Load-bearing columns inspired fabrication of ductile and mechanically enhanced BSA hydrogels.

Currently, protein-based hydrogels are widely applied in soft materials, tissue engineering and implantable scaffolds owing to their excellent biocompatibility, and degradability. However, most protein-based hydrogels are soft brittle. In this study, a ductile and mechanically enhanced bovine serum albumin (BSA) hydrogel is fabricated by soaking the a 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) induced BSA hydrogel in (NH4)2SO4 solution. An EDC/NHS coupling reaction induce protein coupling reactions that cause the BSA skeleton to resemble architectural load-bearing walls, protecting the integrity of the hydrogel and preventing collapse. The effects of the BSA and (NH4)2SO4 concentrations on the hydrogel mechanics are evaluated, and the possible strengthening mechanism is discussed. Besides, the highly kosmotropic ions greatly enhance the hydrophobic interaction within BSA gels and dehydration effect and their mechanical properties were significantly enhanced. The various mechanical properties of hydrogels can be regulated over a large window by soaking hydrogels into various ions. And most of them can be washed away, maintaining high biocompatibility of the protein. Importantly, the protein hydrogels prepared by this strategy could also be modified as strain sensors. In a word, this work demonstrates a new, universal method to provide multi-functional, biocompatible, strength enhanced and regulable mechanical pure protein hydrogel, combining the Hofmeister effect with -NH2/-COOH association groups.

Integrated network pharmacological analysis revealed that Smilax glabra Roxb. alleviates IMQ-induced psoriatic skin inflammation through regulating T cell immune response.

ETHNOPHARMACOLOGICAL RELEVANCE: Psoriasis is an autoimmune disease characterized by dysfunctional T cells and dysregulated immune responses. Smilax glabra Roxb. (SGR) is a formulation used in Traditional Chinese Medicine for the treatment of inflammatory skin disorders, including psoriasis. This study explores the scientific basis for its use by examining the effects of SGR on T cell differentiation and insulin receptor signaling, relevant pathways implicated in the pathophysiology of psoriasis. AIM OF THE STUDY: This study investigates the therapeutic potential of SGR (a Chinese medicine) in psoriasis and its impact on T cell differentiation. MATERIALS AND METHODS: An integrated network pharmacology and bioinformatics approach was employed to elucidate the mechanisms of SGR in regulating T cell differentiation. A psoriasis mouse model was utilized to evaluate the effects of SGR on T cell subsets. Immunohistochemistry and gene expression analyses were conducted to investigate the modulation of insulin receptor signaling pathways by SGR. RESULTS: SGR treatment effectively reset the expression of various T cell subsets in the psoriasis mouse model, suggesting its ability to regulate T cell differentiation and immune function. Furthermore, SGR treatment inhibited insulin receptor signaling and downstream pathways, including PI3K/AKT and ERK, in psoriatic skin lesions. This indicates that SGR may exert its therapeutic effects through modulation of the insulin receptor signaling pathway. CONCLUSIONS: This study provides novel insights into the therapeutic potential of SGR in psoriasis. By modulating T cell differentiation and targeting the insulin receptor signaling pathway, SGR holds promise as a potential treatment option for psoriasis.

Fabricating the multibranch carboxyl-modified cellulose for hemorrhage control.

Excessive bleeding is associated with a high mortality risk. In this study, citric acid and ascorbic acid were sequentially modified on the surface of microcrystalline cellulose (MCAA) to increase its carboxyl content, and their potential as hemostatic materials was investigated. The MCAA exhibited a carboxylic group content of 9.52 %, higher than that of citric acid grafted microcrystalline cellulose (MCA) at 4.6 %. Carboxyl functionalization of microcrystalline cellulose surfaces not only plays a fundamental role in the structure of composite materials but also aids in the absorption of plasma and stimulation of platelets. Fourier -transform infrared (FT-IR), thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS) spectra confirmed that carboxyl groups were successfully introduced onto the cellulose surface. Physical properties tests indicated that the MCAA possessed higher thermal stability (Tmax = 472.2 °C) compared to microcrystalline cellulose (MCC). Additionally, in vitro hemocompatibility, cytotoxicity and hemostatic property results demonstrated that MCAA displayed good biocompatibility (hemolysis ratio <1 %), optimal cell compatibility (cell viability exceeded 100 % after 72 h incubation), and impressive hemostatic effect (BCIMCAA = 31.3 %). Based on these findings, the hemostatic effect of covering a wound with MCAA was assessed, revealing enhanced hemostatic properties using MCAA in tail-amputation and liver-injury hemorrhage models. Furthermore, exploration into hemostatic mechanisms revealed that MCAA can significantly accelerate coagulation through rapid platelet aggregation and activation of the clotting cascade. Notably, MCAA showed remarkable biocompatibility and induced minimal skin irritation. In conclusion, the results affirmed that MCAA is a safe and potentially effective hemostatic agent for hemorrhage control.

Effect of Imaging Selection Paradigms on Endovascular Thrombectomy Outcomes in Patients With Acute Ischemic Stroke.

BACKGROUND: The effect of imaging selection paradigms on endovascular thrombectomy outcomes in patients with acute ischemic stroke with large vessel occlusion remains uncertain. The study aimed to assess the effect of basic imaging (noncontrast computed tomography with or without computed tomographic angiography) versus advanced imaging (magnetic resonance imaging or computed tomography perfusion) on clinical outcomes following thrombectomy in patients with stroke with large vessel occlusion in the early and extended windows using a pooled analysis of patient-level data from 2 pivotal randomized clinical trials done in China. METHODS: This post hoc analysis used data from 1182 patients included in 2 multicenter, randomized controlled trials in China that evaluated adjunct therapies to endovascular treatment for acute ischemic stroke (Direct Endovascular Treatment for Large Artery Anterior Circulation Stroke performed from May 20, 2018, through May 2, 2020, and Intravenous Tirofiban Before Endovascular Treatment in Stroke from October 10, 2018, through October 31, 2021). Patients with occlusion of the intracranial internal carotid artery or proximal middle cerebral artery (M1/M2 segments) were categorized according to baseline imaging modality (basic versus advanced) as well as treatment time window (early, 0-6 hours versus extended, 6-24 hours from last known well to puncture). The primary outcome was the proportion of patients with functional independence (modified Rankin Scale score of 0-2) at 90 days. Multivariable Poisson regression analysis was performed to determine the association between imaging selection modality and outcomes after endovascular treatment at each time windows. RESULTS: A total of 1182 patients were included in this cohort analysis, with 648 in the early (471 with basic imaging versus 177 advanced imaging) and 534 in the extended (222 basic imaging versus 312 advanced imaging) time window. There were no differences in 90-day functional independence between the advanced and basic imaging groups in either time windows (early window: adjusted relative risk, 0.99 [95% CI, 0.84-1.16]; P=0.91; extended window: adjusted relative risk, 1.00 [95% CI, 0.84-1.20]; P=0.97). CONCLUSIONS: In this post hoc analysis of 2 randomized clinical trial pooled data involving patients with large vessel occlusion stroke, an association between imaging selection modality and clinical or safety outcomes for patients undergoing thrombectomy in either the early or extended windows was not detected. Our study adds to the growing body of literature on simpler imaging paradigms to assess thrombectomy eligibility across both the early and extended time windows. REGISTRATION: URL: http://www.chictr.org.cn; Unique identifiers: ChiCTR-IOR-17013568 and ChiCTR-INR-17014167.

Tetrastigma polysaccharide reprogramming of tumor-associated macrophages via PPARγ signaling pathway to play antitumor activity in breast cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Tetrastigma Hemsleyanum Diels et Gilg (SYQ) is a typical She ethnomedicine that has been used in anti-tumor treatment in Chinese folklore. The polysaccharide of SYQ (SYQ-PA) has been reported to have antioxidant and anti-inflammatory effects, but the effect and mechanism on antitumor is still unclear. AIM OF THE STUDY: To investigate the activity and mechanism of SYQ-PA against breast cancer in vitro and in vivo. MATERIALS AND METHODS: In this study, different stages of MMTV-PYMT mice, which at 4-week-old and 8-week-old representative the transition from hyperplasia to late carcinoma, were used to investigate the potential effect of SYQ-PA of breast cancer development in vivo. The mechanism was explored with IL4/13-induced peritoneal macrophages model. Flow cytometry assay was employed to analysis the change of tumor microenvironment and the macrophages typing. The inhibition of the condition medium from macrophages on breast cancer cells was detected with xCELLigence system detection. The inflammation factors were tested with cytometric bead array. Co-culture system was used to detect the cell migration and invasion. In addition, the underlying mechanism was investigated using RNAseq analysis, Q-PCR and Western blot, and the PPARγ inhibitor was used to verify the mechanism. RESULTS: SYQ-PA significantly attenuated the process of breast primary tumor growth and reduced the infiltration of TAMs accompanied promoting the polarization of M1 phenotype in MMTV-PyMT mice. Then in vitro studies showed that SYQ-PA promoted macrophages polarization form IL4/13 induced M2 toward to the anti-tumor M1 phenotypes, and the conditioned medium (CM) from the induced macrophages inhibited the proliferation of breast cancer cells. At the same time, SYQ-PA treated macrophages inhibited the migration and invasion of 4T1 in the co-culture system. Further results indicated that SYQ-PA suppressed the release of anti-inflammatory factors and promoted the production of inflammatory cytokines which may induce M1 macrophage polarization and inhibit breast cancer cell proliferation. Subsequently, the underlying mechanism analysis based on RNAseq and molecular assays indicated that SYQ-PA inhibited PPARγ expression and regulated downstream NF-κB in macrophages. After treated with PPARγ inhibitor, T0070907, the effect of SYQ-PA was decreased, or even disappeared. As the downstream, the expression of ß-catenin was also inhibited obviously, those above all contribute the process of SYQ-PA induced M1 macrophages polarization. CONCLUSIONS: Collectively, SYQ-PA was observed inhibited breast cancer, at least in part, via PPARγ activation- and ß-catenin-mediated M2 macrophages polarization. These data expound the antitumor effect and mechanism of SYQ-PA, and provide a possible that SYQ-PA can be used as an adjuvant drug for macrophage tumor immunotherapy in breast cancer.

Fabricating Oxidized Cellulose Sponge for Hemorrhage Control and Wound Healing.

Uncontrolled hemorrhage and infection are the main reasons for many trauma-related deaths in both clinic and battlefield. However, most hemostatic materials have various defects and side effects, such as low hemostatic efficiency, poor biocompatibility, weak degradation ability, and lack of antimicrobial properties. Herein, an oxidized cellulose (OC) sponge with antibacterial properties and biosafety was fabricated for hemorrhage control and wound healing. The as-prepared OC sponges were prone to water triggered expansion and superabsorbent capacity, which could facilitate blood component concentration effectively. Importantly, they had significant biodegradability with little irritation to the skin. This hemostat could also reduce the plasma clotting time to 53.54% in vitro and demonstrated less blood loss than commercially available hemostatic agents (GS) in a mouse model of bleeding from liver defects. Furthermore, the biocompatibility antimicrobial properties and possible hemostatic mechanism of the OC sponge were also systematically evaluated. Importantly, the potential wound healing applications have also been demonstrated. Therefore, the materials have broad clinical application prospects.

Panax notoginseng saponins normalises tumour blood vessels by inhibiting EphA2 gene expression to modulate the tumour microenvironment of breast cancer.

BACKGROUND: Panax notoginseng saponins (PNS), the main active component of Panax notoginseng, can promote vascular microcirculation. PNS exhibits antitumor effects in various cancers. However, the molecular basis of the relationship between PNS and tumor blood vessels remains unclear. PURPOSE: To study the relationship between PNS inhibiting the growth and metastasis of breast cancer and promoting the normalization of blood vessels. METHODS: We performed laser speckle imaging of tumor microvessels and observed the effects of PNS on tumor growth and metastasis of MMTV-PyMT (FVB) spontaneous breast cancer in a transgenic mouse model. Immunohistochemical staining of Ki67 and CD31 was performed for tumors, scanning electron microscopy was used to observe tumor vascular morphology, and flow cytometry was used to detect tumor tissue immune microenvironment (TME). RNA-seq analysis was performed using the main vessels of the tumor tissues of the mice. HUVECs were cultured in tumor supernatant in vitro to simulate tumor microenvironment and verify the sequencing differential key genes. RESULTS: After treatment with PNS, we observed that tumor growth was suppressed, the blood perfusion of the systemic tumor microvessels in the mice increased, and the number of lung metastases decreased. Moreover, the vascular density of the primary tumor increased, and the vascular epidermis was smoother and flatter. Moreover, the number of tumor-associated macrophages in the tumor microenvironment was reduced, and the expression levels of IL-6, IL-10, and TNF-α were reduced in the tumor tissues. PNS downregulated the expression of multiple genes associated with tumor angiogenesis, migration, and adhesion. In vitro tubule formation experiments revealed that PNS promoted the formation and connection of tumor blood vessels and normalized the vessel morphology primarily by inhibiting EphA2 expression. In addition, PNS inhibited the expression of tumor vascular marker proteins and vascular migration adhesion-related proteins in vivo. CONCLUSION: In this study, we found that PNS promoted the generation and connection of tumor vascular endothelial cells, revealing the key role of EphA2 in endothelial cell adhesion and tumor blood vessel morphology. PNS can inhibit the proliferation and metastasis of breast cancer by inhibiting EphA2, improving the immune microenvironment of breast cancer and promoting the normalization of tumor blood vessels.

Flavonoid Group of Smilax glabra Roxb. Regulates the Anti-Tumor Immune Response Through the STAT3/HIF-1 Signaling Pathway.

Background: Smilax glabra Roxb. (SGR) is a widely used traditional Chinese medicine, which has known effects of enhancing immunity. However, its anti-tumor effects and mechanism of action are still unclear. Methods: We selected MMTV-PyMT mice to determine the anti-tumor efficacy of SGR ethyl acetate (SGR-EA). First, flow cytometry was used to detect the number of immune cells in the mice tumor microenvironment. Furthermore, M2 polarization of macrophages was stimulated in vitro, and the expressions of macrophage M1/M2 surface markers and mRNA were as determined. Finally, we carried out a network pharmacology analysis on the active components of SGR-EA and in vitro experiments to verify that SGR-EA regulated the hypoxia-inducible factor (HIF)-1 signaling pathway to modulate the anti-tumor immune response by resetting M2 macrophages toward the M1 phenotype which inhibited tumor growth and lung metastasis in the mice. Result: SGR-EA inhibited tumor growth and lung metastasis in the mice. Tumor-associated macrophages switched from M2 to the tumor-killing M1 phenotype and promoted the recruitment of CD4+ and CD8+ T cells in the tumor microenvironment. In vitro, SGR-EA significantly inhibited the polarization of macrophages into M2 macrophages and increased the number of M1 macrophages. In addition, following an intervention with SGR-EA, the expression of the HIF-1 signaling pathway-related proteins stimulated by interleukin-4 in macrophages was significantly inhibited. Conclusion: SGR-EA played an anti-tumor role by inhibiting the activation of the HIF-1 signaling pathway and response by resetting tumor-associated macrophages toward the M1 phenotype.

Total glucosides of paeony inhibit breast cancer growth by inhibiting TAMs infiltration through NF-κB/CCL2 signaling.

PURPOSE: The high density of tumor-associated macrophages (TAMs) and inflammatory factors are crucial elements leading to tumor immune tolerance. Previously, we found that total glucosides of paeony (TGP) have strong inhibitory effects on the release of various inflammatory factors; however, it is unclear whether the inhibitory effects can improve the inflammatory microenvironment of tumors. Therefore, in the present study, we investigated the mechanism via which TGP depresses tumor growth and metastasis via modulation of TAM infiltration in breast cancer. METHODS: We assessed the effects of TGP on various mouse models of tumor. Lung metastasis was detected using hematoxylin and eosin staining. T cell (CD3+CD4+ and CD3+CD8+) effector and memory subsets, and TAM (CD45+CD11b+F4/80+) populations in the tumor microenvironment (TME) were examined using flow cytometry. Lipopolysaccharide (LPS)-stimulated macrophage experiments were used to investigate the TGP anti-inflammatory effects in vitro. Furthermore, conditional medium (CM) was added to detect 4T1 breast cancer cell growth using a Real-Time Cell Analyzer (RTCA) xCELLigence system. Inflammatory cytokine and chemokine levels were measured using cytometric bead array (CBA) kits and quantitative polymerase chain reaction (qPCR). NF-κB expression in the nucleus was examined by immunofluorescence and Western blot analysis. RESULTS: TGP suppressed tumor growth and lung metastasis, decreased CD45+CD11b+F4/80+ (TAMs) population obviously, and increased CD44LowCD62LHi (T memory stem cells) and CD44HiCD62LHi (central memory cells) populations in the tumor-infiltrating CD4+ and CD8+ T cells. In addition, TGP reduced inflammatory factor levels in tumors, thus inhibiting the infiltration of TAMs to improve the inflammation immunosuppressive microenvironment. In the in vitro experiment, TGP inhibited IL-10 and C-C Motif Chemokine Ligand 2 (CCL2) secretion and mRNA expression in LPS-stimulated macrophages to inhibit 4T1 cell growth and restrain macrophages M2 polarization. In addition, TGP can directly inhibit 4T1 cell proliferation by restraining autocrine CCL2 and IL-10. Further mechanistic studies reavealed that TGP inhibited CCL2 secretion by inhibiting NF-κB accumulation in the nucleus in macrophages. CONCLUSION: TGP reduced TAM recruitment mainly through the NF-κB/CCL2 signaling pathway, thereby promoting T cell infiltration in the TME. TGP has a unique advantage in balancing the inflammatory response. Furthermore, our results present novel insights on the mechanisms underlying TAM infiltration that were inhibited by TGP, with potential application in development of novel therapies targeting CCL2 pathways.

Blood Stasis Syndrome Accelerates the Growth and Metastasis of Breast Cancer by Promoting Hypoxia and Immunosuppressive Microenvironment in Mice.

Blood stasis syndromes (BSSs) are closely related to the occurrence and development of tumors, although the mechanism is still unclear. This study was aimed at exploring the effect and mechanism underlying different BSSs on tumor growth and metastasis. We established four BSS mouse models bred with breast cancer: qi deficiency and blood stasis (QDBS), cold coagulation blood stasis (CCBS), heat toxin and blood stasis (HTBS), and qi stagnation and blood stasis (QSBS). The results showed that microcirculation in the lower limb, abdominal wall, and tumor in situ decreased by varying degrees in the BSS groups. In addition, BSS promoted tumor growth and lung metastasis. The ratio of regulatory T cells in the tumor microenvironment was downregulated. Moreover, hypoxia-inducible factor 1-α, Wnt1, ß-catenin, vascular endothelial growth factor, and Cyclin D1 levels increased in the tumors of BSS mice. In conclusion, BSS not only promoted the formation of a hypoxic and immunosuppressive microenvironment but also promoted the neovascularization.

Safflower yellow reduces DEN-induced hepatocellular carcinoma by enhancing liver immune infiltration through promotion of collagen degradation and modulation of gut microbiota.

Safflower yellow (SY) is the main active ingredient isolated from the traditional Chinese medicine Carthamus tinctorius, which is a valuable natural edible pigment that is widely used to treat cerebrovascular and cardiovascular diseases. However, the effect of SY on hepatocellular carcinoma (HCC) remains unclear. In this study, we showed that SY decreased the degree of injury and inhibited the release of inflammatory factors in the liver of a diethylnitrosamine (DEN)-induced HCC mouse model. Flow cytometry and immunoblotting showed that SY increased the infiltration of CD8+ T cells and Gr-1+ macrophages to improve the immune microenvironment by affecting the expression of collagen fibers. Further cellular experiments showed that SY degraded the collagens in the liver cells through the TGF-ß/Smad signalling pathway. SY also regulated the gut microbiota which may contribute to the immune microenvironment. In conclusion, SY exhibited a potent effect on the development of HCC by enhancing liver immune infiltration by promoting collagen degradation and modulating the gut microbiota. This study provides novel insights into the mechanism of SY as a candidate for the treatment of HCC in the future.

Network Pharmacology Analysis of the Identification of Phytochemicals and Therapeutic Mechanisms of Paeoniae Radix Alba for the Treatment of Asthma.

BACKGROUND: Paeoniae Radix Alba (PRA), the root of the plant Paeonia lactiflora Pall., has been suggested to play an important role for the treatment of asthma. A biochemical understanding of the clinical effects of Paeoniae Radix Alba is needed. Here, we explore the phytochemicals and therapeutic mechanisms via a systematic and comprehensive network pharmacology analysis. METHODS: Through TCMSP, PubChem, GeneCards database, and SwissTargetPrediction online tools, potential targets of active ingredients from PRA for the treatment of asthma were obtained. Cytoscape 3.7.2 was used to determine the target of active ingredients of PRA. Target protein interaction (PPI) network was constructed through the STRING database. The Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genes (KEGG) pathway enrichment analysis were analyzed through the biological information annotation database (DAVID). RESULTS: Our results indicate that PRA contains 21 candidate active ingredients with the potential to treat asthma. The enrichment analysis of GO and KEGG pathways found that the treatment of asthma by PRA may be related to the process of TNF (tumor necrosis factor) release, which can regulate and inhibit multiple signaling pathways such as ceramide signaling. CONCLUSIONS: Our work provides a phytochemical basis and therapeutic mechanisms of PRA for the treatment of asthma, which provides new insights on further research on PRA.

Hirsutella sinensis fungus improves cardiac function in mouse model of heart failure.

Cordyceps sinensis, including Hirsutella sinensis, is a highly valuable traditional Chinese medicine and is used to treat patients with pulmonary heart disease in clinical practice. However, the underlying mechanisms of its effects remain unclear. In this study, a mouse model of heart failure established by non-thoracic, transverse aortic constriction (TAC) was developed to determine the underlying mechanisms of therapeutic effects of Hirsutella sinensis fungus (HSF) powder. The results showed that HSF treatment remarkably ameliorated myocardial hypertrophy, collagen fiber hyperplasia, and cardiac function in mice with heart failure. Using transcriptional and epigenetic analyses, we found that the mechanism of HSF mainly involved a variety of signaling pathways related to myocardial fibrosis and determined that HSF could reduce the levels of TGF-ß1 proteins in heart tissue, as well as type I and III collagen levels. These data suggest that HSF alleviates heart failure, inhibits irreversible ventricular remodeling, and improves cardiac function through the regulation of myocardial fibrosis-related signaling pathways, which can provide novel opportunities to improve heart failure therapy.

Enhanced effects of salidroside on erectile function and corpora cavernosa autophagy in a cavernous nerve injury rat model.

We explored the efficacy and mechanisms of salidroside treatment for erectile dysfunction induced by bilateral cavernous nerve injury (BCNI). Forty male rats were divided into four groups as follows: sham (cavernous nerves exposed only) (S); BCNI (M); BCNI + rapamycin (M + rapamycin); and BCNI + salidroside (M + salidroside). Erectile function in the rats was measured by intracavernosal pressure. Penile tissue was harvested for transmission electron microscopy, immunohistochemistry, immunofluorescence, Masson's trichrome staining, haematoxylin-eosin staining, TdT-mediated dUTP Nick End Labeling and western blotting. The M group exhibited a decrease in erectile responses and increased apoptosis and fibrosis compared to these in the S group. Meanwhile, nerve content and the penile atrophy index were also decreased in the M group. Treatment with salidroside and rapamycin for 3 weeks partially restored erectile function and significantly attenuated corporal apoptosis, fibrosis, nerve content and penile atrophy in the M group. Moreover, the autophagy level was further enhanced in the M + salidroside group, which was the same as that in the positive observation group (M + rapamycin). Salidroside treatment not only improved erectile function in rats with BCNI, but also inhibited apoptosis and fibrosis and ameliorated the loss of nerve content and endothelial and corpus cavernosum smooth muscle cells by promoting protective autophagy.

Change in the active component of processed Tetradium ruticarpum extracts leads to improvement in efficacy and toxicity attenuation.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried and nearly ripe fruits of Tetradium ruticarpum (A. Juss.) T.G. Hartley (TR) have long been used in treating headache and gastrointestinal disorders in oriental medicine. TR is usually processed by stir-frying with licorice extract before use. Although processing procedure is considered as the way to relieve pungent smell, reduce toxicity, and improve efficacy, its effects on TR's toxicity and efficacy and bioactive compound profiles are largely unknown. AIM OF THE STUDY: The purposes of the study are to evaluate the acute toxicity, efficacy and variation of toxic and effective components of TR before and after processing, and to explore the possible mechanism of how the processing procedure affect the quality of TR as a herbal medicine. MATERIALS AND METHODS: Volatile oil, aqueous extract and ethanol extract of raw and processed TR were tested for their acute toxicity, analgesic, and anti-inflammatory effects in mouse models, respectively. To identify potential toxic and effective components, the extracts were analyzed with gas chromatography-mass spectrometry and ultra-performance liquid chromatography - quadrupole time-of-flight mass spectrometry, followed by fold-change-filtering analysis. RESULTS: LD50 and LD5 tests indicated that although the aqueous extract has higher toxicity than volatile oil and ethanol extract, the use of TR is safe under the recommended does. The processing procedure could effectively decrease the toxicity of all three extracts with the largest decrease in volatile oil, which is likely due to the loss of volatile compounds during processing. Analgesic and anti-inflammatory studies suggested that volatile oil and ethanol extract of TR have better efficacy than the aqueous extract and the processing procedure significantly enhanced the efficacy of these two former extracts, whereas processing showed no substantially effects on the bioactivities of aqueous extract. Integrated analysis of animal trial and chromatographic analyses indicated that indole and quinolone type alkaloids, limonoids, amides and 18ß-glycyrrhetinic acid were identified as the potential main contributors of TR's efficacy, whereas hydroxy or acetoxy limonoid derivates and coumarins could be the major causes of toxicity. Moreover, the reduced toxicity and improved efficacy of the processed TR are liked due to the licorice ingredients and altered alkaloids with better solubility. CONCLUSIONS: In summary, the integrated toxicity and efficacy analyses of volatile, aqueous and ethanol extracts of TR indicated that the processing procedure could effectively reduce its acute toxicity in all three extracts and enhance its analgesic and anti-inflammatory effects in volatile and ethanol extracts. The promising candidate compounds related to the toxicity and efficacy of TR were also identified. The results could expand our understanding of the value of the standard processing procedure of TR, be valuable to the quality control of TR manufacturing and administration, as well as support clinical rational and safety applications of this medicinal plant.

High-Fat Diet Promotes Macrophage-Mediated Hepatic Inflammation and Aggravates Diethylnitrosamine-Induced Hepatocarcinogenesis in Mice.

It has been reported that diet and nutrition play important roles in the occurrence and development of hepatocellular carcinoma (HCC). In this study, we investigated the potential tumor-promoting mechanisms of a high-fat diet (HFD) in mice with dietondiethylnitrosamine (DEN)-induced hepatocarcinogenesis. HFD significantly decreased the survival rate and induced severe liver dysfunction in DEN-induced mice, as indicated by increased serum glutamic-pyruvic transaminase (ALT), glutamic oxalacetic transaminase (AST), and alkaline phosphatase (ALP) levels and increased liver index, liver nodule count, and γ-glutamyltransferase (γ-GT) activity. Moreover, an increased number of fat droplets and HCCs were found in the livers of the HFD mice, who displayed little collagen in and around the liver cancer groove and the infiltration of large number of inflammatory cells, such as macrophages, compared with the control mice. HFD also significantly increased proliferating cell nuclear antigen (PCNA), nuclear factor-κB (NF-κB), cyclin D1, tumor necrosis factor (TNF), and interleukin-1 (IL-1) expression levels in the liver. In vitro, we found that the inducible nitric oxide synthase (iNOS) percentage increased in macrophages after palmitic acid treatment, as well as the secretion of inflammatory factors and cytokines such as interleukin-6(IL-6), interleukin-10(IL-10), CCL2, Interferon γ (IFN-γ), and TNF. Thus, our results demonstrate that an HFD may promote DEN-induced hepatocarcinogenesis in mice by destroying liver function and enhancing the inflammatory response by recruiting and polarizing macrophages in the liver. This study could therefore provide new insights into the tumor promoting effects of an HFD in HCC.

Crude Radix Aconiti Lateralis Preparata (Fuzi) with Glycyrrhiza Reduces Inflammation and Ventricular Remodeling in Mice through the TLR4/NF-κB Pathway.

Radix Aconiti Lateralis Preparata (Fuzi) is a traditional Chinese medicine. Its alkaloids are both cardiotonic and cardiotoxic; however, the underlying mechanisms are unclear. Compatibility testing and processing are the primary approaches used to reduce the toxicity of aconite preparations. The purpose of this study was to compare the effects of crude Fuzi (CFZ), CFZ combined with Glycyrrhiza (Gancao) (CFZ+GC), and prepared materials of CFZ (PFZ) on heart failure (HF) in C57BL/6J mice and explore the potential mechanisms of action of CFZ. Transverse aortic constriction (TAC) was used to generate the HF state, and CFZ (1.5 g·mL-1), PFZ (1.5 g·mL-1), or CFZ+GC (1.8 g·mL-1) was orally administered to the HF-induced mice daily. For the subsequent 8 weeks, hemodynamic indicators, ventricular pressure indices, and mass indices were evaluated, and histopathological imaging was performed. CFZ, CFZ+GC, and PFZ significantly improved left ventricular function and structure and reduced myocardial damage. CFZ+GC was more effective than CFZ and PFZ, whereas CFZ had higher toxicity than CFZ+GC and PFZ. CFZ and CFZ+GC attenuated ischemia-induced inflammatory responses and also inhibited Toll-like receptor-4 (TLR4) and nuclear factor kappa beta (NF-κB) action in the heart. Moreover, mass spectrometry analysis revealed a decrease in the levels of toxic components of CFZ+GC, whereas those of the protective components were increased. This study suggested that GC reduces the toxicity and increases the efficacy of CFZ on HF induced by TAC. Furthermore, GC+CFZ reduces the risk of HF by ameliorating the inflammation response, which might be partially related to the inhibition of the TLR4/NF-κB pathway.

Gehua Jiecheng Decoction Inhibits Diethylnitrosamine-Induced Hepatocellular Carcinoma in Mice by Improving Tumor Immunosuppression Microenvironment.

Gehua Jiecheng Decoction (GHJCD), a famous traditional Chinese medicine, has been used in the prevention and treatment of precancerous lesion of liver cancer, but its active mechanism has not been reported. This study aimed to evaluate the therapeutic effect of GHJCD on diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in mice and the mechanism of this effect. We found that GHJCD effectively inhibited the occurrence of liver cancer and reduced the tumor area. The ratio of regulatory cells (Tregs), tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs) in HCC microenvironment was down-regulated, whereas that of CD8 T and effective CD8 T cells was up-regulated. In addition, the expression levels of inflammatory factors IL-6, IL-10, TNF-α, and CCL-2 in the liver were inhibited, whereas those of the angiogenesis related molecules CD31 and VEGF were decreased. Moreover, WNT1, ß-catenin, NF-kB, p-MAPK, p-AKT, and p-SRC content in the liver decreased, whereas APC content increased. These results suggested that GHJCD exerted a good inhibitory effect on liver cancer induced by DEN and thus may have a multi-target effect; GHJCD not only antagonized the immunosuppressive effect of the microenvironment of liver cancer but also exerted strong anti-inflammatory and antiangiogenesis effects.

Effect of Hirsutella sinensis Fungus on the Hypothalamic-Pituitary-Adrenal Axis in Lewis Rats with Kidney-Yang Deficiency Syndrome.

Kidney-yang deficiency syndrome (KYDS) is a classic syndrome in traditional Chinese medicine, which is mainly caused by damage to the hypothalamic-pituitary-adrenal (HPA) axis. Hirsutella sinensis fungus (HSF), an artificial substitute of Cordyceps sinensis, has been widely used in TCM. However, the effects and the possible mechanism of HSF on the HPA axis and corresponding KYDS have not yet been investigated. In this study, Lewis rats were used as a spontaneous KYDS model. HSF was intragastrically administered to the Lewis rats at two doses: low dose (1 g/kg) and high dose (2 g/kg). Body weight, temperature, and behavioral tests including grip strength, open field, and Morris water maze (MWM) tests were used to evaluate the KYDS symptoms. Enzyme-linked immunosorbent assay was used to detect the level of circulating adrenocortisol (ACTH), corticosterone (CORT), corticotropin releasing hormone (CRH), cyclic adenosine monophosphate (cAMP), and cyclic guanosine monophosphate (cGMP). In addition, mRNA expression of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin 10 (IL-10), CRH, glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) was detected by quantitative real-time polymerase chain reaction (Q-PCR). The Lewis rats were indicated to have KYDS symptoms and HSF treatment ameliorated these symptoms via enhancement of the HPA axis function, which was evidenced by the increased levels of CRH, ACTH, and CORT in serum and 17-OHCS in urine. HSF also significantly improved the expression of TNF-α, IFN-γ, and IL-2, secreted by Th1 cells, which might accelerate the activation of the immune system related to the HPA axis function. Thus, we conclude that HSF can alleviate KYDS symptoms in Lewis rats by regulating the HPA axis through accelerated immune system activation.

PM2.5 Exposure Induces Inflammatory Response in Macrophages via the TLR4/COX-2/NF-κB Pathway.

Fine particulate matter with an aerodynamic diameter less than 2.5 µm (PM2.5) is a serious air pollutant associated with health problems. Macrophages play an important role in the process of PM2.5-induced inflammation in respiratory diseases. However, the detailed mechanism remains unclear. We aimed to examine the mechanism of PM2.5-induced inflammation and find possible anti-inflammatory inhibitors. PM2.5 was collected in Hangzhou, China, and the composition of adsorbed materials on PM2.5 was characterized. RAW 254.7 cells were then treated with PM2.5. Phagocytosis was observed, and inflammatory response was triggered as demonstrated by the release of high levels of monocyte chemoattractant protein-1(MCP-1), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) and increased mRNA expression of inducible nitric oxide synthase (iNOS) and TNF-α. Treatment with classic inhibitors suppressed the released pro-inflammatory factors in a dose-dependent manner. Using Immunology Inflammation Compound Library, we screened 70 inhibitors and clustered them based on similarities in their inhibitory effects, which we detected using cytometric bead array (CBA) assay. Molecular analysis revealed that the expression of toll-like receptor 4 (TLR4), nuclear factor kappa-B (NF-κB), and cyclooxygenase-2 (COX-2) was increased in PM2.5-stimulated RAW 254.7 cells. Corresponding inhibitors were selected, and the CBA assay verified their anti-inflammatory effects. These inhibitors reduced the expression of pro-inflammatory factors, and this reduction was correlated with the downregulation of the TLR4/NF-κB/COX-2 signaling pathway. In conclusion, PM2.5 induces an inflammatory response in macrophages via activation of TLR4/NF-κB/COX-2 signaling, and the inhibitors of this pathway are potential therapeutic candidates to treat inflammatory disorders.