Interleukin-6 Mice Research Articles

Effect of different ways of ingesting orange essential oil on blood immune index and intestinal microflora in mice.

Studies have found that the addition of plant essential oils to feed had a positive effect on intestinal microflora and immunity in mice. However, the effect of different ways of ingestion of orange essential oil on mice has seldom been reported. In the present study, we investigated the effects of ingestion of orange essential oil by gavage, sniffing and feeding on intestinal microflora and immunity in mice. The results obtained showed that a low concentration of essential oil feeding significantly increased the spleen index of mice (P < 0.05). The effect of different ways of ingestion on the thymus index, immunoglobulin G and immunoglobulin M of mice was not significant (P > 0.05). High and medium concentrations of essential oil feeding increased the level of interleukin-2 in mice (P < 0.05). H+ K+ -ATPase activity was significantly increased in mice fed with gavage and different concentrations of essential oil feed compared to the control group (P < 0.05). The analysis of the results of the microflora in the cecum and colon of mice indicated that the medium concentration of essential oil feeding group and the sniffing group significantly changed the structure of the flora and increased the diversity of the intestinal microflora. All three essential oil ingestion methods increased the abundance of Bacteroidetes and Lactobacillus in the intestine of mice. Compared with gavage and feeding, sniffing had a significant effect on immunoglobulins in mice. All the three ingestion methods could affect the intestinal microflora of mice and increase the abundance of Lactobacillus. © 2022 Society of Chemical Industry.

Activation of GPR81 Aggravates Remote Organ Injury During Hepatic Ischemia-Reperfusion Injury

Hepatic ischemia-reperfusion injury (HIRI) is a serious situation with high morbidity and mortality, which is usually accompanied with hyperlactatemia due to impaired lactate clearance in liver. G-protein-coupled receptor 81 (GPR81) has recently been identified as the bioactive receptor of lactate. GPR81 is profoundly involved in the modulation of metabolism and inflammation, but its significance in HIRI remains unclear. The present study investigated the potential roles of GPR81 in HIRI by using the GPR81 agonist 3-chloro-5-hydroxybenzoic acid (CHBA). The results indicated that treatment with CHBA had no obvious effects on HIRI-induced histologic abnormalities and elevation of serum aspartate aminotransferase, alanine aminotransferase. However, CHBA significantly upregulated the serum level of tumor necrosis factor alpha and interleukin-6 in mice with HIRI. Administration of CHBA also exacerbated HIRI-induced histologic lesions in lung, increased the level of myeloperoxidase in lung tissue and the protein concentration in bronchoalveolar lavage fluid. In addition, the serum levels of brain natriuretic peptide and creatinine also increased in CHBA-treated mice. The results indicate that activation of GPR81 might aggravate HIRI-induced remote organ injury and result in serious outcomes.

Inflammatory cytokines analysis in C57BL/6 mouse skin after dermal exposure with chemical threat agent phosgene oxime

Phosgene Oxime (dichloroform oxime; CX), an urticant categorized as a vesicating agent, is a potential chemical threat agent. Its exposure causes rapid and painful dermal injury and systemic toxic effects. CX exposure can result in enzyme inactivation, corrosive injury, and cell death with rapid tissue destruction, and induce the recruitment of immune cells. However, the mechanism of CX‐induced skin toxicity and inflammation is not known. Studies in our lab have shown that CX exposure causes mast cell degranulation and release of inflammatory mediators like COX‐2, MMP‐9, myeloperoxidase, and an inflammatory response in the skin. In the present study, we analyzed the inflammatory cytokine profile in male and female C57BL/6 mouse skin following dermal CX exposure (neat CX for either 0.5 or 1.0 min using two 12 mm vapor caps on the dorsal skin at MRIGlobal). Cytokine array analyses showed an increase in the pro‐inflammatory cytokines IL‐6 (Interleukin 6) and TNF‐α (Tumor Necrosis Factor Alpha) in both male and female mice at 24h post CX‐exposure. KC (neutrophil chemoattractant), MCP‐1 (Monocyte chemoattractant), MCSF (Macrophage colony‐stimulating factor) and RANTES (T cell, monocyte attractant) increased in both sexes at day 3 post CX‐exposure, showing involvement of immune cells in the CX‐induced injury. IL‐13, an allergic inflammation and several diseases’ mediator was found to be increased at later time point in female mice and at 2 hours after 1.0 min CX exposure in male mice. IL‐4 and IL‐5 levels decreased at 24 h but were increased at day 3 post 0.5 min CX exposure. Altogether, our results show immune cell infiltration with changes in inflammatory cytokines. Similar Inflammatory cytokine changes from CX exposure were observed in male and female mice; however, slightly different patterns were evident in some cytokines in female mice that related to the observed difference in the inflammatory response and somewhat faster dermal wound healing. Further molecular analysis is underway to determine the signaling pathways involved in CX‐induced dermal inflammation and toxicity for identifying the therapeutic targets.

Does G protein‐coupled Estrogen Receptor 1 Contribute to Cisplatin‐induced Acute Kidney Injury?

Cisplatin (Cp) is an effective chemotherapeutic agent used to treat a wide variety of malignancies. Nephrotoxicity is the dose‐limiting side effect of Cp. Acute kidney injury (AKI) following Cp administration is multi‐factorial, involving cellular toxicity, inflammation and proximal tubular injury. Recent evidence points to renal protective actions of G protein‐coupled estrogen receptor 1 (GPER1) in nephropathies evoked by salt loading, hypertension, methotrexate and ischemia reperfusion. Additionally, GPER1 abundance increases after renal ischemia reperfusion injury. However, the involvement of GPER1 signaling in Cp‐induced nephrotoxicity remains unclear. Therefore, this study was designed to test whether genetic deletion of GPER1 exacerbates Cp‐induced AKI in male mice. We subjected 14‐16 weeks‐old male mice homozygous (homo), heterozygous (het) for GPER1 gene and wild‐type (WT) littermates to Cp or saline injections and assessed markers for renal injury on the third day after injections. Specifically, we measured serum creatinine (sCr), urea, neutrophil gelatinase‐associated lipocalin (NGAL), histological indexes of renal tubular injury and abundance of kidney injury molecule‐1 (KIM‐1) and NGAL in the kidney. We determined also serum levels of proinflammatory markers in saline and Cp‐treated mice. A rise sCr was evident in all Cp‐treated mice, compared to saline‐treated mice (WT: 1.72±0.24 vs. 0.12±0.01; het: 1.14±0.30 vs. 0.12±0.01; homo: 1.28±0.31 vs. 0.14±0.01 mg/dl, p&lt;0.0001, p=0.0149, p=0.0028, respectively, n=9‐13). Serum urea and NGAL levels followed the same pattern as sCr. Cp‐induced AKI resulted in comparable histological evidence of injury in all genotypes. Kidney KIM‐1 abundance was higher in saline‐treated homo mice, compared to corresponding WT mice (14.00±2.30 vs. 7.62±0.70 AU, respectively, p=0.0188, n=8, 13). Kidney KIM‐1 abundance was markedly upregulated in response to Cp in all groups, however, no genotypic differences were observed in kidney KIM‐1 in Cp‐treated mice. Cytokine panel assessment demonstrated increased circulating levels of interleukin‐2 (IL‐2), IL‐5, IL‐10 and tumor necrosis factor‐a in Cp‐treated mice. GPER1 deletion lowered plasma IL‐12p70 (p=0.0176). Given the protective role of heme oxygenase‐1 (HO‐1) in Cp‐mediated cell injury, we also investigated genotypic differences in renal HO‐1 abundance. WT and homo mice showed greater renal HO‐1 abundance in response to Cp. Interestingly, renal HO‐1 abundance was lower in Cp‐treated homo, compared to Cp‐treated WT mice (65.74±6.70 vs. 87.83±5.11 AU, respectively, p=0.0083, n=8, 13), despite the lack of genotypic differences in kidney HO‐1 levels in saline‐treated mice. Overall, we demonstrate that GPER1 homo, het male mice and WT littermates elicit comparable renal injury in response to Cp. We show also that GPER1 deletion diminishes HO‐1 induction in response Cp, suggesting that GPER1 may play a permissive role for HO‐1 cytoprotective actions in AKI. GPER1‐induced regulation of HO‐1 after Cp treatment may offer a novel therapeutic target for the treatment of AKI.

Meprin β Modulates Cellular Proliferation Through The IL‐6 Signaling Pathway In IRInduced Kidney Injury

Inflammation plays a central role in the progression of kidney injury induced by ischemia/reperfusion (IR). Interleukin‐6 (IL‐6) trans‐signaling has been shown to play a protective role by promoting repair processes in IR‐induced renal injury. Meprin metalloproteases have been implicated in inflammation‐induced kidney injury. Meprins proteolytically process the proinflammatory cytokine, IL‐6, causing IL‐6 inactivation. It was recently reported that meprins also cleave the IL‐6 receptor (IL‐6R). IL‐6 binds to its receptor (IL‐6R) in two ways, membrane bound (mbIL‐6R), activating the classic IL‐6 signaling pathway, or the soluble form (sIL‐6R), activating the IL‐6 trans‐signaling pathway. IL‐6 trans‐signaling induces proliferation through either MAPK/ERK or PI3K/AKT pathway or in crosstalk with AKT/ERK. We previously showed that meprin β modulates cellular survival (BCL‐2) through IL‐6/JAK/STAT signaling pathway in IRinduced kidney injury. However, it’s not known how meprin β modulation of the IL‐6 signaling pathway impacts the cellular proliferation in IR‐induced acute kidney injury. Previous studies have shown that IL‐6 binds to its receptor forming IL‐6/IL6R complex, which binds to the membranebound gp130 dimer. This leads to activation phosphorylation of ERK and AKT pathways, which in turn leads to induction of cellular proliferation. PCNA is a cellular proliferation marker that is induced through activation of the IL‐6 signaling pathway. The goal of the current study was to determine how meprin β modulation of the IL‐6 signaling pathway impacts downstream cellular proliferation in IR‐induced kidney injury. We used the unilateral IR as a model of renal inflammation in wild‐type (WT) and meprin β knockout (βKO) male mice, with the contralateral kidneys serving as controls. The mice were sacrificed at 96 h post‐IR, and kidney tissue processed for evaluation by RT‐PCR and immunohistochemistry (IHC). To determine staining intensity, the tissue sections were evaluated for IL‐6 and PCNA levels using light microscopy and imaged using Image J analysis Software. Statistical analysis of the optical density (OD) data utilized two‐way ANOVA. Our PCR data showed a significant increase in mRNA levels for IL‐6 and PCNA in WT and βKO mice (P ≤ 0.01) at 96 h‐post IR when compared to WT control kidneys. However, the baseline mRNA levels for PCNA were significantly higher in βKO (P ≤ 0.01) when compared to WT kidneys. Immunohistochemical data showed significant increases (P&lt;0.05) in IL‐6 and PCNA in select tubules in both genotypes at 96 h post‐IR when compared to control kidneys for each genotype. Data from immunofluorescence counterstaining of kidney tissues showed that the levels of IL‐6, and PCNA were higher in meprin β‐expressing proximal tubules (PTs), at 96 h post‐IR when compared to the distal kidney tubules (DTs), which lack meprins. High levels of IL‐6 were also present in the lumen of PTs and DTs from WT and βKO kidneys at 96 h post‐IR, suggesting increased release into filtrate and subsequently into urine. However, high levels of PCNA were present in the lumen of PTs only from both genotypes at 96 h post‐IR. In conclusion, our data shows that meprin β expression modulates cellular proliferation through the IL‐6 signaling pathway in IR‐induced kidney injury.

Recombinant Enterococcus faecium expressing porcine lactoferricin exerts bactericidal effects and protects against enterotoxigenic Escherichia coli in mice

Porcine lactoferricin functions as an antibacterial peptide as well as an antiviral, antifungal, tumor-growth inhibiting, and immune-enhancing agent. Herein, the optimized and non-optimized amino-acid coding sequences for porcine lactoferricin were cloned into pNZ8112 expression vector, generating the recombinant plasmids pNZ8112-pLFcin (pNZ8112 cin) and pNZ8112-pLFcin’ (pNZ8112 cin’), respectively. The plasmids were then electroporated into Enterococcus faecium cells, resulting in recombinant strains pNZ8112 cin/E. faecium and pNZ8112 cin’/E. faecium, respectively. Subsequent studies revealed that the recombinant strains exerted antibacterial effects against 12 types of pathogenic bacteria, with strongest activity against Staphylococcus aureus. The studies involving oral immunization of 4–6 weeks old female BALB/c mice with the two recombinant strains revealed an increased average daily weight gain, feed intake, and level of lactic acid bacteria and decreased feed-to-weight ratio. Furthermore, these effects were concomitant with a significant increase in the level of serum IgG, intestinal mucus sIgA, and interleukin-2 in immunized mice as compared to those in non-immunized mice. Moreover, the challenge experiment indicated that pre-immunization with the two strains could provide 83.3% protection against enterotoxigenic Escherichia coli in mice, which implicates the potential application of the recombinant E. faecium strains in the development of antibiotic-free domestic livestock feed additives.

Mechanism and experimental verification of "Polygoni Cuspidati Rhizoma et Radix-Ligustri Lucidi Fructus" combination in treatment of acute gouty arthritis based on network pharmacology

Based on network pharmacology, the mechanism of Polygoni Cuspidati Rhizoma et Radix-Ligustri Lucidi Fructus(PL) combination against acute gouty arthritis(AGA) was explored and preliminarily verified by animal experiment. The chemical components and corresponding targets of PL were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). The active components with oral bioavailability(OB)≥30% and drug-likeness(DL)≥0.18 were screened based on literature, and the related protein targets were collected. Then the protein targets were standardized with the help of UniProt database. The AGA-related targets were searched from GeneCards, NCBI, and DrugBank. The common targets of the disease and the medicinals were yielded by FunRich V3, and the protein-protein interaction(PPI) network was constructed to screen the key targets, followed by Gene Ontology(GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis of the key targets. Afterwards, some of the key targets were verified by sodium urate crystal-induced AGA mouse model. A total of 25 active components and 287 targets of PL, 811 targets of AGA, and 88 common targets were screened out. PPI network analysis showed that tumor necrosis factor(TNF), interleukin-6(IL-6), and interleukin-1β(IL-1β) may be the core targets of PL in the treatment of AGA. The key targets were mainly involved in 566 GO terms(P&lt;0.05), including multiple biological processes such as inflammatory response and immune response. Moreover, they were related to 116 KEGG pathways and these pathways were involved in inflammation and immunity, mainly including NOD-like receptor signaling pathway and TNF signaling pathway. Animal experiment confirmed that PL can alleviate ankle swelling, improve abnormal gait, and down-regulate the protein expression of TNF-α, IL-6, and IL-1β in AGA mice, indicating that PL can treat AGA through TNF-α, IL-6, and IL-1β and the feasibility of network pharmacology to predict drug targets. This study preliminarily discussed the key targets and biological signaling pathways involved in the treatment of AGA with PL combination, which reflected the multi-pathway and multi-target action characteristics of Chinese medicine. Moreover, this study laid a scientific basis for research on the treatment of AGA with PL combination, as well as the mechanism of action.

Comparative Study of Radiation-induced Lung Injury Model in Two Strains of Mice.

Radiation-induced lung injury (RILI) is a common complication of radiotherapy for thoracic tumor. Its incidence rate is as high as 20%. At present, there is no effective treatment in clinical practice. However, to study the mechanism of radiation-induced lung injury, we should first establish an appropriate animal model. In a series of scientific studies on RILI, mice are the animals most often chosen by researchers. However, there are few reports on which strain of mice is more suitable as a model of RILI. In this study, Kunming (KM) and C57BL/6 strains of mice were used as research objects to find the most suitable mice to replicate the RILI model. C57BL/6 mice and KM mice were exposed to irradiation at a dose of 20 Gy. The lung tissue of C57BL/6 mice exposed to radiation showed dilation and hyperemia of capillaries, infiltration of inflammatory cells, and thickening of alveolar septum, while the lung tissue of KM mice exposed to radiation was not as obvious as that of C57BL/6 mice. After irradiation, the expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the lung tissue of C57BL/6 mice was significantly increased, while the expression of IL-6 and TNF-α in KM mice was almost unchanged. These studies showed that C57BL/6 mice are more suitable for the model of radiation-induced lung injury because of sensitive inflammatory reaction and the pathological changes of lung tissue.

Resolution of Cisplatin-Induced Fatigue Does Not Require Endogenous Interleukin-10 in Mice

Resolution of Cisplatin-Induced Fatigue Does Not Require Endogenous Interleukin-10 in Mice

Aqueous and alcoholic extracts of Artemisia annua L. improved insulin resistance via decreasing TNF-alpha, IL-6 and free fatty acids in high-fat diet/streptozotocin-induced diabetic mice.

Objective:Type 2 diabetes mellitus (T2DM) is a metabolic disease that influences many people worldwide. Management of insulin resistance in T2DM without side effects of chemical drugs, is the ultimate goal of the medical community. Artemisia annua L. is used for the treatment of diabetes in folkloric medicine. The present study investigated the effects of aqueous and alcoholic extracts of A. annua (AA) on insulin resistance in high-fat diet/STZ-induced diabetic mice. Material and Methods: Mice were divided into groups including control with a normal diet, un-treated high-fat diet/streptozotocin-induced diabetic mice, and diabetic mice treated by oral administration of 100, 200, and 400 mg/kg body weight of water (hot and cold) and alcoholic extracts of AA. After four weeks of treatment with AA, blood sampling was carried out to measure factors involved in insulin resistance such as low-density lipoprotein/ High-density lipoprotein (LDL/HDL) ratio, free fatty acids, Tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and homeostasis model assessment of insulin resistance (HOMA-IR) as an index of insulin resistance. Results:The results showed that all AA extracts (100, 200, and 400 mg/kg) and metformin (250 mg/kg) significantly reduced the serum levels of free fatty acids, TNF-alpha, IL-6, LDL/HDL ratio, and HOMA-IR in diabetic mice compared to untreated diabetic mice (p<0.0001). Notably, the 400 mg/kg dose of cold-water extract was more effective than metformin in reduction of TNF-alpha and IL-6 (p<0.01 and p<0.05, respectively). Conclusion:These data illustrated that AA extracts attenuated insulin resistance by reducing the lipid profile and adipocytokines.

Neutrophil Gelatinase-Associated Lipocalin From Macrophages Plays a Critical Role in Renal Fibrosis Via the CCL5 (Chemokine Ligand 5)-Th2 Cells-IL4 (Interleukin 4) Pathway.

NGAL (neutrophil gelatinase-associated lipocalin; or lipocalin 2, Lcn2) is a novel mineralocorticoid target in the cardiovascular system. We showed that Lcn2 gene invalidation protects against proteinuria and renal injury upon mineralocorticoid excess and we hypothesized that NGAL produced from macrophages promotes the expression of chemoattractant molecules involved these renal lesions. The role of NGAL was analyzed using myeloid-specific (MΦ KO NGAL) Lcn2 knockout mice challenged with uni-nephrectomy, aldosterone, and salt (NAS) for 6 weeks. The role of the CCL5 (chemokine ligand 5) and IL4 (interleukin 4) in kidney fibrosis was studied by administration of the CCL5 receptor antagonist maraviroc or by injections of an anti-IL4 neutralizing antibody. In CTL mice, NAS increased the renal expression of extracellular matrix proteins, such as collagen I, αSMA, and fibronectin associated with interstitial fibrosis which were blunted in MΦ KO NGAL mice. The expression of CCL5 was blunted in sorted macrophages from MΦ KO NGAL mice challenged by NAS and in macrophages obtained from KO NGAL mice and challenged ex vivo with aldosterone and salt. The pharmacological blockade of the CCL5 receptor reduced renal fibrosis and the CD4+ Th cell infiltration induced by NAS. Neutralization of IL4 in NAS mice blunted kidney fibrosis and the overexpression of profibrotic proteins, such as collagen I, αSMA, and fibronectin. In conclusion, NGAL produced by macrophages plays a critical role in renal fibrosis and modulates the CCL5/IL4 pathway in mice exposed to mineralocorticoid excess.

HLA-Restriction of Human Treg Cells Is Not Required for Therapeutic Efficacy of Low-Dose IL-2 in Humanized Mice.

Regulatory T (Treg) cells are essential to maintain immune homeostasis in the intestine and Treg cell dysfunction is associated with several inflammatory and autoimmune disorders including inflammatory bowel disease (IBD). Efforts using low-dose (LD) interleukin-2 (IL-2) to expand autologous Treg cells show therapeutic efficacy for several inflammatory conditions. Whether LD IL-2 is an effective strategy for treating patients with IBD is unknown. Recently, we demonstrated that LD IL-2 was protective against experimental colitis in immune humanized mice in which human CD4+ T cells were restricted to human leukocyte antigen (HLA). Whether HLA restriction is required for human Treg cells to ameliorate colitis following LD IL-2 therapy has not been demonstrated. Here, we show that treatment with LD IL-2 reduced 2,4,6-trinitrobenzensulfonic acid (TNBS) colitis severity in NOD.PrkdcscidIl2rg-/- (NSG) mice reconstituted with human CD34+ hematopoietic stem cells. These data demonstrate the utility of standard immune humanized NSG mice as a pre-clinical model system to evaluate therapeutics targeting human Treg cells to treat IBD.

Protective effect of Lactobacillus plantarum YS3 on dextran sulfate sodium-induced colitis in C57BL/6J mice.

The protective effect of Lactobacillus plantarum YS3 (LP-YS3) on ulcerative colitis (UC) was assessed using a mouse model of dextran sodium sulfate (DSS)-induced colitis. Different concentrations of LP-YS4 were administered to the experimental mice by daily gavage. Several inflammatory and biochemical indices, such as interleukin-2 (IL-2), interleukin-10 (IL-10), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), glutathione (GSH), malondialdehyde (MDA), myeloperoxidase (MPO), and nitric oxide (NO), were examined in mouse serum and colon tissue. The mRNA and protein expression levels of c-Kit, CXC chemokine receptor type 2 (CXCR2), interleukin-8 (IL-8), and stem cell factor (SCF) in mouse colon tissue were assessed using Western blot and quantitative polymerase chain reaction (qPCR) assays. The findings indicated that LP-YS3 remarkably decreased the disease activity index (DAI) of UC mice (p<.05), inhibited colon length shortening induced by UC, and elevated the value of colon weight/length ratio. LP-YS3 could also markedly reduce (p<.05) the activities of MDA, MPO, and NO; while an increase in the GSH content in the colonic tissue of UC mice. Moreover, LP-YS3 remarkably increased (p<.05) the serum level of IL-2 in UC mice, while reduced those of IL-10, IL-6, IL-1β, TNF-α cytokines. qPCR data revealed that LP-YS3 could markedly upregulate the expression levels of c-Kit and SCF, while downregulate those of CXCR2 and IL-8 in the colonic tissue of UC mice (p<.05). LP-YS3 exerted an outstanding protective effect on DSS-induced colitis in C57BL/6J mice, especially at higher concentrations. PRACTICAL APPLICATIONS: Lactobacillus plantarum YS3 is a newly isolated and identified lactic acid bacteria. This study confirmed that L. plantarum YS3 can inhibit colitis and has good probiotic potential, which needs further development and utilization.

Buyang Huanwu Decoction Enhances Revascularization via Akt/GSK3β/NRF2 Pathway in Diabetic Hindlimb Ischemia.

Background Peripheral arterial disease (PAD) is a typical disease of atherosclerosis, most commonly influencing the lower extremities. In patients with PAD, revascularization remains a preferred treatment strategy. Buyang Huanwu decoction (BHD) is a popular Chinese herbal prescription which has showed effects of cardiovascular protection through conducting antioxidant, antiapoptotic, and anti-inflammatory effects. Here, we intend to study the effect of BHD on promoting revascularization via the Akt/GSK3β/NRF2 pathway in diabetic hindlimb ischemia (HLI) model of mice. Materials and Methods All db/db mice (n = 60) were randomly divided into 6 groups by table of random number. (1) Sham group (N = 10): 7-0 suture thread passed through the underneath of the femoral artery and vein without occlusion. The remaining 5 groups were treated differently on the basis of the HLI (the femoral artery and vein from the inguinal ligament to the knee joint were transected and the vascular stump was ligated with 7-0 silk sutures) model: (2) HLI+NS group (N = 15): 0.2 ml NS was gavaged daily for 3 days before modeling and 14 days after occlusion; (3) HLI+BHD group (N = 15): 0.2 ml BHD (20 g/kg/day) was gavaged daily for 3 days before modeling and 14 days after occlusion; (4) HLI+BHD+sh-NC group (N = 8): local injection of adenovirus vector carrying the nonsense shRNA (Ad-GFP) in the hindlimbs of mice before treatment; (5) HLI+BHD+sh-NRF2 group (N = 8): knockdown of NRF2 in the hindlimbs of mice by local intramuscular injection of adenovirus vector carrying NRF2 shRNA (Ad-NRF2-shRNA) before treatment; and (6) HLI+BHD+LY294002 group (N = 4): intravenous injection of LY294002 (1.5 mg/kg) once a day for 14 days on the basis of the HLI+BHD group. Laser Doppler examination, vascular cast, and immunofluorescence staining were applied to detect the revascularization of lower limbs in mice. Western blot analysis was used to detect the expression of vascular endothelial growth factor (VEGF), interleukin-1beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor- (TNF-) α, heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone-1 (NQO-1), catalase (CAT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphorylated protein kinase B (p-AKT), and phosphorylated glycogen synthase kinase-3 beta (p-GSK3β). HE staining was used to assess the level of muscle tissue damage and inflammation in the lower extremities. Local multipoint injection of Ad-NRF2-shRNA was used to knock down NRF2, and qPCR was applied to detect the mRNA level of NRF2. The blood glucose, triglyceride, cholesterol, MDA, and SOD levels of mice were tested using corresponding kits. The SPSS 20.0 software and GraphPad Prism 6.05 were used to do all statistics. Values of P < 0.05 were considered as statistically significant. Results and Conclusions. BHD could enhance the revascularization of lower limbs in HLI mice, while BHD has no effect on blood glucose and lipid level in db/db mice (P > 0.05). BHD could elevate the protein expression of VEGF, HO-1, NQO-1, and CAT (P < 0.05) and decrease the expression of IL-1β, IL-6, and TNF-α (P < 0.05) in HLI mice. Meanwhile, BHD could activate NRF2 and promote the phosphorylation of AKT/GSK3β during revascularization (P < 0.05). In contrast, knockdown of NRF2 impaired the protective effects of BHD on HLI (P < 0.05). LY294002 inhibited the upregulation of NRF2 activated by BHD through inhibiting the phosphorylation of the AKT/GSK3β pathway (P < 0.05). The present study demonstrated that BHD could promote revascularization on db/db mice with HLI through targeting antioxidation, anti-inflammation, and angiogenesis via the AKT/GSK3β/NRF2 pathway.

BTB and CNC homology 1 inhibition ameliorates fibrosis and inflammation via blocking ERK pathway in pulmonary fibrosis

Objective Patients with idiopathic pulmonary fibrosis (IPF) are still suffering from unfavorable survival. BTB and CNC homology1 (Bach1) is a regulator of oxidative stress and participates in the pathogenesis of multiple lung diseases. Thus, this study aimed to determine the effect of Bach1 knockdown on fibrosis and inflammation in pulmonary fibrosis (PF) mice and cell models. Methods Bleomycin induced PF mice were constructed and treated with Bach1 siRNA adenovirus (BLM + Bach1 siRNA group), control siRNA adenovirus (BLM + Control siRNA group) or normal saline (BLM group), then lung tissues were collected for Bach1 expression detection, H&E staining and Masson’s trichrome staining. Afterwards, collagen type I alpha 1 chain (COL1A1) and interleukin-6 (IL-6) expressions in serum and bronchoalveolar lavage fluid (BALF) were examined. Subsequently, mouse lung fibroblasts (MLFs) were collected from PF mice and treated with TGF-β1 to construct PF cell model, which was treated with Bach1 siRNA adenovirus (TGF-β1 + Bach1 siRNA group) and MAP kinase (ERK) inhibitor U0126 alone (TGF-β1 + U0126 group) or in combination (TGF-β1 + U0126 + Bach1 siRNA group), then alpha-smooth muscle actin (α-SMA), fibronectin 1 (Fn1), COL1A1, IL-6 expressions and cell viability were detected. Results Lung tissue Bach1 mRNA and protein expressions were upregulated in PF mice compared to control mice. Bach1 knockdown reduced lung fibrosis (displayed by Masson’s trichrome staining) and inflammation (displayed by H&E staining), then downregulated serum and BALF expressions of COL1A1 and IL-6 in PF mice. Subsequently, in PF cell model, Bach1 knockdown blocked ERK pathway, but did not affect Smads, c-Jun N-terminal kinase (JNK) or thymoma viral proto-oncogene 1 (Akt) pathways. Further experiments revealed that Bach1 knockdown repressed cell viability, α-SMA, Fn1, IL-6 and COL1A1 expressions in PF cell model, then ERK inhibition by U0126 enhanced these effects. Conclusions Bach1 is involved in the PF pathogenesis via modulating ERK signaling pathway.

Inhibition of inflammatory signaling in Pax5 mutant cells mitigates B-cell leukemogenesis

PAX5 is one of the most frequently mutated genes in B-cell acute lymphoblastic leukemia (B-ALL), and children with inherited preleukemic PAX5 mutations are at a higher risk of developing the disease. Abnormal profiles of inflammatory markers have been detected in neonatal blood spot samples of children who later developed B-ALL. However, how inflammatory signals contribute to B-ALL development is unclear. Here, we demonstrate that Pax5 heterozygosis, in the presence of infections, results in the enhanced production of the inflammatory cytokine interleukin-6 (IL-6), which appears to act in an autocrine fashion to promote leukemia growth. Furthermore, in vivo genetic downregulation of IL-6 in these Pax5 heterozygous mice retards B-cell leukemogenesis, and in vivo pharmacologic inhibition of IL-6 with a neutralizing antibody in Pax5 mutant mice with B-ALL clears leukemic cells. Additionally, this novel IL–6 signaling paradigm identified in mice was also substantiated in humans. Altogether, our studies establish aberrant IL6 expression caused by Pax5 loss as a hallmark of Pax5-dependent B-ALL and the IL6 as a therapeutic vulnerability for B-ALL characterized by PAX5 loss.

Synergistic Effect of Endurance Training Combined with Curcumin on Intratumoral Expression of Interleukin-4 (Il4) and Stat-6 in Female Mice with Breast Cancer

Synergistic Effect of Endurance Training Combined with Curcumin on Intratumoral Expression of Interleukin-4 (Il4) and Stat-6 in Female Mice with Breast Cancer

Anti-Inflammatory Activity of Chitosan and 5-Amino Salicylic Acid Combinations in Experimental Colitis.

Chitosan is used in various drug delivery approaches as a pharmaceutical excipient. Although its potential as an immunomodulatory agent has been reported, its use in this capacity has not been fully explored. The efficacy of chitosan as an active pharmacological agent, particularly in anti-inflammatory therapy in inflammatory bowel diseases (IBD), was investigated in this study. The potential impact of the molecular weight (MW) and degree of deacetylation (DD) of chitosan was investigated together with 5-amino salicylic acid (5-ASA) for its efficacy in a combination anti-inflammatory therapy in murine experimental colitis. Such a combination would potentially be developed into novel dual strategies whereby chitosan acts as a mucoadhesive excipient as well as provide an additional anti-inflammatory benefit. Chitosan grades with different MW and DD were administered intrarectally alone or in combination with 5-ASA to colitis mice for 3 days. Myeloperoxidase (MPO) and alkaline phosphatase (ALP) activity and tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and nuclear factor kappa-B (NF-κB) levels were assessed from the colon. Intrarectal treatment of colitis with 30 mg/kg chitosan alone and with 30 mg/kg 5-ASA for 3 days led to a significant decrease in MPO, ALP, TNF-α, IL-6, IL-1β and NF-κB in colitis mice compared to untreated mice. Surprisingly, the efficacy of chitosan as an anti-inflammatory polymer was relatively independent from its structural properties, namely DD and MW. However, combinations of chitosan with 5-ASA showed a significant pharmacological improvement, whereby the additive anti-inflammatory efficacy observed shows the possibility of finetuning chitosan by combining it with anti-inflammatory agents to optimize its anti-inflammatory potential.

Innate immune activation and antitumor effects of Lactobacillus-fermented Sparassis crispa extract in mice

• Lacto -fermented Sparassis crispa (LFSC) extract has high β(1–3)-glucan content. • LFSC’s antitumor activity has a different mechanism from that of fluorouracil. • NK cells, neutrophils, and M1 macrophages increased after LFSC oral treatment. • LFSC promoted apoptosis and reactive oxygen species generation in tumors. Sparassis crispa (SC), a mushroom abundantly containing β(1–3)-glucan, is known to activate the innate immunity. Lactic acid bacteria are also known to activate the immunity. Here, we investigated the effects of SC and Lactobacillus -fermented SC (LFSC) on the innate immunity and tumor growth in mice. LFSC treatment promoted the higher accumulation of innate immune cells than the SC treatment. However, the antitumor activity of the LFSC was lower than that of the fluorouracil (5-FU). Furthermore, LFSC significantly reduced the serum levels of interleukin-6 in mice and enhanced the apoptosis rate and the production of reactive oxygen species in mouse tumor tissues. These effects were not observed in 5-FU-treated mice. LFSC also significantly enhanced the phagocytic activity of and the mRNA expression of thrombotic/inflammatory factors in human monocytes. These findings suggest that LFSC significantly enhances the innate immunity and exhibits antitumor effects by a mechanism different from that of 5-FU.

Ganoderic acid B attenuates LPS-induced lung injury

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a serious respiratory disease, the mechanism is unclear. This paper revealed the mechanism of ganoderic acid B (BB) on lipopolysaccharide-induced pneumonia in mice. Pneumonia model was induced by LPS in mice and A549 cells. Lung dry/wet weight (W/D) and myeloperoxidase (MPO) activity in lung were examined. Lung histopathological changes was observed by HE staining. Superoxide dismutase (SOD), malondialdehyde (MDA) and proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in mice and A549 cells were detected. Rho/NF-κB pathway in mice and A549 cells were examined by Western Blot. BB significantly reduced W/D and MPO activity, restored lung histopathological changes. BB also increased SOD, decreased MDA, TNF-α, IL-1β and IL-6 in mice and A549 cells. In addition, BB inhibited Rho/NF-κB pathway in mice and A549 cells. BB has protective effect on LPS-induced pneumonia in mice, and its mechanism is related to the regulation of Rho/NF-κB signaling pathway.