The Alleviation of Gut Microbiota-Induced Depression and Colitis in Mice by Anti-Inflammatory Probiotics NK151, NK173, and NK175

Gut microbiota dysbiosis is strongly associated with psychiatric disorders and inflammatory bowel disease (IBD). Herein, we examined whether the fecal microbiota of IBD patients with depression (IBDD) and their gut microbiota culture (iGm) could cause depression and colitis in mice and anti-inflammatory probiotics could mitigate depression in iGm-transplanted or immobilization stress (IS)-exposed mice. Fecal microbiota transplantation (FMT) from IBDD patients, which exhibited Enterobacteriaceae-rich gut microbiota, and its gut microbiota culture (iGm) increased depression-like behaviors in mice. Their treatments heightened the blood lipopolysaccharide (LPS) level and colonic IL-1β and IL-6 expression. However, FMT from healthy volunteers or sulfasalazine treatment alleviated cGm-induced depressive-like behaviors and hippocampal and colonic inflammation in mice. Moreover, oral administration of Lactobacillus plantarum NK151, Bifidobacterium longum NK173, and Bifidobacterium bifidum NK175, which inhibited LPS-induced IL-6 expression in macrophages, alleviated cGm-induced depression-like behaviors, hippocampal NF-κB⁺Iba1⁺ cell numbers and IL-1β and IL-6 expression, blood LPS, IL-6, and creatinine levels, and colonic NF-κB⁺CD11c⁺ number and IL-1β and IL-6 expression in mice. Treatment with NK151, NK173, or NK175 mitigated immobilization stress (IS)-induced depressive-like behaviors, neuroinflammation, and gut inflammation in mice. NK151, NK173, or NK175 also decreased IS-induced blood LPS, IL-6, and creatinine levels. The transplantation of Enterobacteriaceae-rich gut microbiota can cause depression and colitis, as IS exposure, and anti-inflammatory NK151, NK173, and NK175, may alleviate stress-induced fatigue, depression, and colitis by regulating the expression of proinflammatory and anti-inflammatory cytokines through the suppression of gut bacterial LPS.     

Inhibitory action of water soluble fraction of Terminalia chebula on systemic and local anaphylaxis

We investigated the effects of the water soluble fraction of Terminalia chebula (Combretaceae) (WFTC) on systemic and local anaphylaxis. WFTC administered 1h before compound 48/80 injection inhibited compound 48/80-induced anaphylactic shock 100% with doses of 0.01-1.0 g/kg. When WFTC was administered 5 or 10 min after compound 48/80 injection, the mortality also decreased in a dose-dependent manner. Passive cutaneous anaphylaxis was inhibited by 63.5+/-7.8% by oral administration of WFTC (1.0 g/kg). When WFTC was pretreated at concentrations ranging from 0.005 to 1.0 g/kg, the serum histamine levels were reduced in a dose-dependent manner. WFTC (0.01-1.0 mg/ml) also significantly inhibited histamine release from rat peritoneal mast cells (RPMC) by compound 48/80. However, WFTC (1.0 mg/ml) had a significant increasing effect on anti-dinitrophenyl IgE-induced tumor necrosis factor-alpha production from RPMC. These results indicate that WFTC may possess a strong antianaphylactic action.     

Combined enzymatic hydrolysis and herbal extracts fortification to boost in vitro antioxidant activity of edible bird’s nest solution

Objective Edible bird’s nest (EBN) is a popular traditional tonic food in Chinese population for centuries. Malaysia is one of the main EBN suppliers in the world. This study aims to explore the best strategy to boost the antioxidant potential of EBN solution. Methods In this study, the raw EBN (4%, mass to volume ratio) was initially enzymatic hydrolyzed using papain enzyme to produce EBN hydrolysate (EBNH), then spray-dried into powdered form. Next, 4% (mass to volume ratio) of EBNH powder was dissolved in ginger extract (GE), mulberry leaf extract (MLE) and cinnamon twig extract (CTE) to detect the changes of antioxidant activities, respectively. Results Results obtained suggest that enzymatic hydrolysis significantly reduced the viscosity of 4% EBN solution from (68.12 ± 0.69) mPa.s to (7.84 ± 0.31) mPa.s. Besides, the total phenolic content (TPC), total flavonoid content (TFC), total soluble protein, DPPH scavenging activity and ferric reducing antioxidant power (FRAP) were substantially increased following EBN hydrolysis using papain enzyme. In addition, fortification with GE, MLE and CTE had further improved the TPC, TFC, DPPH scavenging activity and FRAP of the EBNH solution. Among the samples, MLE-EBNH solution showed the most superior antioxidant potential at (86.39 ± 1.66)% of DPPH scavenging activity and (19.79 ± 2.96) mmol/L FeSO4 of FRAP. Conclusion This study proved that combined enzymatic hydrolysis and MLE fortification is the best strategy to produce EBN product with prominent in vitro antioxidant potential. This preliminary study provides new insight into the compatibility of EBN with different herbal extracts for future health food production.     

Inhibition of homodimerization of Toll-like receptor 4 by curcumin

Toll-like receptors play a key role in sensing microbial components and inducing innate immune responses. Ligand-induced dimerization of TLR4 is required for the activation of downstream signaling pathways. Thus, the receptor dimerization may be one of the first lines of regulation in activating TLR-mediated signaling pathways and induction of subsequent immune responses. LPS induces the activation of NF-kappaB and IRF3 through MyD88- or TRIF-dependent pathways. Curcumin, a polyphenol found in the plant Curcuma longa, has been shown to suppress the activation of NF-kappaB induced by various pro-inflammatory stimuli by inhibiting IKKbeta kinase activity in MyD88-dependent pathway. Curcumin also inhibited LPS-induced IRF3 activation. These results imply that curcumin inhibits both MyD88- and TRIF-dependent pathways in LPS-induced TLR4 signaling. However, in TRIF-dependent pathway, curcumin did not inhibit IRF3 activation induced by overexpression of TRIF in 293T cells. These results suggest that TLR4 receptor complex is the molecular target of curcumin in addition to IKKbeta. Here, we report biochemical evidence that phytochemicals (curcumin and sesquiterpene lactone) inhibit both ligand-induced and ligand-independent dimerization of TLR4. Furthermore, these results demonstrate that small molecules with non-microbial origin can directly inhibit TLRs-mediated signaling pathways at the receptor level. These results imply that the activation of TLRs and subsequent immune/inflammatory responses induced by endogenous molecules or chronic infection can be modulated by certain dietary phytochemicals we consume daily.     

Combinational treatment of 5‐fluorouracil and casticin induces apoptosis in mouse leukemia WEHI‐3 cells in vitro

Leukemia is one of the major diseases causing cancer‐related deaths in the young population, and its cure rate is unsatisfying with side effects on patients. Fluorouracil (5‐FU) is currently used as an anticancer drug for leukemia patients. Casticin, a natural polymethoxyflavone, exerts anticancer activity against many human cancer cell lines in vitro, but no other reports show 5‐FU combined with casticin increased the mouse leukemia cell apoptosis in vitro. Herein, the antileukemia activity of 5‐FU combined with casticin in WEHI‐3 mouse leukemia cells was investigated in vitro. Treatment of two‐drug combination had a higher decrease in cell viability and a higher increase in apoptotic cell death, the level of DNA condensation, and the length of comet tail than that of 5‐FU or casticin treatment alone in WEHI‐3 cells. In addition, the two‐drug combination has a greater production rate of reactive oxygen species but a lower level of Ca²⁺ release and mitochondrial membrane potential (ΔΨ_m) than that of 5‐FU alone. Combined drugs also induced higher caspase‐3 and caspase‐8 activities than that of casticin alone and higher caspase‐9 activity than that of 5‐FU or casticin alone at 48 hours treatment. Furthermore, 5‐FU combined with casticin has a higher expression of Cu/Zn superoxide dismutase (SOD [Cu/Zn]) and lower catalase than that of 5‐FU or casticin treatment alone. The combined treatment has higher levels of Bax, Endo G, and cytochrome C of proapoptotic proteins than that of casticin alone and induced lower levels of B‐cell lymphoma 2 (BCL‐2) and BCL‐X of antiapoptotic proteins than that of 5‐FU or casticin only. Furthermore, the combined treatment had a higher expression of cleaved poly (ADP‐ribose) polymerase (PARP) than that of casticin only. Based on these findings, we may suggest that 5‐FU combined with casticin treatment increased apoptotic cell death in WEHI‐3 mouse leukemia cells that may undergo mitochondria and caspases signaling pathways in vitro.     

Effect of sex hormones on lipid peroxidation in rat liver

The role of sex hormones in hepatic lipid peroxidation, and in hepatic aldehyde oxidase and xanthine oxidase activities were investigated using rat liver homogenates. It was observed that male rat had a significantly greater content of malondialdehyde in liver than female. Among the sex hormones tested, estradiol, one of female hormones, markedly inhibited the formation of lipid peroxides in liver tissues in vitro. Especially, the inhibitory effect of estradiol appeared more remarkably in Fe+2-induced lipid peroxidation. The hepatic xanthine oxidase activity was decreased about 15% by 10(-6) M estradiol, whereas, the aldehyde oxidase activity was almost completely disappeared at the same concentration of estradiol. It implies that sex differences in lipid peroxidation is attributed to the suppression of free radical generating system by estradiol.     

Evaluation of the sub-acute toxicity of the sclerotium of Lignosus rhinocerus (Cooke), the Tiger Milk mushroom

Ethnopharmacological relevance

Lignosus rhinocerus (known locally as ‘Tiger Milk mushroom’) is the most important medicinal mushroom used by the indigenous communities of Malaysia to treat fever, cough, asthma, cancer, food poisoning and as a general tonic. The sclerotium of the mushroom is the part with medicinal value. Lignosus rhinocerus was hitherto unexploited commercially because of limited supply. Recently, the mushroom was successfully cultivated.

Materials and methods

Sprague Dawley rats (5 rats/group/sex) were fed orally with 250, 500 and 1000 mg/kg TM02, 1000 mg/kg TM03 as well as 1000 mg/kg wild type Lignosus rhinocerus sclerotial powder. Sclerotial powder was orally administered once daily and consecutively for 28 days. Body weight of each animal was measured and any gross behavioral change was observed daily. Hematological and clinical biochemical parameters as well as histopathological analysis were carried out on 29th day.

Results

The results showed that oral administration of the sclerotial powder at daily dose of up to 1000 mg/kg had no adverse effect on the growth rate, hematological and clinical biochemical parameters (including renal and liver function parameters). Histological studies showed that the treatments did not induce any pathological changes in the liver, kidney, heart, spleen and lung of the animals.

Conclusion

In conclusion, our results show that there was no treatment-related sub-acute toxicity in rats following 28-days oral administration of 250, 500 and 1000 mg/kg TM02, 1000 mg/kg TM03 as well as 1000 mg/kg wild type Lignosus rhinocerus sclerotial powder. As the highest tested dose of 1000 mg/kg was not associated with any toxicity concern, the NOAEL dose is higher than 1000 mg/kg.     

Inhibitory effects of casticin on migration of eosinophil and expression of chemokines and adhesion molecules in A549 lung epithelial cells via NF-κB inactivation

Ethnopharmacological relevance

The fruits of Vitex rotundifolia L. have long been used for the treatment of inflammation of the respiratory tract in East Asia.

Aim

To determine if casticin, one of the constituents of Vitex rotundifolia L., has anti-allergic and anti-inflammatory effects in asthma.

Materials and methods

The in vitro anti-inflammatory activity of casticin was studied in A549 human type II-like epithelial lung cells using an eotaxin inhibition assay. Additionally, its effects on eotaxin, regulated on activation normal T cell expressed and secreted (RANTES), vascular cell adhesion molecule (VCAM)-1, and inter-cellular adhesion molecule (ICAM)-1 expression were investigated by real time-polymerase chain reaction (real time-PCR). The inhibition of nuclear factor κB (NF-κB) activity in the presence of casticin was determined by analyzing confocal microscopy images of fluorescence immunocytochemical analysis while the suppression of inhibitory κB (IκB)-α phosphorylation was studied using Western blot analysis. Finally, the inhibitory effect of casticin on eosinophil migration toward prestimulated A549 cell media was measured using the human eosinophilic leukemia cell line.

Results and discussion

Casticin significantly suppressed eotaxin production in cytokine activated A549 lung epithelial cells. Casticin also suppressed the mRNA expression levels of eotaxin, RANTES, VCAM-1, and ICAM-1, which subsequently contributed to the inhibition of eosinophil migration. Furthermore, casticin inhibited IκB-α phosphorylation and nuclear translocation of p65 in A549 cells.

Conclusion

Casiticin inhibited the eosinophil migration and activity of chemokines and adhesion molecules involved in the inflammatory process of asthma by suppressing the NF-κB pathway. These results suggest that casticin has the potential for use in the treatment of allergic asthma.