Yanan Li

Bio: Yanan Li is an academic researcher from China Agricultural University. The author has contributed to research in topics: Inflammasome & Phosphorylation. The author has an hindex of 1, co-authored 5 publications receiving 2 citations.

Lactobacillus rhamnosus GR-1 Prevents Escherichia coli-Induced Apoptosis Through PINK1/Parkin-Mediated Mitophagy in Bovine Mastitis

Abstract: Escherichia coli is one of the most important pathogens that cause clinical mastitis in dairy cattle worldwide and lead to severe economic losses. Antibiotics are often used to treat this inflammatory disease; however, antimicrobial resistance and environmental pollution cannot be ignored. Probiotic is the best alternative; however, its mechanisms of action to prevent mastitis remain unclear. Moreover, the role of probiotics in regulating mitophagy, a selective autophagy that maintains mitochondrial quality, needs to be explored. E. coli infection induced NOD-like receptor family member pyrin domain-containing protein 3 (NLRP3) inflammasome assembly, Caspase-1 activation, and apoptosis in MAC-T cells. Infection also resulted in mitochondrial damage and subsequent increase in reactive oxygen species (ROS) production. Moreover, inhibition of ROS release by scavenger N-acetyl-L-cysteine (NAC) abrogated the importance of ROS in NLRP3 assembly and apoptosis in MAC-T cells. Pretreatment with Lactobacillus rhamnosus GR-1 (LGR-1), a probiotic, alleviated E. coli-induced NLRP3 inflammasome activation and apoptosis via ROS inhibition. Besides, E. coli infection inhibited mitophagy while LGR-1 pretreatment augmented PINK1/Parkin-mediated mitophagy activation, which further blocked ROS generation. To explore the effect of LGR-1 in vivo, a mouse mastitis model was established. The results showed that LGR-1 pretreatment had preventive and protective effects on E. coli induced mastitis, and could reduce cytokines levels such as IL-1β and TNF-α. In accordance with the results in vitro, E. coli can inhibit mitophagy and activate NLRP3 inflammasome and apoptosis, while LGR-1 can weaken the effect of E. coli. Taken together, our data indicated that LGR-1 pretreatment induced PINK1/Parkin-mediated mitophagy that eliminated damaged mitochondria and reduced ROS production and NLRP3 inflammasome activation, which subsequently decreased E. coli-induced apoptosis. To conclude, our study suggests that therapeutic strategies aiming at the upregulation of mitophagy under E. coli-induced mastitis may preserve mitochondrial function and provide theoretical support for the application of probiotics in bovine mastitis.

Lactobacillus johnsonii L531 Alleviates the Damage Caused by Salmonella Typhimurium via Inhibiting TLR4, NF-κB, and NLRP3 Inflammasome Signaling Pathways.

Abstract: Salmonella Typhimurium (S. Typhimurium) is an aggressive zoonotic pathogen that causes enteritis and diarrhea. Antibiotic therapy is still the primary method at present. However, the increasing emergence of multi-drug resistant bacteria weakens the therapeutic efficacy of antibiotics. Probiotics have been widely studied as an alternative antibiotic therapy. In this study, we established an IPEC-J2 cell model of S. Typhimurium infection, aiming to determine the protective effect of Lactobacillus johnsonii L531 (L. johnsonii L531) on S. Typhimurium infection. As our data showed, S. Typhimurium infection resulted in a robust inflammatory response demonstrated by promoted protein levels of the inflammatory-related pathway (TLR4, MyD88, p-IκBα, and p-p65), increased cytokine levels of IL-6, IL-1β, IL-18, and TNF-α, and activated the NLRP3 inflammasome via promoting its assembly. However, L. johnsonii L531 pre-incubation inhibited the activation of the above inflammatory signaling pathways and reduced the expression levels of pro-inflammatory cytokines. In addition, L. johnsonii L531 alleviated the damage of S. Typhimurium to tight junctions ZO-1, Occludin, and Claudin-1. In summary, our findings suggested that L. johnsonii L531 alleviated S. Typhimurium-induced tight junction injury by inhibiting the TLR4/NF-κB/NLRP3 inflammasome signaling pathway.

Exopolysaccharides from Bifidobacterium animalis Ameliorate Escherichia coli-Induced IPEC-J2 Cell Damage via Inhibiting Apoptosis and Restoring Autophagy

Abstract: Enteropathogenic Escherichia coli (EPEC) is a common zoonotic pathogen that causes acute infectious diarrhea. Probiotics like Bifidobacterium are known to help prevent pathogen infections. The protective effects of Bifidobacterium are closely associated with its secretory products exopolysaccharides (EPS). We explored the effects of the EPS from Bifidobacterium animalis subsp. lactis (B. lactis) on ameliorating the damage of an intestinal porcine epithelial cell line (IPEC-J2) during EPEC infection. Pretreatment with EPS alleviated EPEC-induced apoptosis through the restoration of cell morphology and the downregulation of protein expressions of cleaved-caspase 8, cleaved-caspase 3, and cleaved-PARP. EPS-mediated remission of apoptosis significantly improved cell viability during EPEC infection. EPEC infection also resulted in impaired autophagy, as demonstrated by decreased expressions of autophagy-related proteins Beclin 1, ATG5, and microtubule-binding protein light chain-3B (LC3B) and the increased expression of p62 through western blot analysis. However, EPS reversed these effects which indicated that EPS promoted autophagosome formation. Furthermore, EPS prevented the lysosome damage induced by EPEC as it enhanced lysosomal acidification and raised lysosome-associated protein levels, thus promoted autophagosome degradation. Our findings suggest that the amelioration of EPEC-induced cell damages by EPS is associated with the limitation of detrimental apoptosis and the promotion of autophagy flux.

Salmonella Infantis Delays the Death of Infected Epithelial Cells to Aggravate Bacterial Load by Intermittent Phosphorylation of Akt With SopB

Abstract: Salmonella Infantis has emerged as a major clinical pathogen causing gastroenteritis worldwide in recent years. As an intracellular pathogen, Salmonella has evolved to manipulate and benefit from the cell death signaling pathway. In this study, we discovered that S. Infantis inhibited apoptosis of infected Caco-2 cells by phosphorylating Akt. Notably, Akt phosphorylation was observed in a discontinuous manner: immediately 0.5 h after the invasion, then before peak cytosolic replication. Single-cell analysis revealed that the second phase was only induced by cytosolic hyper-replicating bacteria at 3-4 hpi. Next, Akt-mediated apoptosis inhibition was found to be initiated by Salmonella SopB. Furthermore, Akt phosphorylation increased mitochondrial localization of Bcl-2 to prevent Bax oligomerization on the mitochondrial membrane, maintaining the mitochondrial network homeostasis to resist apoptosis. In addition, S. Infantis induced pyroptosis, as evidenced by increased caspase-1 (p10) and GSDMS-N levels. In contrast, cells infected with the ΔSopB strain displayed faster but less severe pyroptosis and had a less bacterial load. The results indicated that S. Infantis SopB mediated Akt phosphorylation delayed pyroptosis, but aggravated its severity. The wild-type strain also caused more severe diarrhea and intestinal inflammatory damage than the ΔSopB strain in mice. These findings revealed that S. Infantis delayed the cells death by intermittent activation of Akt, allowing sufficient time for replication, thereby causing more severe inflammation.

Lactobacillus Rhamnosus GR-1 Ameliorates Trueperella Pyogenes–Induced Barrier Dysfunction of Bovine Endometrial Epithelial Cells

Abstract: Trueperella pyogenes is a common opportunistic pathogen which is one of the main causes of postpartum endometritis in dairy cows. As a substitute for antibiotics, the probiotic Lactobacillus rhamnosus GR-1 has been used in a wide range of clinical treatments. Our experiments were designed to establish a model of anti-damage which LGR-1 was used to protect bovine endometrial epithelial cells (BEECs) from inflammatory damage and cell destruction caused by T. pyogenes. Increased expression of NLRP3 inflammasomes and cytokines was observed following T. pyogenes challenge, but this increase was relieved by LGR-1 pretreatment. Immunofluorescence and Western blot analyses revealed that T. pyogenes infection also results in the damage of tight junction proteins in BEECs. The expression levels of Claudin-1, Occludin, and ZO-1 were decreased in cells only infected with T. pyogenes but not in cells pretreated with LGR-1. Moreover, the detection of the anti-apoptotic protein Bcl-2 and apoptotic proteins BAX, cytochrome c, as well as the activating effector caspase-3 revealed that T. pyogenes induced apoptosis of BEECs, which was also confirmed by DAPI staining to observe the morphological changes of the nuclei of cell apoptosis and by TUNEL staining to locate the cells undergoing apoptosis. Our data indicate that LGR-1 ameliorates the T. pyogenes–induced barrier dysfunction of BEECs and pre-application of LGR-1 could be an effective strategy for controlling T. pyogenes infection.

Antimicrobial resistance and presence of virulence factor genes in Arcanobacterium pyogenes isolated from the uterus of postpartum dairy cows

Abstract: Arcanobacterium pyogenes is considered the most significant bacterium involved in the pathogenesis of metritis in cattle. Infections caused by antimicrobial-resistant bacteria are a great challenge in both human and veterinary medicine. The purpose of this study was to present an overview of antimicrobial resistance in A. pyogenes isolated from the uteruses of postpartum Holstein dairy cows and to identify virulence factors. Seventy-two A. pyogenes isolates were phenotypically characterized for antimicrobial resistance to amoxicillin, ampicillin, ceftiofur, chloramphenicol, florfenicol, oxytetracycline, penicillin, spectinomycin, streptomycin and tetracycline by the broth microdilution method. Presence of virulence factor genes of A. pyogenes was investigated. Isolates exhibited resistance to all antimicrobial agents tested; high levels of resistance were found to amoxicillin (56.9%); ampicillin (86.1%), chloramphenicol (100%), florfenicol (59.7%), oxytetracycline (54.2%), penicillin (86.1%) and tetracycline (50%). Of all isolates, 69 (95.8%) were resistant to at least 2 of the antimicrobial agents tested and multidrug resistance (resistant to at least 3 antimicrobials) was observed in 64 (88.9%) of the A. pyogenes isolates. The major multidrug resistance profile was found for chloramphenicol-ampicillin-penicillin-florfenicol-amoxicillin-tetracycline, which was observed in 21 (29.2%) multidrug resistant isolates. No isolate was resistant to all nine antimicrobial agents tested but four isolates (5.6%) were resistant to eight antimicrobials. The information highlights the need for prudent use of specific antimicrobial agents. All four virulence factor genes occurred in isolates from normal puerperium and clinical metritis; however, the fimA gene was present in significantly higher frequency in isolates from metritis cows.

Modulatory Properties of Food and Nutraceutical Components Targeting NLRP3 Inflammasome Activation

Abstract: Inflammasomes are key intracellular multimeric proteins able to initiate the cellular inflammatory signaling pathway. NLRP3 inflammasome represents one of the main protein complexes involved in the development of inflammatory events, and its activity has been largely demonstrated to be connected with inflammatory or autoinflammatory disorders, including diabetes, gouty arthritis, liver fibrosis, Alzheimer’s disease, respiratory syndromes, atherosclerosis, and cancer initiation. In recent years, it has been demonstrated how dietary intake and nutritional status represent important environmental elements that can modulate metabolic inflammation, since food matrices are an important source of several bioactive compounds. In this review, an updated status of knowledge regarding food bioactive compounds as NLRP3 inflammasome modulators is discussed. Several chemical classes, namely polyphenols, organosulfurs, terpenes, fatty acids, proteins, amino acids, saponins, sterols, polysaccharides, carotenoids, vitamins, and probiotics, have been shown to possess NLRP3 inflammasome-modulating activity through in vitro and in vivo assays, mainly demonstrating an anti-NLRP3 inflammasome activity. Plant foods are particularly rich in important bioactive compounds, each of them can have different effects on the pathway of inflammatory response, confirming the importance of the nutritional pattern (food model) as a whole rather than any single nutrient or functional compound.

Probiotic Bacillus subtilis LF11 Protects Intestinal Epithelium Against Salmonella Infection

Abstract: Enteric diseases caused by Salmonella are prevalent in poultry farming. With the forbiddance of antibiotics in feedstuff industry, Bacillus subtilis (B. subtilis) preparation as antibiotic alternatives against Salmonella infection has gained increasing attention recently. However, the protection modes of B. subtilis against Salmonella infection in broilers are strain-specific. In this study, probiotic B. subtilis LF11 significantly reduced diarrhea and mortality of broilers caused by Salmonella braenderup (S. braenderup) in spite of no inhibition effect on it in vitro. Here, the intestinal epithelial cells NCM460 were incubated to explore the protection of B. subtilis LF11 on intestinal epithelium against Salmonella. The results revealed that B. subtilis LF11 showed obvious exclusion activity with the decrease of adhesion and invasion of S. braenderup to NCM460 cells, accordingly with the increase of NCM460 cell survival compared with S. braenderup challenge alone. Meanwhile, RT-PCR and Western blot proved that the gene transcription and expression levels of four tight junction proteins in NCM 460 cells were upregulated, which was further confirmed by immunofluorescence observation. Besides, B. subtilis LF11 downregulated the gene transcription levels of the proinflammatory cytokines IL-6, IL-8, and TNF-α induced by S. braenderup H9812. ELISA analysis also verified that B. subtilis LF11 reduced the IL-8 production significantly. In general, B. subtilis LF11 has the ability to protect the intestinal epithelium against Salmonella infection by reducing the Salmonella adhesion and invasion, enhancing the intestinal barrier and attenuating the enterocyte inflammatory responses, and has the potential as probiotics to prevent enteric diseases in broilers.

The NLRP3 inflammasome as a new target in respiratory disorders treatment

Abstract: In recent years a continuous increase in new cases of respiratory disorders, such as rhinitis, asthma, and chronic obstructive pulmonary disease (COPD), has been observed. The exact pathomechanism of these diseases is still blurry, resulting in the lack of targeted and effective therapy. The conventional use of treatment strategies, such as antihistamine drugs and/or glucocorticosteroids act mainly symptomatically and have significant side effects. Specific allergen immunotherapy is only useful in the management of specific allergies and selected patients. Therefore, new therapeutic solutions are constantly being sought. The novelty of recent years has been the association between NLRP3 inflammasome activation and the development of airway inflammatory diseases. This seems to be an interesting therapeutic target that may support or even replace traditional therapies in the future. The review presented, discusses the contribution of NLRP3 inflammasome to the development of allergic rhinitis, allergic asthma, and COPD. Moreover, the modulatory properties of probiotics as potential inhibitors of NLRP3 inflammasome are emphasised.

Exopolysaccharides from Bifidobacterium animalis Ameliorate Escherichia coli-Induced IPEC-J2 Cell Damage via Inhibiting Apoptosis and Restoring Autophagy

Abstract: Enteropathogenic Escherichia coli (EPEC) is a common zoonotic pathogen that causes acute infectious diarrhea. Probiotics like Bifidobacterium are known to help prevent pathogen infections. The protective effects of Bifidobacterium are closely associated with its secretory products exopolysaccharides (EPS). We explored the effects of the EPS from Bifidobacterium animalis subsp. lactis (B. lactis) on ameliorating the damage of an intestinal porcine epithelial cell line (IPEC-J2) during EPEC infection. Pretreatment with EPS alleviated EPEC-induced apoptosis through the restoration of cell morphology and the downregulation of protein expressions of cleaved-caspase 8, cleaved-caspase 3, and cleaved-PARP. EPS-mediated remission of apoptosis significantly improved cell viability during EPEC infection. EPEC infection also resulted in impaired autophagy, as demonstrated by decreased expressions of autophagy-related proteins Beclin 1, ATG5, and microtubule-binding protein light chain-3B (LC3B) and the increased expression of p62 through western blot analysis. However, EPS reversed these effects which indicated that EPS promoted autophagosome formation. Furthermore, EPS prevented the lysosome damage induced by EPEC as it enhanced lysosomal acidification and raised lysosome-associated protein levels, thus promoted autophagosome degradation. Our findings suggest that the amelioration of EPEC-induced cell damages by EPS is associated with the limitation of detrimental apoptosis and the promotion of autophagy flux.