPRS was the direct target of CA, and according to in vivo findings, exposure to CA reduced the PTPRS content. Furthermore, PTPRS overexpression inhibited CA's enhancement of axon regeneration marker content and neuronal axon lengths. CA improves SCI by increasing axon development through regulating PTPRS expression.

Calycosin Enhances Heat Shock Related-Proteins in H9c2 Cells to Modulate Survival and Apoptosis against Heat Shock.

Heat shock proteins (HSPs), which function as chaperones, are activated in response to various environmental stressors. In addition to their role in diverse aspects of protein production, HSPs protect against harmful protein-related stressors. Calycosin exhibits numerous beneficial properties. This study aims to explore the protective effects of calycosin in the heart under heat shock and determine its underlying mechanism. H9c2 cells, western blot, TUNEL staining, flow cytometry, and immunofluorescence staining were used. The time-dependent effects of heat shock analyzed using western blot revealed increased HSP expression for up to 2[Formula: see text]h, followed by protein degradation after 4[Formula: see text]h. Hence, a heat shock damage duration of 4[Formula: see text]h was chosen for subsequent investigations. Calycosin administered post-heat shock demonstrated dose-dependent recovery of cell viability. Under heat shock conditions, calycosin prevented the apoptosis of H9c2 cells by upregulating HSPs, suppressing p-JNK, enhancing Bcl-2 activation, and inhibiting cleaved caspase 3. Calycosin also inhibited Fas/FasL expression and activated cell survival markers (p-PI3K, p-ERK, p-Akt), indicating their cytoprotective properties through PI3K/Akt activation and JNK inhibition. TUNEL staining and flow cytometry confirmed that calycosin reduced apoptosis. Moreover, calycosin reversed the inhibitory effects of quercetin on HSF1 and Hsp70 expression, illustrating its role in enhancing Hsp70 expression through HSF1 activation during heat shock. Immunofluorescence staining demonstrated HSF1 translocation to the nucleus following calycosin treatment, emphasizing its cytoprotective effects. In conclusion, calycosin exhibits pronounced protective effects against heat shock-induced damages by modulating HSP expression and regulating key signaling pathways to promote cell survival in H9c2 cells.

Calycosin inhibited MIF-mediated inflammatory chemotaxis of macrophages to ameliorate ischemia reperfusion-induced acute kidney injury.

BACKGROUND: Inflammatory macrophage infiltration plays a critical role in acute kidney disease induced by ischemia-reperfusion (IRI-AKI). Calycosin is a natural flavone with multiple bioactivities. This study aimed to investigate the therapeutic role of calycosin in IRI-AKI and its underlying mechanism. METHODS: The renoprotective and anti-inflammatory effects of calycosin were analyzed in C57BL/6 mice with IRI-AKI and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. RNA-seq was used for mechanism investigation. The molecular target of calycosin was screened by in silico methods and validated by surface plasmon resonance (SPR). Macrophage chemotaxis was analyzed using Transwell and agarose gel spot assays. RESULTS: Calycosin treatment significantly reduced serum creatinine and urea nitrogen and attenuated tubular destruction in IRI-AKI mice. Additionally, calycosin markedly suppressed NF-κB signaling activation and the expression of inflammatory mediators IL-1ß and TNF-α in IRI-AKI kidneys and LPS-stimulated RAW 264.7 cells. Interestingly, RNA-seq revealed calycosin remarkably downregulated chemotaxis-related pathways in RAW 264.7 cells. Among the differentially expressed genes, Ccl2/MCP-1, a critical chemokine mediating macrophage inflammatory chemotaxis, was downregulated in both LPS-stimulated RAW 264.7 cells and IRI-AKI kidneys. Consistently, calycosin treatment attenuated macrophage infiltration in the IRI-AKI kidneys. Importantly, in silico target prediction, molecular docking, and SPR assay demonstrated that calycosin directly binds to macrophage migration inhibitory factor (MIF). Functionally, calycosin abrogated MIF-stimulated NF-κB signaling activation and Ccl2 expression and MIF-mediated chemotaxis in RAW 264.7 cells. CONCLUSIONS: In summary, calycosin attenuates IRI-AKI by inhibiting MIF-mediated macrophage inflammatory chemotaxis, suggesting it could be a promising therapeutic agent for the treatment of IRI-AKI.

Calycosin prevents NLRP3-induced gut fibrosis by regulating IL-33/ST2 axis.

Intestinal interstitial fibrosis is a core event of inflammatory bowel disease (IBD) development. Calycosin has been recognized to carry various therapeutic bioactivities. However, the role of calycosin in intestinal interstitial fibrosis remains to be illustrated. This aim of this study was to explore the effects of calycosin on intestinal interstitial fibrosis in IBD and the underlying mechanisms. The in vitro and in vivo models were established by using TNBS-induced mouse IBD model and co-culture of intestinal epithelial cells and intestinal interstitial cells; moreover, lentivirus-mediated knockdown of NLRP3 expression was applied. The results showed that calycosin significantly improved the intestinal interstitial fibrosis of TNBS-induced IBD. Mechanistically, calycosin downregulated NLRP3 expression and inhibited the activation of IL-33/ST2 signaling in intestinal epithelial cells, which subsequently impedes intestinal interstitial cell migration and activation by regulating the secretion of IL-33/ST2 signaling-induced fibrosis mediators. Notably, combination of calycosin and NLRP3 signaling blockade improved the intestinal interstitial fibrosis extent. Altogether, this study suggests calycosin can improve intestinal interstitial fibrosis by downregulating NLRP3-IL-33/ST2 signaling, reducing inflammation and decreasing pro-fibrotic factors' secretion, which provides a new perspective for therapeutic options of IBD.

Calycosin action against atherosclerosis: integrating network pharmacology and in-silico investigation.

Atherosclerosis, caused by lipid deposit in the arterial wall for narrowing the arteries, is an increased risk factor of developing heart failure. Presently, clinical first-line drug therapy can be found with side effects, and thus new substitute medication should be developed needfully. Calycosin is one of the most bioactive products refined from natural plant, and it exerts promising cardiovascular protective effect. However, the pharmacological mechanisms of calycosin against atherosclerosis have not been elaborated. In this study, a systematic network pharmacology combined with molecular docking analysis was used to reveal the interaction activity and biological target in calycosin against atherosclerosis. We screened all preparative targets linked to calycosin and atherosclerosis from the available public databases. These results indicated total 409 putative targets in calycosin action, 71 of which were interacted with atherosclerosis. Further biological docking analysis suggested that calycosin displayed the powerful binding affinities with target proteins, including interleukin-6 (IL6) and mitogen-activated protein kinase 3 (MAPK3) MAPK3. Then enrichment findings revealed that calycosin action to treat atherosclerosis might be related to inhibition of inflammatory reaction and oxidative stress through modulating nucleolus transcription factor for improving lipid metabolism. In conclusion, the anti-atherosclerotic targets and molecular mechanisms in calycosin action were revealed systematically through preclinical evaluation. And calycosin may be a potential natural compound for the treatment of atherosclerosis.

Exploring the effects of calycosin on anthracycline-induced cardiotoxicity: a network pharmacology, molecular docking, and experimental study.

Background: Chemotherapy with anthracyclines can cause cardiotoxicity, possibly leading to stopping treatment in some cancer patients. In cardio-oncology research, preventing and minimizing anthracycline-induced cardiotoxicity (AIC) is a hot issue. For the treatment of AIC, calycosin (CA), an isoflavone component in astragali radix (AR), has become a research focus. However, the elaborate mechanisms of calycosin treating AIC remain to be unrevealed. Aim of the study: To explore the effects of CA on AIC through multiple dimensions concerning network pharmacology, molecular docking, and experimental evaluations. Methods: The study evaluated calycosin's potential targets and mechanisms for treating AIC using network pharmacology and molecular docking. The candidate genes/targets of CA and AIC were screened using the online-available database. Protein-protein interactions (PPI) between the common targets were constructed using the STRING platform, and the results were then visualized using Cytoscape. Molecular docking was used to evaluate the strength of the binding force between CA and the common targets. The possible pharmacological mechanisms of CA were explained by pathway enrichment and GSEA. Subsequently, the candidate targets were identified in vitro experiments. Results: Network pharmacology effectively discovered the CA's multitarget intervention in AIC, including TNF, ABCC1, TOP2A, ABCB1, and XDH. CA binds to the ATP-binding cassette subfamily B member 1(ABCB1) had the highest binding energy (-7.5 kcal/mol) according to the molecular docking analysis and was selected and visualized for subsequent analysis. In vitro experiments showed that ABCB1 exhibited significant time-curve changes under different doses of doxorubicin (DOX) compared with DMSO control experiments. The anti-AIC pharmacological mechanism of CA were revealed by highlighting the biological processes of oxidative stress (OR) and inflammation. Conclusions: We employed a practicable bioinformatics method to connect network and molecular docking to determine the calycosin's therapeutic mechanism against AIC and identified some bioinformatics results in in vitro experiments. The results presented show that CA may represent an encouraging treatment for AIC.

Blocking group 2 innate lymphoid cell activation and macrophage M2 polarization: potential therapeutic mechanisms in ovalbumin-induced allergic asthma by calycosin.

BACKGROUND: Calycosin, a flavonoid compound extracted from Astragalus membranaceus, has shown anti-asthma benefits in house dust mite-induced asthma. Recent studies have suggested that innate-type cells, including group 2 innate lymphoid cells (ILC2s) and macrophages, serve as incentives for type 2 immunity and targets for drug development in asthma. This work focuses on the effects of calycosin on the dysregulated ILC2s and macrophages in allergic asthma. METHODS: In vivo, the asthmatic mouse model was established with ovalbumin (OVA) sensitization and challenge, and calycosin was intraperitoneally administered at doses of 20 and 40 mg/kg. In vivo, mouse primary ILC2s were stimulated with interleukin (IL)-33 and mouse RAW264.7 macrophages were stimulated with IL-4 and IL-13 to establish the cell models. Cells were treated with calycosin at doses of 5 and 10 µM. RESULTS: In vivo, we observed significantly reduced numbers of eosinophils, neutrophils, monocyte macrophages and lymphocytes in the bronchoalveolar lavage fluid (BALF) of OVA-exposed mice with 40 mg/kg calycosin. Histopathological assessment showed that calycosin inhibited the airway inflammation and remodeling caused by OVA. Calycosin markedly decreased the up-regulated IL-4, IL-5, IL-13, IL-33, and suppression tumorigenicity 2 (ST2) induced by OVA in BALF and/or lung tissues of asthmatic mice. Calycosin repressed the augment of arginase 1 (ARG1), IL-10, chitinase-like 3 (YM1) and mannose receptor C-type 1 (MRC1) levels in the lung tissues of asthmatic mice. In vivo, calycosin inhibited the IL-33-induced activation as well as the increase of IL-4, IL-5, IL-13 and ST2 in ILC2s. Calycosin also repressed the increase of ARG1, IL-10, YM1 and MRC1 induced by IL-4 and IL-13 in RAW264.7 macrophages. In addition, we found that these changes were more significant in 40 mg/kg calycosin treatment than 20 mg/kg calycosin. CONCLUSIONS: Collectively, this study showed that calycosin might attenuate OVA-induced airway inflammation and remodeling in asthmatic mice via preventing ILC2 activation and macrophage M2 polarization. Our study might contribute to further study of asthmatic therapy.

Calycosin (CA) inhibits proliferation, migration and invasion by suppression of CXCL10 signaling pathway in glioma.

Glioblastoma is the most common malignant tumor in the central nervous system and its occurrence and development is involved in various molecular abnormalities. C-X-C chemokine ligand 10 (CXCL10), an inflammatory chemokine, has been reported to be related to the pathogenesis of cancer while it has not yet been linked to glioma. Calycosin, a bioactive compound derived from Radix astragali, has demonstrated anticancer properties in several malignancies, including glioma. Nonetheless, its underlying mechanisms are not fully understood. This study explores CXCL10 as a potential therapeutic target for calycosin in the suppression of glioblastoma. We observed that CXCL10 expression correlates positively with glioma malignancy and inversely with patient prognosis, highlighting its potential as a glioblastoma treatment target. Furthermore, we found that calycosin inhibited proliferation, migration, and invasion in U87 and U251 glioma cells, and decreased CXCL10 expression in a dose-dependent manner, along with its downstream effectors such as NLRP3, NF-κB, and IL-1ß. Additionally, molecular docking experiments demonstrated that calycosin exhibits a notable binding affinity to CXCL10. Overexpression of CXCL10 counteracted the inhibitory effects of calycosin on cell proliferation, migration, and invasion, while CXCL10 knockdown enhanced these effects. Finally, we verified that calycosin inhibited glioma growth in a xenograft mouse model and downregulated CXCL10 and its downstream molecules. These findings suggest that targeting CXCL10 may be an effective strategy in glioblastoma treatment, and calycosin emerges as a potential therapeutic agent.

Calycosin alleviates titanium particle-induced osteolysis by modulating macrophage polarization and subsequent osteogenic differentiation.

Periprosthetic osteolysis (PPO) caused by wear particles is one of the leading causes of implant failure after arthroplasty. Macrophage polarization imbalance and subsequent osteogenic inhibition play a crucial role in PPO. Calycosin (CA) is a compound with anti-inflammatory and osteoprotective properties. This study aimed to evaluate the effects of CA on titanium (Ti) particle-induced osteolysis, Ti particle-induced macrophage polarization and subsequent osteogenic deficits, and explore the associated signalling pathways in a Ti particle-stimulated calvarial osteolysis mouse model using micro-CT, ELISA, qRT-PCR, immunofluorescence and western blot techniques. The results showed that CA alleviated inflammation, osteogenic inhibition and osteolysis in the Ti particle-induced calvarial osteolysis mouse model in vivo. In vitro experiments showed that CA suppressed Ti-induced M1 macrophage polarization, promoted M2 macrophage polarization and ultimately enhanced osteogenic differentiation of MC3T3-E1 cells. In addition, CA alleviated osteogenic deficits by regulating macrophage polarization homeostasis via the NF-κB signalling pathway both in vivo and in vitro. All these findings suggest that CA may prove to be an effective therapeutic agent for wear particle-induced osteolysis.

Calycosin enhances Treg differentiation for alleviating skin inflammation in atopic dermatitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Atopic dermatitis (AD) is a prevalent chronic inflammatory skin disorder that poses a significant global health challenge. There is a lack of safe and effective medications to treat AD. Astragalus membranaceous is a traditional Chinese medicine widely used in clinical treatment of skin diseases. Calycosin (CA), derived from the root of Astragalus membranaceous, exhibits dual attributes of anti-inflammatory and antioxidant properties, suggesting its promise for addressing cutaneous inflammation. Nonetheless, the precise mechanisms underlying CA's therapeutic actions in AD remain elusive. AIM OF THE STUDY: This study aimed to evaluate the efficacy and safety of CA in treating AD while also delving into the mechanistic underpinnings of CA's action in AD. MATERIALS AND METHODS: The cell viability and anti-inflammatory impacts of CA in vitro were first gauged using CCK-8 and RT-qPCR. The potential mechanisms of CA were then probed using modular pharmacology. Flow cytometry was employed to ascertain the differentiation of Treg and Th17 cells derived from naïve T cells, as well as the proportions and mean fluorescence intensity (MFI) of human iTreg cells. The expressions of IL-10 and TGF-ß1 were measured and Treg suppression assay was performed. The in vivo therapeutic efficacy of topical CA application was assessed using a calcipotriol (MC903)-induced AD mouse model. The expression metrics of inflammatory cytokines, IL-17A, FOXP3, and RORγt were authenticated via immunohistochemistry, RT-qPCR, Western blot, and ELISA. RESULTS: CA exhibited a favorable safety profile and reduced the mRNA expressions of Th2 inflammatory cytokines in HaCaT cells. Modular pharmacology analysis pinpointed Th17 differentiation as the pivotal mechanism behind CA's therapeutic effect on AD. In vitro, CA fostered the differentiation of naïve T cells into Tregs while inhibiting their differentiation into Th17 cells. Furthermore, CA augmented the proliferation of human iTregs. In vivo, CA alleviated skin manifestations and decreased the levels of inflammatory mediators (IL-4, IL-5, IL-13, TSLP, and NF-κB related cytokines) in AD-like mouse models. Simultaneously, it regulated Treg/Th17 balance through suppressing IL-17A and RORγt expressions and bolstering FOXP3 expression. CONCLUSIONS: The study provides insights into the mechanistic pathways through which CA exerts its anti-inflammatory effects, particularly through promoting Treg cell differentiation and inhibiting Th17 cell differentiation. Furthermore, CA emerges as an alternative or adjunctive treatment strategy for managing AD.

Unveiling the potential anti-cancer activity of calycosin against multivarious cancers with molecular insights: A promising frontier in cancer research.

BACKGROUND: Calycosin may be a potential candidate regarding chemotherapeutic agent, because already some studies against multivarious cancer have been made with this natural compound. AIM: This review elucidated a brief overview of previous studies on calycosin potential effects on various cancers and its potential mechanism of action. METHODOLOGY: Data retrieved by systematic searches of Google Scholar, PubMed, Science Direct, Web of Science, and Scopus by using keywords including calycosin, cancer types, anti-cancer mechanism, synergistic, and pharmacokinetic and commonly used tools are BioRender, ChemDraw Professional 16.0, and ADMETlab 2.0. RESULTS: Based on our review, calycosin is available in nature and effective against around 15 different types of cancer. Generally, the anti-cancer mechanism of this compound is mediated through a variety of processes, including regulation of apoptotic pathways, cell cycle, angiogenesis and metastasis, oncogenes, enzymatic pathways, and signal transduction process. These study conducted in various study models, including in silico, in vitro, preclinical and clinical models. The molecular framework behind the anti-cancer effect is targeting some oncogenic and therapeutic proteins and multiple signaling cascades. Therapies based on nano-formulated calycosin may make excellent nanocarriers for the delivery of this compound to targeted tissue as well as particular organ. This natural compound becomes very effective when combined with other natural compounds and some standard drugs. Moreover, proper use of this compound can reverse resistance to existing anti-cancer drugs through a variety of strategies. Calycosin showed better pharmacokinetic properties with less toxicity in human bodies. CONCLUSION: Calycosin exhibits excellent potential as a therapeutic drug against several cancer types and should be consumed until standard chemotherapeutics are available in pharma markets.

Calycosin ameliorates osteoarthritis by regulating the imbalance between chondrocyte synthesis and catabolism.

Osteoarthritis (OA) is a severe chronic inflammatory disease. As the main active component of Astragalus mongholicus Bunge, a classic traditional ethnic herb, calycosin exhibits anti-inflammatory action and its mechanism of exact targets for OA have yet to be determined. In this study, we established an anterior cruciate ligament transection (ACLT) mouse model. Mice were randomized to sham, OA, and calycosin groups. Cartilage synthesis markers type II collagen (Col-2) and SRY-Box Transcription Factor 9 (Sox-9) increased significantly after calycosin gavage. While cartilage matrix degradation index cyclooxygenase-2 (COX-2), phosphor-epidermal growth factor receptor (p-EGFR), and matrix metalloproteinase-9 (MMP9) expression were decreased. With the help of network pharmacology and molecular docking, these results were confirmed in chondrocyte ADTC5 cells. Our results indicated that the calycosin treatment significantly improved cartilage damage, this was probably attributed to reversing the imbalance between chondrocyte synthesis and catabolism.

The effect of different LED wavelengths on the components and biosynthesis of isoflavonoid in sprout Astragalus membranaceus.

An artificial light source is the optimal element for studying the usability of the medicinal plant Astragalus membranaceus as a sprout vegetable. Based on artificial light source conditions, formononetin (FO) level was the highest (2.6 mg/L) in A. membranaceus exposed to white light emitting diode (LED) light, and calycosin (CA) level was the highest (3.09 mg/L) in the plant exposed to red LED light. According to the publicly available transcriptome data of LED-exposed sprout A. membranaceus LED, reference genes related to the content enhancement of FO, an isoflavone compound, and those related to the content enhancement of CA were selected. The expression patterns of these genes were assayed using qPCR. Among the genes related to FO enhancement, Gene-225190T showed the highest mRNA levels in cells of LED-white light-exposed sprout A. membranaceus; among the genes related to CA enhancement, Gene_042770T showed the highest expression under red LED light. Most genes related to the overall biosynthesis regulation of flavonoids of the upper concept of isoflavone were highly expressed in response to red LED light, and the transcriptional level of 4CL in response to red LED light was the highest. Based on these results, the artificial light sources that regulated the FO and CA contents in sprouts A. membranaceus were white and red LED lights, and the selected reference genes were capable of regulating isoflavone biosynthesis.

ER-α36 is involved in calycosin inhibition of IL-6 production in macrophages.

The tumour microenvironment (TME) is crucial for tumour development and progression. Tumour-associated macrophages (TAMs) in the TME can promote tumour progression and metastasis by releasing cytokines, such as IL-6. Calycosin, a phytoestrogen that is one of the active compounds in Radix Astragali, has been shown to inhibit tumour growth and metastasis. However, the underlying mechanism by which calycosin inhibits tumour growth remains unclear. Thus, this study aimed to investigate the effect of calycosin on IL-6 production in peripheral blood mononuclear cell (PBMC)- and THP-1-derived macrophages and explore its potential mechanisms using co-immunoprecipitation, western blotting, immunofluorescence, chromatin immunoprecipitation and luciferase assays. We found that calycosin treatment substantially upregulated the expression of ER-α36, a variant of the ER, and reduced IL-6 production in macrophages. Mechanistically, ER-α36 physically interacted with NF-κBp65 and retained p65 in the cytoplasm to attenuate NF-κB function as an IL-6 transcriptional inducer. In conclusion, our result indicated that calycosin inhibited IL-6 production by enhancing ER-α36 expression and its interaction with p65, which attenuated NF-κB function as an IL-6 inducer. Therefore, calycosin can be developed as an effective agent for cancer therapy by targeting TAMs.

Rehabilitation training enhanced the therapeutic effect of calycosin on neurological function recovery of rats following spinal cord injury.

BACKGROUND: Calycosin (CA), a flavonoids component, has demonstrated potential neuroprotection effects by inhibiting oxidative stress in spinal cord injury (SCI) models. This study aims to investigate the impact of combined rehabilitation training (RT) and calycosin therapy on neurological function following SCI, primarily by assessing changes in motor function recovery, neuronal survival, neuronal oxidative stress levels, and neural proliferation, in order to provide novel insights for the treatment of SCI. MATERIALS AND METHODS: The SCI model was constructed by compressing the spinal cord using vascular clamps. Calycosin was injected intraperitoneally into the SCI model rats, and a group of 5 rats underwent RT. The motor function of rats after SCI was evaluated using the Basso Beattle Bresnaha (BBB) score and the inclined plate test. Histopathological changes were evaluated by NeuN immunohistochemistry, HE and Nissl staining. Apoptosis was detected by TUNEL staining. The antioxidant effect of combined treatment was assessed by measuring changes in oxidative stress markers after SCI. Western blot analysis was conducted to examine changes in Hsp90-Akt/ASK1-p38 pathway-related proteins. Finally, cell proliferation was detected by BrdU and Ki67 assays. RESULTS: RT significantly improved the BBB score and angle of incline promoted by calycosin, resulting in enhanced motor function recovery in rats with SCI. Combining rehabilitation training with calycosin has a positive effect on morphological recovery. Similarly, combined RT enhanced the Nissl and NeuN staining signals of spinal cord neurons increased by calycosin, thereby increasing the number of neurons. TUNEL staining results indicated that calycosin treatment reduced the apoptosis signal in SCI, and the addition of RT further reduced the apoptosis. Moreover, RT combined with calycosin reduced oxidative stress by increasing SOD and GSH levels, while decreasing MDA, NO, ROS, and LDH expressions compared to the calycosin alone. RT slightly enhanced the effect of calycosin in activating Hsp90 and Akt and inhibiting the activation of ASK1 and p38, leading to enhanced inhibition of oxidative stress by calycosin. Additionally, the proliferation indexes (Ki67 and BrdU) assays showed that calycosin treatment alone increased both, whereas the combination treatment further promoted cell proliferation. CONCLUSION: Our research findings demonstrate that rehabilitation training enhances the ability of calycosin to reduce oxidative stress, resulting in a decrease in neuronal apoptosis and an increase in proliferation, ultimately promoting neuronal survival.

Natural products in traditional Chinese medicine: molecular mechanisms and therapeutic targets of renal fibrosis and state-of-the-art drug delivery systems.

Renal fibrosis (RF) is the end stage of several chronic kidney diseases. Its series of changes include excessive accumulation of extracellular matrix, epithelial-mesenchymal transition (EMT) of renal tubular cells, fibroblast activation, immune cell infiltration, and renal cell apoptosis. RF can eventually lead to renal dysfunction or even renal failure. A large body of evidence suggests that natural products in traditional Chinese medicine (TCM) have great potential for treating RF. In this article, we first describe the recent advances in RF treatment by several natural products and clarify their mechanisms of action. They can ameliorate the RF disease phenotype, which includes apoptosis, endoplasmic reticulum stress, and EMT, by affecting relevant signaling pathways and molecular targets, thereby delaying or reversing fibrosis. We also present the roles of nanodrug delivery systems, which have been explored to address the drawback of low oral bioavailability of natural products. This may provide new ideas for using natural products for RF treatment. Finally, we provide new insights into the clinical prospects of herbal natural products.

Calycosin protects against chronic prostatitis in rats via inhibition of the p38MAPK/NF-κB pathway.

Currently, the effect and molecular mechanism of calycosin, the main active ingredient of Qinshi Simiao San, which can alleviate chronic prostatitis (CP), on CP remain unclear. This study aimed to elucidate the potential mechanism of action of calycosin in CP in a rat CP model. The prostate tissue morphology was evaluated based on hematoxylin-eosin staining. Enzyme-linked immunosorbent assay was conducted to evaluate inflammatory cytokine and immune factor levels (secretory immunoglobulin A [SIgA]; immunoglobulin G [IgG]) in prostate tissues and serum. Additionally, representative biomarkers of oxidative stress, including malondialdehyde, superoxide dismutase, and catalase were detected using detection kits, and reactive oxygen species release was evaluated using immunofluorescence staining. Furthermore, the p38 mitogen-activated protein kinase (p38MAPK)/NF-kappaB (NF-κB) signaling pathway was analyzed by western blotting. The results showed that calycosin substantially ameliorated the pathological damage to prostate tissues of the CP rats. Moreover, calycosin significantly downregulated interleukin (IL)-1ß, IL-6, and tumor necrosis factor-alpha, IgG, and SIgA levels. Furthermore, we found that calycosin considerably suppressed oxidative stress and inhibited the activation of the p38MAPK/NF-κB signaling pathway in rats with CP. In summary, our findings revealed that calycosin protects against CP in rats by inhibiting the p38MAPK/NF-κB pathway.

Calycosin inhibits gemcitabine-resistant lung cancer cells proliferation through modulation of the LDOC1/GNL3L/NFκB.

Lung cancer is the most common malignant cancer worldwide. Combination therapies are urgently needed to increase patient survival. Calycosin is a phytoestrogen isoflavone that has been reported previously to inhibit tumor cell growth, although its effects on lung cancer remain unclear. The aim of this study was to investigate the effects of calycosin on cell proliferation and apoptosis of gemcitabine-resistant lung cancer cells. Using calycosin to treat human lung cancer cells (CL1-0) and gemcitabine-resistant lung cancer cells (CL1-0 GEMR) and examine the effects on the cells. Cultured human lung cancer cells (CL1-0) and gemcitabine-resistant lung cancer cells (CL1-0 GEMR) were treated with increasing concentrations of calycosin. Cell viability and apoptosis were studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide, flow cytometry, and TUNEL assays. Western blots were used to measure the expression levels of proliferation-related proteins and cancer stem cell proteins in CL1-0 GEMR cells. The results showed that calycosin treatment inhibited cell proliferation, decreased cell migration ability, and suppressed cancer stem cell properties in CL1-0 GEMR cells. Interestingly, in CL1-0 GEMR cells, calycosin treatment not only increased LDOC1 but also decreased GNL3L/NFκB protein levels and mRNA levels, in concentration-dependent manners. We speculate that calycosin inhibited cell proliferation of the gemcitabine-resistant cell line through regulating the LDOC1/GNL3L/NFκB pathway.

Calycosin attenuates the inflammatory damage of microglia induced by oxygen and glucose deprivation through the HMGB1/TLR4/NF-κB signaling pathway.

Stroke seriously threatens human life and health worldwide, but only very few effective stroke medicines are currently available. Our previous studies have indicated that the phytoestrogen calycosin exerts neuroprotective effects in cerebral ischemia and reperfusion injury rats. Therefore, the objective of this study is to further explore the protective effect of calycosin on inflammatory injury in microglia after oxygen-glucose deprivation/reoxygenation (OGD/R) and to clarify whether its protective effect is related to the HMGB1/TLR4/NF-κB signaling pathway. Here, the OGD/R model of rodent microglia is established in vitro to simulate cerebral ischemia-reperfusion injury. Through the CCK-8 test, ELISA, qRT-PCR, and western blot analysis, we find that the activity of microglia is decreased, the expressions of HMGB1 and TLR4 and the phosphorylation of NF-κB (p-NF-κB) are increased, and the releases of the inflammatory factors IL-6, IL-1ß, and TNF-α are increased after OGD/R. Pretreatment with calycosin could ameliorate these states, increase cell viability, reduce HMGB1, TLR4 and p-NF-κB expression, and reduce inflammatory cytokine production. In addition, the effect of calycosin is similar to that of TAK-242 (an inhibitor of TLR4), and the effect of the combined treatment is better than that of the single treatment. The results indicate that calycosin protects microglia from OGD/R injury and reduces the inflammatory response. Calycosin might alleviate cerebral ischemia-reperfusion injury by inhibiting the HMGB1/TLR4/NF-κB pathway.

Identification and Functional Characterization of UDP-Glycosyltransferases Involved in Isoflavone Biosynthesis in Astragalus membranaceus.

Isoflavones are rich natural compounds present in legumes and are essential for plant growth and development. Moreover, they are beneficial for animals and humans. Isoflavones are primarily found as glycoconjugates, including calycosin-7-O-ß-d-glucoside (CG) in Astragalus membranaceus, a legume. However, the glycosylation mechanism of isoflavones in A. membranaceus remains unclear. In the present study, three uridine diphosphate (UDP)-glycosyltransferases (UGTs) that may be involved in the biosynthesis of isoflavone were identified in the transcriptome of A. membranaceus. Enzymatic analysis revealed that AmUGT88E29 and AmUGT88E30 had high catalytic activity toward isoflavones in vitro. In addition, AmUGT88E29 and AmUGT88E30 could accept various flavones, flavanones, flavonols, dihydroflavonols, and dihydrochalcones as substrates. AmUGT71G10 was only active against phloretin and dihydroresveratrol. Overexpression of AmUGT88E29 significantly increased the contents of CG, an isoflavone glucoside, in the hairy roots of A. membranaceus. This study provided candidate AmUGT genes for the potential metabolic engineering of flavonoid compounds in plants and a valuable resource for studying the calycosin glycosides biosynthesis pathway.