[Banxia Xiexin Decoction inhibiting colitis-associated colorectal cancer infected with Fusobacterium nucleatum by regulating Wnt/ß-catenin pathway].

This paper investigates the intervention effect and mechanism of Banxia Xiexin Decoction(BXD) on colitis-associated colorectal cancer(CAC) infected with Fusobacterium nucleatum(Fn). C57BL/6 mice were randomly divided into a control group, Fn group, CAC group [azoxymethane(AOM)/dextran sulfate sodium salt(DSS)](AOM/DSS), model group, and BXD group. Except for the control and AOM/DSS groups, the mice in the other groups were orally administered with Fn suspension twice a week. The AOM/DSS group, model group, and BXD group were also injected with a single dose of 10 mg·kg~(-1) AOM combined with three cycles of 2.5% DSS taken intragastrically. The BXD group received oral administration of BXD starting from the second cycle until the end of the experiment. The general condition and weight changes of the mice were monitored during the experiment, and the disease activity index(DAI) was calculated. At the end of the experiment, the colon length and weight of the mice in each group were compared. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in the colon tissue. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of interleukin(IL)-2, IL-4, and IL-6 inflammatory factors in the serum. Immunohistochemistry(IHC) was used to detect the expression of Ki67, E-cadherin, and ß-catenin in the colon tissue. Western blot was used to detect the protein content of Wnt3a, ß-catenin, E-cadherin, annexin A1, cyclin D1, and glycogen synthase kinase-3ß(GSK-3ß) in the colon tissue. The results showed that compared with the control group, the Fn group had no significant lesions. The mice in the AOM/DSS group and model group had decreased body weight, increased DAI scores, significantly increased colon weight, and significantly shortened colon length, with more significant lesions in the model group. At the same time, the colon histology of the model group showed more severe adenomas, inflammatory infiltration, and cellular dysplasia. The levels of IL-4 and IL-6 in the serum were significantly increased, while the IL-2 content was significantly decreased. The IHC results showed low expression of E-cadherin and high expression of Ki67 and ß-catenin in the model group, with a decreased protein content of E-cadherin and GSK-3ß and an increased protein content of Wnt3a, ß-catenin, annexin A1, and cyclin D1. After intervention with BXD, the body weight of the mice increased; the DAI score decreased; the colon length increased, and the tumor decreased. The histopathology showed reduced tumor proliferation and reduced inflammatory infiltration. The levels of IL-6 and IL-4 in the serum were significantly decreased, while the IL-2 content was increased. Meanwhile, the expression of E-cadherin was upregulated, and that of Ki67 and ß-catenin was downregulated. The protein content of E-cadherin and GSK-3ß increased, while that of Wnt3a, ß-catenin, annexin A1, and cyclin D1 decreased. In conclusion, BXD can inhibit CAC infected with Fn, and its potential mechanism may be related to the inhibition of Fn binding to E-cadherin, the decrease in annexin A1 protein level, and the regulation of the Wnt/ß-catenin pathway.

Effect of bovine colostrum liposomes on the bioavailability of immunoglobulin G and their immunoregulatory function in immunosuppressed BALB/c mice.

Bovine colostrum (BC) has high nutritional value; however, the low bioavailability of immune active substances in BC may affect their immunoregulatory function. Our previous studies indicated that encapsulating bovine colostrum with liposomes could enable the sustained release of immunoglobulin G in vitro; however, the effect of bovine colostrum liposomes (BCLs) on the bioavailability of immunoglobulins in vivo is still unknown. In addition, the immunoregulatory function of BCLs on immunosuppressed mice is still unclear. Therefore, our current study aimed to explore the effect of BCLs on the bioavailability of immunoglobulins, and further explore their immunoregulatory effect on immunosuppressed BALB/c mice. Through metabolic cage experiments, it was shown that BCLs decreased the urine and fecal concentrations of IgG and exhibited a higher bioavailability of IgG in mice than BC (about 2-fold). In addition, by establishing an immunosuppressed animal model, it was found that BCLs could increase the body weight, spleen weight, and thymus weight in immunosuppressed BALB/c mice, which further restored the serum levels of interleukin-4 (IL-4), interleukin-10 (IL-10), tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ). Through histology analysis, it was suggested that BCLs restored the structure of jejunal epithelial cells, which was accompanied by an improvement in intestinal cytokine levels (IL-4, IL-10, TNF-α, and IFN-γ). Finally, BCLs increased serum and intestine concentrations of immunoglobulin G (IgG) and immunoglobulin A (IgA) in immunosuppressed BALB/c mice, which further indicated that BCLs had a sustained-release effect for immunoglobulin G in vivo. Our current research will provide a basis for understanding the role of BCLs on the bioavailability of IgG and their immunoregulatory effect on immunosuppressed mice, which might further provide some reference for the application of BCLs.

Dietary restriction-induced alterations on estrogen receptor alpha expression in regulating fertility in male Coturnix coturnix japonica: Relevance of Withania somnifera in modulation of inflammation and oxidative stress in testis.

PROBLEM: Reproductive performance of animals gets affected by nutritional restrictions which act as potential stressors leading to hormonal imbalance and testicular inflammation, the major causes of infertility. Withania somnifera (WS), well-known traditional medicinal plant, has been used as antistress and infertility treatment. Therefore, the present study looks into the ameliorative effects of WS on the reproductive and immune system of male Coturnix coturnix japonica in stressed conditions like water and food restriction focussing on the modulation in estrogen receptor alpha (ERα). METHOD OF STUDY: Biochemical estimations for oxidative stress, histological alterations, immuno-fluorescent localization of ERα, interleukin (IL)-1ß, IL-4, and interferon gamma (IFN-γ) in testicular cells were performed. RESULTS: Nutritional restriction declines endogenous estradiol, ERα in testicular cells while it elevates corticosterone leading to oxidative stress in testis thereby reducing fertility by decrease in sperm. Results indicate significant reversal in all the parameters after the administration of WS by improving testicular cell morphology, increased superoxide and catalase activity thus reducing oxidative stress. WS increases spermatogenesis and enhances expression of ERα in testicular cells in quail. Further, WS increases IL-4, decreases IL-1ß and IFN-γ expression in testis, thereby improving immune profile contrary to stressed conditions. CONCLUSION: WS stimulates HPG-axis even after stress resulting in increased endogenous estradiol which stimulates the expression of ERα in testis; increases sperm count and immunity thereby improving the reproductive performance. WS may be the best therapy against nutritional-restriction stress induced reproductive toxicity by reducing oxidative stress mediated inflammatory response via increased testicular expression of ERα in quail.

[Xinfeng Capsule alleviates interleukin-1ß-induced chondrocyte inflammation and extracellular matrix degradation by regulating the miR-502-5p/TRAF2/NF-κB axis].

OBJECTIVE: To investigate the mechanism that mediates the inhibitory effect of Xinfeng Capsule (XFC) on interleukin (IL)-1ß-induced impairment of chondrocytes. METHODS: XFC-medicated serum was collected from SD rats with XFC gavage, and its optimal concentration for chondrocyte treatment was determined using Cell Counting Kit-8 assay and flow cytometry. Dual luciferase reporter analysis was performed to analyze the targeting relationship between miR-502-5p and TRAF2. In cultured human chondrocytes induced with IL-1ß, the effects of transfection with miR-502-5p inhibitor and XFC-medicated serum, alone or in combination, on expression levels of IL-1ß, tumor necrosis factor-α (TNF-α), IL-4, and IL-10 were examined with ELISA, and the changes in the expressions of collagen type Ⅱ alpha 1 (COL2A1), matrix metalloproteinase 13 (MMP13), adisintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and miR-502-5p/TRAF2/NF-κB axis gene expression were detected using RT-qPCR, Western blotting, and immunofluorescence assay. RESULTS: In cultured human chondrocytes, treatment with IL-1ß significantly decreased the cell viability, increased cell apoptosis rate, lowered miR-502-5p, IL-4, IL-10, and COL2A1 expressions, and enhanced IL-1ß, TNF-α, ADAMTS5, MMP13, TRAF2, and NF-κB p65 expressions (P < 0.05), and these changes were significantly improved by treatment with XFC-medicated serum at the optimal concentration of 20% (P < 0.05). Transfection of the chondrocytes with miR-502-5p inhibitor resulted in elevated expressions of IL-1ß, TNF-α, ADAMTS5, MMP13, TRAF2, and NF-κB p65 and lowered expressions of miR-502-5p, IL-4, IL-10, and COL2A1, and XFC-medicated serum obviously reversed the effects of miR-502-5p inhibitor. CONCLUSION: XFC can inhibit IL-1ß-induced inflammatory response and ECM degradation in cultured human chondrocytes possibly by regulating the miR-502-5p/TRAF2/NF-κB axis.

Ozonated Sunflower Oil (OSO) Alleviates Inflammatory Responses in Oxazolone-Induced Atopic Dermatitis (AD)-Like Mice and LPS-Treated RAW 264.7 Cells.

Ozone, a highly reactive oxidant molecule, is widely used as a complementary therapy for various skin diseases, including wound healing, pressure ulcers, diabetic foot, and infections. However, there is limited research on the effectiveness of ozone for atopic dermatitis (AD). Ozonated sunflower oil (OSO) is an active ingredient obtained from partially ozonated sunflower oil (SO). OSO markedly reduced the LPS-induced increase in IL-1ß and nitric oxide (NO) levels in RAW 264.7 mouse macrophage cells. Oxazolone (OXZ) was applied to hairless mice to induce AD-like skin symptoms and immune response. OSO significantly alleviated the OXZ-induced increases in the number of infiltrating mast cells, epidermal thickness, AD symptoms, thymic stromal lymphopoietin (TSLP), and filaggrin, as well as the serum levels of NO, IgE, IL-1ß, and TNF-α. Furthermore, OSO inhibited the IL-4/STAT3/MAPK pathway and the expression of NF-κB. Our results suggest that OSO treatment could relieve AD-mediated skin damage through its anti-inflammatory and antioxidant activities. Therefore, it can be used as a therapeutic agent against AD-related skin diseases.

Multi-omics analysis of lung tissue metabolome and proteome reveals the therapeutic effect of Shegan Mahuang Decoction against asthma in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Shegan Mahuang Decoction (SMD) is a classic traditional Chinese medicine (TCM) formula for asthma treatment, but the anti-asthma mechanism of SMD is still not fully studied. AIMS OF THE STUDY: In this study, we established an ovalbumin (OVA)-induced asthma rat model and treated it with SMD to observe its anti-asthma effect and explore the related mechanism. MATERIALS AND METHODS: We evaluated the anti-inflammatory effect of SMD via testing the levels of immunoglobulin E (IgE), C-reactive protein (CRP), interleukin-4 (IL-4), interleukin-6 (IL-6) in serum and performing the hematoxylin-eosin (H&E) staining of lung tissue slices. We analyzed the variations of metabolites and proteins in the lung tissue of different groups using liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics and TMT-based proteomics approaches. The metabolic biomarkers and differentially expressed proteins (DEPs) were picked, and the related signal transduction pathways were also investigated. In addition, the key proteins on the signaling pathway were validated through western blotting (WB) experiment to reveal the anti-asthma mechanism of SMD. RESULTS: The results showed that the SMD could significantly reduce the serum levels of IgE, CRP, IL-4, and IL-6 and attenuate the OVA-induced pathological changes in lung tissue. A total of 34 metabolic biomarkers and 84 DEPs were screened from rat lung tissue, which were mainly associated with lipid metabolism, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, the excessive production of reactive oxygen species (ROS), and lysosome pathway. Besides, SMD could inhibit the myeloid differentiation factor 88 (MyD88)/inhibitor of kappa B kinase (IKK)/nuclear factor-kappa B (NF-κB) signaling pathway to exhibit anti-inflammatory activities. CONCLUSIONS: SMD exhibited a therapeutic effect on asthma, which possibly be exerted by inhibiting the MyD88/IKK/NF-κB signaling pathway.

Effects of Aeriscardovia aeriphila on growth performance, antioxidant functions, immune responses, and gut microbiota in broiler chickens. / Aeriscardovia aeriphilaå¯¹è‚‰é¸¡ç”Ÿé•¿æ€§èƒ½ã€æŠ—æ°§åŒ–åŠŸèƒ½ã€å ç–«åº”ç­”å’Œè‚ é“èŒç¾¤çš„å½±å“.

Aeriscardovia aeriphila, also known as Bifidobacterium aerophilum, was first isolated from the caecal contents of pigs and the faeces of cotton-top tamarin. Bifidobacterium species play important roles in preventing intestinal infections, decreasing cholesterol levels, and stimulating the immune system. In this study, we isolated a strain of bacteria from the duodenal contents of broiler chickens, which was identified as A. aeriphila, and then evaluated the effects of A. aeriphila on growth performance, antioxidant functions, immune functions, and gut microbiota in commercial broiler chickens. Chickens were orally gavaged with A. aeriphila (1×109 CFU/mL) for 21 d. The results showed that A. aeriphila treatment significantly increased the average daily gain and reduced the feed conversion ratio (P<0.001). The levels of serum growth hormone (GH) and insulin-like growth factor 1 (IGF-1) were significantly increased following A. aeriphila treatment (P<0.05). Blood urea nitrogen and aspartate aminotransferase levels were decreased, whereas glucose and creatinine levels increased as a result of A. aeriphila treatment. Furthermore, the levels of serum antioxidant enzymes, including catalase (P<0.01), superoxide dismutase (P<0.001), and glutathione peroxidase (P<0.05), and total antioxidant capacity (P<0.05) were enhanced following A. aeriphila treatment. A. aeriphila treatment significantly increased the levels of serum immunoglobulin A (IgA) (P<0.05), IgG (P<0.01), IgM (P<0.05), interleukin-1 (IL-1) (P<0.05), IL-4 (P<0.05), and IL-10 (P<0.05). The broiler chickens in the A. aeriphila group had higher secretory IgA (SIgA) levels in the duodenum (P<0.01), jejunum (P<0.001), and cecum (P<0.001) than those in the control group. The messenger RNA (mRNA) relative expression levels of IL-10 (P<0.05) and IL-4 (P<0.001) in the intestinal mucosa of chickens were increased, while nuclear factor-|κB (NF|-|κB) (P<0.001) expression was decreased in the A. aeriphila group compared to the control group. Phylum-level analysis revealed Firmicutes as the main phylum, followed by Bacteroidetes, in both groups. The data also found that Phascolarctobacterium and Barnesiella were increased in A. aeriphila-treated group. In conclusion, oral administration of A. aeriphila could improve the growth performance, serum antioxidant capacity, immune modulation, and gut health of broilers. Our findings may provide important information for the application of A. aeriphila in poultry production.

The JAK/STAT/NF-κB signaling pathway can be regulated by rosemary essential oil, thereby providing a potential treatment for DNCB-induced in mice.

OBJECTIVE: The purpose of this study was to investigate the mechanism through which rosemary essential oil treats atopic dermatitis. METHODS: A dinitrochlorobenzene (DNCB)-induced atopic dermatitis mouse model was established and treated with low (1%), medium (2%), and high (4%) doses of Rosmarinus officinalis essential oil (EORO). Serum levels of interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) in each group were determined using enzyme-linked immunosorbent assay (ELISA). Skin tissues were stained with hematoxylin-eosin and toluidine blue. We used network pharmacology and molecular docking techniques to verify the biological activity of essential proteins and their corresponding compounds in the pathway. Gas chromatography-mass spectrometry (GC-MS) was used for metabolomics analysis and multivariate statistical analysis of mouse serum to screen differential metabolites and metabolic pathway analysis. Protein expression of p-JAK1, CD4+ cells, and IL-4 in the skin tissue was detected by immunohistochemistry analysis. Protein levels of STAT3, p-STAT3, P65, and p-P65 in damaged skin tissues were detected using western blotting. RESULT: The skin of mice in the model group showed different degrees of erythema, dryness, scratches, epidermal erosion and shedding, and crusting. After treatment, the serum levels of IL-6 and TNF-α in EORO group were significantly decreased, and the expression of p-JAK1,CD4 + cells, IL-4, p-P65 / P65 and p-STAT3 / STAT3 proteins in skin tissues were decreased. CONCLUSION: EORO can effectively improve DNCB-induced AD-like skin lesions in mice by regulating the JAK/STAT/NF-κB signaling pathway, thereby reducing the production of downstream arachidonic acid metabolites, inhibiting skin inflammation, and restoring epidermal barrier function.

Association of Nonpuerperal Mastitis with Cytokines Related to Helper T Cells TH1/TH2 and TH17/Treg.

Objective: To investigate the association of nonpuerperal mastitis with cytokines related to the helper T cells TH1/TH2 and TH17/Treg and associated immune balance. Methods: From 2016 to 2021, we included 40 patients with non-puerperal mastitis who underwent surgery at China-Japan Friendship Hospital and compared them with 40 control patients with benign non-infectious breast disease. Hematoxylin-eosin staining detects inflammatory infiltrates of breast tissue. The expression of interferon γ and interleukin 4 in breast tissue was detected by immunofluorescence imaging, and the relative protein expression of TH1/TH2 and TH17/Treg cell-associated cytokines in CD4+ T cells was detected by western blotting. CD4+ T cells were isolated by fluorescence-activated cell sorting for detection of the relative protein expression of interferon γ and interleukin 4 in CD4+ T cells. Results: Hematoxylin-eosin staining showed that the nonpuerperal mastitis group had significantly greater inflammatory infiltration than the control group. Immunofluorescence images showed the relative fluorescence intensity of interferon γ was significantly higher in the nonpuerperal mastitis group than in the control group (P < .001), but the relative fluorescence intensity of interleukin 4 did not significantly differ between the 2 groups (P = .0686). Western blotting revealed that the relative protein expression of interferon γ, interleukin 2, and interleukin 17 was significantly higher in the nonpuerperal mastitis group than in the control group (P < .001), but the relative protein expression of interleukin 4 (P = .0512), interleukin 10 (P = .3088), and transforming growth factor ß (P = .0653) did not significantly differ between the 2 groups. Flow cytometry of isolated CD4+ T cells showed the relative protein expression of interferon γ was significantly higher in the nonpuerperal mastitis group than in the control group (P < .001), but the relative protein expression of interleukin 4 did not significantly differ between the 2 groups (P = .0680). Conclusion: The expression of the TH1 cytokines interferon γ and interleukin 2 and the TH17 cytokine interleukin 17 was significantly higher in patients with nonpuerperal mastitis, while the TH2 cytokine interleukin 4 and the Treg cytokines interleukin 10 and transforming growth factor ß were expressed at lower levels. This study provides new research ideas for the treatment of mastitis.

Black Ginseng Extract Exerts Potentially Anti-Asthmatic Activity by Inhibiting the Protein Kinase Cθ-Mediated IL-4/STAT6 Signaling Pathway.

Asthma is a chronic inflammatory lung disease that causes respiratory difficulties. Black ginseng extract (BGE) has preventative effects on respiratory inflammatory diseases such as asthma. However, the pharmacological mechanisms behind the anti-asthmatic activity of BGE remain unknown. To investigate the anti-asthmatic mechanism of BGE, phorbol 12-myristate 13-acetate plus ionomycin (PMA/Iono)-stimulated mouse EL4 cells and ovalbumin (OVA)-induced mice with allergic airway inflammation were used. Immune cells (eosinophils/macrophages), interleukin (IL)-4, -5, -13, and serum immunoglobulin E (IgE) levels were measured using an enzyme-linked immunosorbent assay. Inflammatory cell recruitment and mucus secretion in the lung tissue were estimated. Protein expression was analyzed via Western blotting, including that of inducible nitric oxide synthase (iNOS) and the activation of protein kinase C theta (PKCθ) and its downstream signaling molecules. BGE decreased T helper (Th)2 cytokines, serum IgE, mucus secretion, and iNOS expression in mice with allergic airway inflammation, thereby providing a protective effect. Moreover, BGE and its major ginsenosides inhibited the production of Th2 cytokines in PMA/Iono-stimulated EL4 cells. In EL4 cells, these outcomes were accompanied by the inactivation of PKCθ and its downstream transcription factors, such as nuclear factor of activated T cells (NFAT), nuclear factor kappa B (NF-κB), activator of transcription 6 (STAT6), and GATA binding protein 3 (GATA3), which are involved in allergic airway inflammation. BGE also inhibited the activation of PKCθ and the abovementioned transcriptional factors in the lung tissue of mice with allergic airway inflammation. These results highlight the potential of BGE as a useful therapeutic and preventative agent for allergic airway inflammatory diseases such as allergic asthma.

Anandamide improves food intake and orexinergic neuronal activity in the chronic sleep deprivation induction model in rats by modulating the expression of the CB1 receptor in the lateral hypothalamus.

Sleep deprivation alters orexinergic neuronal activity in the lateral hypothalamus (LH), which is the main regulator of sleep-wake, arousal, appetite, and energy regulation processes. Cannabinoid receptor (CBR) expression in this area is involved in modulating the function of orexin neurons. In this study, we investigated the effects of endocannabinoid anandamide (AEA) administration on improving food intake and appetite by modulating the activity of orexin neurons and CB1R expression after chronic sleep deprivation. Adult male Wistar rats (200-250 g) were randomly divided into three groups: control + vehicle (Control), chronic sleep deprivation + vehicle (SD), and chronic sleep deprivation +20 mg/kg AEA (SD + A). For SD induction, the rats were kept in a sleep deprivation device for 18 h (7 a.m. to 1 a.m.) daily for 21 days. Weight gain, food intake, the electrical power of orexin neurons, CB1R mRNA expression in hypothalamus, CB1R protein expression in the LH, TNF-α, IL-6, IL-4 levels and antioxidant activity in hypothalamus were measured after SD induction. Our results showed that AEA administration significantly improved food intake (p < 0.01), Electrical activity of orexin neurons (p < 0.05), CB1R expression in the hypothalamus (p < 0.05), and IL-4 levels (p < 0.05). AEA also reduced mRNA expression of OX1R and OX2R (p < 0.01 and p < 0.05 respectively), also IL-6 and TNF-α (p < 0.01) and MDA level (p < 0.05) in hypothalamic tissue. As a consequence, AEA modulates orexinergic system function and improves food intake by regulating the expression of the CB1 receptor in the LH in sleep deprived rats.

Garcinone E suppresses breast cancer growth and metastasis by modulating tumor-associated macrophages polarization via STAT6 signaling.

Cancer metastasis remains the most common cause of death in breast cancer patients. Tumor-associated macrophages (TAMs) are a novel therapeutic target for the treatment of metastatic breast cancer. Despite the good anti-cancer activity of garcinone E (GE), there are no reports on its therapeutic effects on breast cancer metastasis. The objective of this study was to examine the anti-cancer effects of GE on metastatic breast cancer. RAW 264.7 and THP-1 cells were polarized to M2 macrophages by IL-4/IL-13 in vitro. A 4T1 mouse breast cancer model and the tail vein breast cancer metastasis model were used to explore the effect of GE on breast cancer growth and metastasis in vivo. In vitro studies showed that GE dose-dependently suppressed IL-4 + IL-13-induced expression of CD206 in both RAW 264.7 cells and differentiated THP-1 macrophages. However, GE did not affect the LPS + IFN-γ-induced polarization to the M1-like macrophages in vitro. GE inhibited the expression of the M2 macrophage specific genes in RAW 264.7 cells, and simultaneously impaired M2 macrophage-induced breast cancer cell proliferation and migration, and angiogenesis. In animal studies, GE significantly suppressed tumor growth, angiogenesis, and lung metastasis in 4T1 tumor-bearing mice, without causing toxicity. In both tumor and lung tissues, the proportion of M2-like TAMs was significantly decreased while the proportion of M1-like TAMs was markedly increased by GE treatment. Mechanistically, GE inhibited phosphorylation of STAT6 in vitro and in vivo. Our results demonstrate for the first time that GE suppresses breast cancer growth and pulmonary metastasis by modulating M2-like macrophage polarization through the STAT6 signaling pathway.

Alpha-linolenic acid improves nasal mucosa epithelial barrier function in allergic rhinitis by arresting CD4+ T cell differentiation via IL-4Rα-JAK2-STAT3 pathway.

BACKGROUND: Allergic rhinitis (AR) defined as inflammation and tissue remodeling of the nasal mucosa in atopic individuals after allergen exposure. Alpha-linolenic acid [cis-9, cis-12, cis-15-octadecatrienoic acid (18:3)] (ALA) as dietary supplementation can reduce inflammation and allergic symptoms. OBJECTIVE: To evaluate the potential therapeutic effect and mechanism of ALA in AR mouse model. METHODS: Ovalbumin sensitized AR mouse model were challenged with oral ALA administration. Nasal symptoms, tissue pathology, immune cell infiltration and goblet cell hyperplasia were investigated. Levels of IgE, TNF-ß, IFN-γ, IL-2, IL-4, IL-5, IL-12, IL-13 and IL-25 were determined by ELISA in serum and nasal fluid. Quantitative RT-PCR and immunofluorescence were performed for occludin and zonula occludens-1 expression. CD3+CD4+ T-cells from peripheral blood and splenic lymphocytes were isolated and Th1/Th2 ratio were determined. Mouse naive CD4+ T cell were isolated and Th1/Th2 ratio, IL-4Rα expression, and IL5/IL13 secretion were determined. IL-4Rα-JAK2-STAT3 pathway change in AR mice were performed by western blot. RESULTS: Ovalbumin induced AR, nasal symptoms, pathological performance, IgE, and cytokine production. ALA treated mice showed reduced nasal symptoms, nasal inflammation, nasal septum thickening, goblet cell hyperplasia, and eosinophil infiltration. In serum and nasal fluid of ovalbumin challenged mice, ALA decreased IgE, IL-4 levels, and the increase of Th2-cells. ALA prevented the disruption of the epithelial cell barrier in ovalbumin-challenged AR mice. Simultaneously, ALA prevents IL-4 induced barrier disruption. ALA treatment of AR by affecting the differentiation stage of CD4+T cells and block IL-4Rα-JAK2-STAT3 pathway. CONCLUSION: This study suggests that ALA has the potential therapeutic effect to ovalbumin-induced AR. ALA can affect the differentiation stage of CD4+T cells and improve epithelial barrier functions through IL-4Rα-JAK2-STAT3 pathways. CLINICAL IMPLICATION: ALA might be considered as drug candidate for improving epithelial barrier function through Th1/Th2 ratio recovery in AR.

[Acupoint injection ameliorates Th1/Th2 imbalance through Toll-like receptor 4/activator protein-1 signal pathway and improves inflammatory response in rats with allergic rhinitis].

OBJECTIVE: To observe the effect of acupoint injection on serum T helper (Th)1/Th2 related cytokines, and the expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and activator protein-1 (AP-1) of nasal mucosa in allergic rhinitis (AR) rats, so as to explore its mechanism underlying improvement of AR. METHODS: Thirty-two SD rats were randomly divided into normal, model, non-acupoint injection and acupoint injection groups (n=8 in each group). The AR model was established by ovalbumin sensitization. In the acupoint injection group, "Yintang" (GV24+) and bilateral "Yingxiang" (LI20) were selected for injection of mixture solution of dexamethasone and lidocaine (0.05 mL/acupoint), once every 4 days for a total of 4 times. The non-acupoints, located at the midpoint between the "Houhai" and "Huantiao" (GB30) on the bilateral hips and the sites 5 cm inferior to the axillary were injected with the same dose of mixture solution as that in the acupoint injection group. The AR severity was assessed by cumulative quantification scoring methods (including the numbers of nose-catching and sneezes, and the amount of nasal secretions in 30 min). The pathological changes of nasal mucosa were observed by HE staining. The contents of immunoglobulin E (IgE), interleukin (IL)-4 and interferon (IFN)-γ in serum were detected by ELISA. The expressions of TLR4 and MyD88 in nasal mucosa was detected by immunofluorescence. The expression of AP-1 in nasal mucosa was detected by Western blot. RESULTS: Following modeling, the AR symptom score, serum IgE and IL-4 contents and expression of TLR4, MyD88 and AP-1 of nasal mucosa were significantly increased in the model group than those in the normal group (P<0.01), while the serum IFN-γ content was significantly decreased (P<0.01). Compared with the model group and non-acupoint injection group, the AR symptom score, the serum contents of IgE and IL-4 and the expressions of TLR4, MyD88 and AP-1 in nasal mucosa were significantly decreased in the acupoint injection group (P<0.01, P<0.05), while the serum IFN-γ content was significantly increased (P<0.01). H.E. staining of the nasal mucosa showed that most of the epithelium fell off, the lamina propria vessels expanded, the glands proliferated, and eosinophils and lymphocytes infiltrated in the model and non-acupoint injection groups, and those were significantly improved in the acupoint injection group. CONCLUSION: Acupoint injection can effectively improve allergic inflammation of the nose in AR rats, which may be related with its function in inhibiting the abnormal activation of TLR4/AP-1 signaling pathway and regulating the imbalance of Th1/Th2.

[Protective effects of three kinds of borneol on different brain regions in acute cerebral ischemia/reperfusion model rats].

This study compared the ameliorating effects of L-borneol, natural borneol, and synthetic borneol on the injury of different brain regions in the rat model of acute phase of cerebral ischemia/reperfusion(I/R) for the first time, which provides a reference for guiding the rational application of borneol in the early treatment of ischemic stroke and has important academic and application values. Healthy specific pathogen-free(SPF)-grade SD male rats were randomly assigned into 13 groups: a sham-operation group, a model group, a Tween model group, a positive drug(nimodipine) group, and high-, medium-, and low-dose(0.2, 0.1, and 0.05 g·kg~(-1), respectively) groups of L-borneol, natural borneol, and synthetic borneol according to body weight. After 3 days of pre-administration, the rat model of I/R was established by suture-occluded method and confirmed by laser speckle imaging. The corresponding agents in different groups were then administered for 1 day. The body temperature was monitored regularly before pre-administration, days 1, 2, and 3 of pre-administration, 2 h after model awakening, and 1 d after model establishment. Neurological function was evaluated based on Zea-Longa score and modified neurological severity score(mNSS) 2 h and next day after awakening. The rats were anesthetized 30 min after the last administration, and blood was collected from the abdominal aorta. Enzyme-linked immunoassay assay(ELISA) was employed to determine the serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-4, and transforming growth factor-beta1(TGF-ß1). The brain tissues were stained with triphenyltetrazolium chloride(TTC) for the calculation of cerebral infarction rate, and hematoxylin-eosin(HE) staining was used for observing and semi-quantitatively evaluating the pathological damage in different brain regions. Immunohistochemistry was employed to detect the expression of ionized calcium binding adapter molecule 1(IBA1) in microglia. q-PCR was carried out to determine the mRNA levels of iNOS and arginase 1(Arg1), markers of polarization phenotype M1 and M2 in microglia. Compared with the sham-operation group, the model group and the Tween model group showed significantly elevated body temperature, Zea-Longa score, mNSS, and cerebral infarction rate, severely damaged cortex, hippocampus, and striatum, increased serum levels of IL-6 and TNF-α, and decreased serum levels of IL-4 and TGF-ß1. The three borneol products had a tendency to reduce the body temperature of rats 1 day after modeling. Synthetic borneol at the doses of 0.2 and 0.05 g·kg~(-1), as well as L-borneol of 0.1 g·kg~(-1), significantly reduced Zea-Longa score and mNSS. The three borneol products at the dose of 0.2 g·kg~(-1) significantly reduced the cerebral infarction rate. L-borneol at the doses of 0.2 and 0.1 g·kg~(-1) and natural borneol at the dose of 0.1 g·kg~(-1) significantly reduced the pathological damage of the cortex. L-borneol and natural borneol at the dose of 0.1 g·kg~(-1) attenuated the pathological damage of hippocampus, and 0.2 g·kg~(-1) L-borneol attenuated the damage of striatum. The 0.2 g·kg~(-1) L-borneol and the three doses of natural borneol and synthetic borneol significantly reduced the serum level of TNF-α, and the 0.1 g·kg~(-1) synthetic borneol reduced the level of IL-6. L-borneol and synthetic borneol at the dose of 0.2 g·kg~(-1) significantly inhibited the activation of cortical microglia, and 0.2 g·kg~(-1) L-borneol up-regulated the expression of Arg1 and down-regulated the expression level of iNOS. In conclusion, the three borneol products may alleviate inflammation to ameliorate the pathological damage of brain regions of rats in the acute phase of I/R by inhibiting the activation of microglia and promoting the polarization of microglia from M1 type to M2 type. The protective effect on brain followed a trend of L-borneol > synthetic borneol > natural borneol. We suggest L-borneol the first choice for the treatment of I/R in the acute phase.

[Bombyx Batryticatus extract promotes microglia polarization to improve neuron injury and behaviors of cerebral ischemia/reperfusion rats].

This study aims to investigate the effect of Bombyx Batryticatus extract(BBE) on behaviors of rats with global cerebral ischemia reperfusion(I/R) and the underlying mechanism. The automatic coagulometer was used to detect the four indices of human plasma coagulation after BBE intervention for quality control of the extract. Sixty 4-week-old male SD rats were randomized into sham operation group(equivalent volume of normal saline, ip), model group(equivalent volume of normal saline, ip), positive drug group(900 IU·kg~(-1) heparin, ip), and low-, medium-, and high-dose BBE groups(0.45, 0.9, and 1.8 mg·g~(-1)·d~(-1) BBE, ip). Except the sham operation group, rats were subjected to bilateral common carotid artery occlusion followed by reperfusion(BCCAO/R) to induce I/R. The administration lasted 7 days for all the groups. The behaviors of rats were examined by beam balance test(BBT). Morphological changes of brain tissue were observed based on hematoxylin-eosin(HE) staining. Immunofluorescence method was used to detect common leukocyte antigen(CD45), leukocyte differentiation antigen(CD11b), and arginase-1(Arg-1) in cerebral cortex(CC). The protein expression of interleukin-1ß(IL-1ß), interleukin-4(IL-4), interleukin-6(IL-6), and interleukin-10(IL-10) was detected by enzyme-linked immunosorbent assay(ELISA). The non-targeted metabonomics was employed to detect the levels of metabolites in plasma and CC of rats after BBE intervention. The results of quality control showed that the BBE prolonged the activated partial thromboplastin time(APTT), prothrombin time(PT), and thrombin time(TT) of human plasma, which was similar to the anticoagulation effect of BBE obtained previously. The results of behavioral test showed that the BBT score of the model group increased compared with that of the sham operation group. Compared with the model group, BBE reduced the BBT score. As for the histomorphological examination, compared with the sham operation group, the model group showed morphological changes of a lot of nerve cells in CC. The nerve cells with abnormal morphology in CC decreased after the intervention of BBE compared with those in the model group. Compared with the sham operation group, the model group had high average fluorescence intensity of CD45 and CD11b in the CC. The average fluorescence intensity of CD11b decreased and the average fluorescence intensity of Arg-1 increased in CC in the low-dose BBE group compared with those in the model group. The average fluorescence intensity of CD45 and CD11b decreased and the average fluorescence intensity of Arg-1 increased in medium-and high-dose BBE groups compared with those in the model group. The expression of IL-1ß and IL-6 was higher and the expression of IL-4 and IL-10 was lower in the model group than in the sham operation group. The expression of IL-1ß and IL-6 was lower and the expression of IL-4 and IL-10 was higher in the low-dose, medium-dose, and high-dose BBE groups than in the model group. The results of non-targeted metabonomics showed that 809 metabolites of BBE were identified, and 57 new metabolites in rat plasma and 45 new metabolites in rat CC were found. BBE with anticoagulant effect can improve the behaviors of I/R rats, and the mechanism is that it promotes the polarization of microglia to M2 type, enhances its anti-inflammatory and phagocytic functions, and thus alleviates the damage of nerve cells in CC.

Cassia absus-mediated upregulation of IL-4, IL-10 and downregulation of IL-1ß, IL-6, TNF- α, NF-κB, IFN-γ in CFA-induced arthritis model.

Traditional use of Cassia absus as an anti-inflammatory in conjunctivitis and bronchitis is well reported. Owing to its anti-inflammatory potential, the current study appraised in vivo anti-arthritic activity of n-hexane and aqueous extracts of Cassia absus seeds (200 mg/kg) using Complete Freund's Adjuvant (CFA) rat model of arthritis. Changes in paw size (mm), joint diameter (mm), and pain response (sec) were recorded at the baseline and then after CFA induction at the interval of 4 days till the 28th day. Blood samples of anesthetized rats were collected for the estimation of hematological, oxidative, and inflammatory biomarkers. Results showed percent inhibition in paw edema (45.09% and 60.79%) with both n-hexane and aqueous extracts, respectively. Significant reduction in paw size and ankle joint diameter (P < 0.01) was seen in extracts treated rats. Erythrocyte Sedimentation rate, C-Reactive Protein, White Blood Cell levels significantly lowered, and Hemoglobin, Platelets and Red Blood Cell count significantly increased post-treatments. Superoxide Dismutase, Catalase, and Glutathione were significantly improved (P < 0.0001) in treated groups as compared to CFA induced arthritic control. Real-time polymerase chain reaction investigation showed significant downregulation (P < 0.05) of Interleukin-1ß, Tumor Necrosis Factor-α, Interleukin-6, Cycloxygenase-2, Nuclear Factor-κB, Prostaglandin E Synthase 2, Interferon Gamma and upregulation of Interleukin-4, Interleukin-10 in both n-hexane and aqueous extract-treated groups. It is thereby concluded that Cassia absus can significantly attenuate CFA-induced arthritis by modulation of oxidative and inflammatory biomarkers.

Glycyrrhetinic acid suppresses breast cancer metastasis by inhibiting M2-like macrophage polarization via activating JNK1/2 signaling.

BACKGROUND: Breast cancer metastasis is leading cause of cancer death among women worldwide. Tumor-associated macrophages (TAMs) have been considered as potential targets for treating breast cancer metastasis because they promote tumor growth and development. Glycyrrhetinic acid (GA) is one of the most important phytochemicals of licorice which has shown promising anti-cancer efficacies in pre-clinical trials. However, the regulatory effect of GA on the polarization of TAMs remains elusive. PURPOSE: To investigate the role of GA in regulating the polarization of M2 macrophages and inhibiting breast cancer metastasis, and to further explore its underlying mechanisms of action. STUDY DESIGN: IL-4 / IL-13-treated RAW 264.7 and THP-1 cells were used as the M2-polarized macrophages in vitro. A 4T1 mouse breast cancer model and the tail vein breast cancer metastasis model were applied to study the effect of GA on breast cancer growth and metastasis in vivo. RESULTS: In vitro studies showed that GA significantly inhibited IL-4 / IL 13-induced M2-like polarization in RAW 264.7 and THP-1 macrophages without affecting M1-like polarization. GA strongly decreased the expression of M2 macrophage markers CD206 and Arg-1, and reduced the levels of the pro-angiogenic molecules VEGF, MMP9, MMP2 and IL-10 in M2 macrophages. GA also increased the phosphorylation of JNK1/2 in M2 macrophages. Moreover, GA significantly suppressed M2 macrophage-induced cell proliferation and migration in 4T1 cancer cells and HUVECs. Interestingly, the inhibitory effects of GA on M2 macrophages were abolished by a JNK inhibitor. Animal studies showed that GA significantly suppressed tumor growth, angiogenesis, and lung metastasis in BALB/c mice bearing breast tumor. In tumor tissues, GA reduced the number of M2 macrophages but elevated the proportion of M1 macrophages, accompanied by activation of JNK signaling. Similar results were found in the tail vein breast cancer metastasis model. CONCLUSION: This study demonstrated for the first time that GA could effectively suppress breast cancer growth and metastasis by inhibiting macrophage M2 polarization via activating JNK1/2 signaling. These findings indicate that GA could be served as the lead compound for the future development of anti-breast cancer drug.

Therapeutic effects of Pulsatilla koreana Nakai extract on ovalbumin-induced allergic rhinitis by inhibition of Th2 cell activation and differentiation via the IL-4/STAT6/GATA3 pathway.

Allergic rhinitis (AR), caused by immunoglobulin E (IgE)-mediated inflammation, generally occurs in the upper respiratory tract. T helper type 2 (Th2) cell-mediated cytokines, including interleukin (IL)-4, IL-5, and IL-13, are important factors in AR pathogenesis. Despite various treatment options, the difficulty in alleviating AR and pharmacological side effects necessitate development of new therapies. The root of Pulsatilla koreana Nakai (P. koreana), a pasque flower, has been used as a herbal medicine. However, its effects on AR remain unclear; therefore, we aimed to explore this subject in the current study. The therapeutic effects of P. koreana water extract (PKN) on the pathophysiological functions of the nasal mucosa was examined in ovalbumin (OVA)-induced AR mice. The effect of PKN on Th2 activation and differentiation was evaluated using concanavalin A-induced splenocytes and differentiated Th2 cells from naïve CD4+ T cells. We also investigated the effect of changes in JAK/STAT6/GATA3 signaling on IL-4-induced Th2 cells. In OVA-induced AR mice, PKN administration alleviated allergic nasal symptoms and decreased the total number of immune cells, lymphocytes, neutrophils, and eosinophils in nasal lavage fluid; serum levels of OVA-specific IgE, histamine, and IL-13 were also significantly reduced. PKN also ameliorated OVA-induced nasal mucosal tissue thickening by inhibiting inflammation and goblet cell hyperplasia. PKN treatment significantly inhibited Th2 activity and differentiation through the IL-4/STAT-6/GATA3 pathway in Th2 cells. PKN is an effective AR treatment with the potential to improve patients' daily lives by regulating the allergic inflammatory response induced by Th2 cells.

Increased Expression of Hepatic Stearoyl-CoA Desaturase (SCD)-1 and Depletion of Eicosapentaenoic Acid (EPA) Content following Cytotoxic Cancer Therapy Are Reversed by Dietary Fish Oil.

Cancer treatment evokes impediments to liver metabolism that culminate in fatty liver. This study determined hepatic fatty acid composition and expression of genes and mediators involved in lipid metabolism following chemotherapy treatment. Female rats bearing the Ward colon tumor were administered Irinotecan (CPT-11) +5-fluorouracil (5-FU) and maintained on a control diet or a diet containing eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) (2.3 g/100 g fish oil). Healthy animals provided with a control diet served as a reference group. Livers were collected one week after chemotherapy. Triacylglycerol (TG), phospholipid (PL), ten lipid metabolism genes, leptin, and IL-4 were measured. Chemotherapy increased TG content and reduced EPA content in the liver. Expression of SCD1 was upregulated by chemotherapy, while dietary fish oil downregulated its expression. Dietary fish oil down-regulated expression of the fatty acid synthesis gene FASN, while restoring the long chain fatty acid converting genes FADS2 and ELOVL2, and genes involved in mitochondrial ß-oxidation (CPT1α) and lipid transport (MTTP1), to values similar to reference animals. Neither leptin nor IL-4 were affected by chemotherapy or diet. Depletion of EPA is associated with pathways evoking enhanced TG accumulation in the liver. Restoring EPA through diet may pose a dietary strategy to attenuate chemotherapy-associated impediments in liver fatty acid metabolism.