Selective targeting of the TLR2/MyD88/NF-κB pathway reduces α-synuclein spreading in vitro and in vivo

Pathways to control the spreading of α-synuclein (α-syn) and associated neuropathology in Parkinson’s disease (PD), multiple system atrophy (MSA) and dementia with Lewy bodies (DLB) are unclear. Here, we show that preformed α-syn fibrils (PFF) increase the association between TLR2 and MyD88, resulting in microglial activation. The TLR2-interaction domain of MyD88 (wtTIDM) peptide-mediated selective inhibition of TLR2 reduces PFF-induced microglial inflammation in vitro. In PFF-seeded A53T mice, the nasal administration of the wtTIDM peptide, NEMO-binding domain (wtNBD) peptide, or genetic deletion of TLR2 reduces glial inflammation, decreases α-syn spreading, and protects dopaminergic neurons by inhibiting NF-κB. In summary, α-syn spreading depends on the TLR2/MyD88/NF-κB pathway and it can be reduced by nasal delivery of wtTIDM and wtNBD peptides.

NEMO-Binding Domain Peptide ameliorates alveolar hypercoagulation and fibrinolytic inhibition in lipopolysaccharide-induced acute respiratory distress syndrome via NF-κB signaling pathway in mice

Background It was confirmed that alveolar hypercoagulation and fibrinolytic inhibition were associated with refractory hypoxemia in acute respiratory distress syndrome (ARDS), and NF-κB pathway was involved in this process. The purpose of the present study is to explore the effects and relevant mechanisms exerted by NEMO-binding domain peptide (NBDP) to alleviate alveolar hypercoagulation and fibrinolytic inhibition aroused by lipopolysaccharide in ARDS mice. Materials and Methods Adult male BALB/c mice inhaled lipopolysaccharide (LPS, mg/L) to induce ARDS. 30 minutes before LPS administration, we treated the mice with increasing concentrations of intratracheally inhaled NBDP or saline aerosol. Six hours after LPS treatment, bronchoalveolar lavage fluids (BALF) were collected and then all mice were executed. We checked coagulation and fibrinolysis associated factors in lung tissues and in BALF as well. We simultaneously observed the activation of NF-κB signaling pathway as well. Results NBDP pretreatment dose-dependently inhibited either the expressions of tissue factor (TF) and plasminogen activator inhibitor (PAI) 1 in lung tissues or the secretions of TF, PAI-1, thrombin-antithrombin complex (TAT) and promoted activated protein C (APC) secretion in BALF induced by LPS. LPS-induced high expression of pulmonary procollagen peptide type Ⅲ (PⅢP) was also declined by NBDP pretreatment in dose-dependent manner. Western blotting showed that NBDP pretreatment obviously attenuated LPS-induced IKKα/β, Iκα and NF-κB p65 activation. LPS-induced p65 DNA binding activity was inhibited by NBDP pretreatment either. We also noticed NBDP protected mice against LPS-induced lung injury in a dose-dependent manner. Conclusions Our experimental findings demonstrate that NBDP dose-dependently ameliorated LPS-induced alveolar hypercoagulation and fibrinolytic inhibition through inhibiting NF-κB signaling pathway. NBDP is expected to be a new therapeutic target to correct the abnormalities of alveolar coagulation and fibrinolysis in ARDS.

NEMO-Binding Domain Peptide Ameliorates Alveolar Hypercoagulation and Fibrinolytic Inhibition in Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome via NF-κB Signaling Pathway in Mice

Background It was confirmed that alveolar hypercoagulation and fibrinolytic inhibition were associated with refractory hypoxemia in acute respiratory distress syndrome (ARDS), and NF-κB pathway was involved in this process. The purpose of the present study is to explore the effects and relevant mechanisms exerted by NEMO-binding domain peptide (NBDP) to alleviate alveolar hypercoagulation and fibrinolytic inhibition aroused by lipopolysaccharide in ARDS mice. Materials and Methods Adult male BALB/c mice inhaled lipopolysaccharide (LPS, mg/L) to induce ARDS. 30 minutes before LPS administration, we treated the mice with increasing concentrations of intratracheally inhaled NBDP or saline aerosol. Six hours after LPS treatment, bronchoalveolar lavage fluids (BALF) were collected and then all mice were executed. We checked coagulation and fibrinolysis associated factors in lung tissues and in BALF as well. We simultaneously observed the activation of NF-κB signaling pathway as well. Results NBDP pretreatment dose-dependently inhibited either the expressions of tissue factor (TF) and plasminogen activator inhibitor (PAI) 1 in lung tissues or the secretions of TF, PAI-1, thrombin-antithrombin complex (TAT) and promoted activated protein C (APC) secretion in BALF induced by LPS. LPS-induced high expression of pulmonary procollagen peptide type Ⅲ (PⅢP) was also declined by NBDP pretreatment in dose-dependent manner. Western blotting showed that NBDP pretreatment obviously attenuated LPS-induced IKKα/β, Iκα and NF-κB p65 activation. LPS-induced p65 DNA binding activity was inhibited by NBDP pretreatment either. We also noticed NBDP protected mice against LPS-induced lung injury in a dose-dependent manner.Conclusions Our experimental findings demonstrate that NBDP dose-dependently ameliorated LPS-induced alveolar hypercoagulation and fibrinolytic inhibition through inhibiting NF-κB signaling pathway. NBDP is expected to be a new therapeutic target to correct the abnormalities of alveolar coagulation and fibrinolysis in ARDS.

Targeting NF-κB by the Cell-Permeable NEMO-Binding Domain Peptide Improves Albuminuria and Renal Lesions in an Experimental Model of Type 2 Diabetic Nephropathy

Diabetic nephropathy (DN) is a multifactorial disease characterized by hyperglycemia and close interaction of hemodynamic, metabolic and inflammatory factors. Nuclear factor-κB (NF-κB) is a principal matchmaker linking hyperglycemia and inflammation. The present work investigates the cell-permeable peptide containing the inhibitor of kappa B kinase γ (IKKγ)/NF-κB essential modulator (NEMO)-binding domain (NBD) as therapeutic option to modulate inflammation in a preclinical model of type 2 diabetes (T2D) with DN. Black and tan, brachyuric obese/obese mice were randomized into 4 interventions groups: Active NBD peptide (10 and 6 µg/g body weight); Inactive mutant peptide (10 µg/g); and vehicle control. In vivo/ex vivo fluorescence imaging revealed efficient delivery of NBD peptide, systemic biodistribution and selective renal metabolization. In vivo administration of active NBD peptide improved albuminuria (>40% reduction on average) and kidney damage, decreased podocyte loss and basement membrane thickness, and modulated the expression of proinflammatory and oxidative stress markers. In vitro, NBD blocked IKK-mediated NF-κB induction and target gene expression in mesangial cells exposed to diabetic-like milieu. These results constitute the first nephroprotective effect of NBD peptide in a T2D mouse model that recapitulates the kidney lesions observed in DN patients. Targeting IKK-dependent NF-κB activation could be a therapeutic strategy to combat kidney inflammation in DN.

Efficacy of NEMO-binding domain peptide used to treat experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus: an in-vivo study

Purpose Treatment of chronic osteomyelitis (bone infection) remains a clinical challenge. Our previous study had demonstrated that NEMO-binding domain (NBD) peptide effectively ameliorates the inhibition of osteoblast differentiation by TNF-α in vitro. In this work, NBD peptide was evaluated in vivo for treating chronic osteomyelitis induced by methicillin-resistant Staphylococcus aureus (MRSA) in a rabbit model. Methods Tibial osteomyelitis was induced in 50 New Zealand white rabbits by tibial canal inoculation of MRSA strain. After 3 weeks, 45 rabbits with osteomyelitis were randomly divided into four groups that correspondingly received the following interventions: 1) Control group (9 rabbits, no treatment); 2) Van group (12 rabbits, debridement and parenteral treatment with vancomycin); 3) NBD + Van group (12 rabbits, debridement and local NBD peptide injection, plus parenteral treatment with vancomycin); 4) NBD group (12 rabbits, debridement and local NBD peptide injection). Blood samples were collected weekly for the measurement of leucocyte count, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels. The rabbits in all four groups were sacrificed 6 weeks after debridement; the anti-infective efficacy was evaluated by radiological, histological, and microbiological examination, and promotion of bone remodeling was quantified by micro-CT using the newly formed bone. Results Except two rabbits in the Control group and one in the NBD group that died from severe infection before the end point, the remaining 42 animals (7, 12, 12, 11 in the Control, Van, NBD + Van, and NBD group respectively) were sacrificed 6 weeks after debridement. In general, there was no significant difference in the leucocyte count, and ESR and CRP levels, although there were fluctuations throughout the follow-up period after debridement. MRSA was still detectable in bone tissue samples of all animals. Interestingly, treatment with NBD peptide plus vancomycin significantly reduced radiological and histological severity scores compared to that in other groups. The best therapeutic efficacy in bone defect repair was observed in the NBD peptide + Van group. Conclusions In a model of osteomyelitis induced by MRSA, despite the failure in demonstrating antibacterial effectiveness of NBD peptide in vivo, the results suggest antibiotics in conjunction with NBD peptide to possibly have promising therapeutic potential in osteomyelitis.