Baicalin alleviates Mycoplasma gallisepticum-induced oxidative stress and inflammation via modulating NLRP3 inflammasome-autophagy pathway
Introduction
Mycoplasma gallisepticum (MG) is the smallest prokaryotic microorganism, and one of the primary etiologies of chronic respiratory disease in chickens [1], [2]. The disease develops slowly, causing immune dysregulation and severe inflammation of the respiratory tract in avian species [3], [4], [5]. The pathogen was reported to cause latent infections and persisted in a flock for a long time [6], [7]. Previous studies reported that MG infection caused significant economic losses in terms of weight gain, reduced egg production, hatchability and low carcass quality, and infected flocks becomes susceptible to other diseases [8], [9]. MG mainly reside in the respiratory tract of chickens and infect the mucosal respiratory surface epithelium of chickens [3], [10]. A previous study cocultured chicken tracheal epithelial cells with chicken-like macrophages (HD11 cells) exposed to live mycoplasma stimulated macrophages with the upregulation of several proinflammatory genes. MG induced inflammatory response in HD11 cells and increased the expression of several cytokines such as IL-6, IL-1β, CCL-20, CXCL-13, macrophage inflammatory protein 1β (MIP-1β) and RANTES. These results showed that MG stimulated macrophages and results in a strong inflammatory response [11]. HD11 cells were first reported in 1979 derived from chicken hematopoietic cells transformed by seven strain of defective avian leukemia viruses both in vivo and in vitro [12]. HD11 cells are considered as an accurate representation of primary avian macrophages [13], and were used in several studies to scrutinize the effect of various bacteria and viruses on these cells [14], [15], [16]. Therefore, in the present study, HD11 cells were used to study MG-induced inflammatory response in order to explore the therapeutic targets that could be helpful in the prevention of MG infection.
Inflammation is considered an essential immune response that resist a wide range of pathogens and repair damaged tissue. However, inflammation may also have harmful effects including exacerbating tissue damage and cell death [17]. Inflammatory response is mediated by a range of pathogen recognition receptors located on the outer membrane of the cells and inside the cells [18]. Among these receptors, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) located in the cytosol of the cells and often recognized danger signals associated with endogenous stress and triggers downstream inflammatory pathways, thereby repairing damaged tissues and eliminating microbial infections [19]. Importantly, NLRP3 recruit’s apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) with pro-caspase-1 to form a multi-protein complex also called NLRP3 inflammasome. Pro-caspase-1 was then activated after autocatalytic cleavage into active caspase-1 and cleaves pro-IL-1β and pro-IL-18 into mature IL-1β and IL-18 [20]. Although, the activation of NLRP3 inflammasome may play a protective role in mediating inflammation, however, the excessive and continuous activation of NLRP3 inflammasome during infection is harmful to the body and a leading cause of immune dysregulation [21], [22]. Therefore, it is of prime importance to inhibit the excessive activation of NLRP3 inflammasome to prevent immune dysregulation during MG infection.
Autophagy (macroautophagy) is a degradative process by which the cells remove harmful and damaged organelles or proteins [23]. Autophagy plays a crucial role in infection and immunity by limiting inflammatory responses and simultaneously limiting the burden of infectious agents [24]. Previous research reported that NF-κB and its downstream signaling pathways linked autophagy and inflammation. During inflammation, NF-κB was overexpressed, followed by the activation of inflammatory cytokines like TNF-α and IL-1 sub-family at the inflamed cites [25]. The expression of P62/SQSTM1, an adaptor protein was found dependent on NF-κB [26], and silencing of p62 leads to the inhibition of NF-κB degradation [27]. In addition, p62-mediated autophagy results in the degradation of mitochondria (mitophagy) [28]. Meanwhile, damaged mitochondria result in the release of excessive inflammasome activation. While, p62 prevents excessive inflammasomes activation by eliminating the damaged mitochondria [29]. Thus, autophagy lies at the center of several cellular responses and interconnects these pathways including immune-related signaling pathways and inflammation [30]. Autophagy was reported to play a crucial role in maintaining quality control systems and homeostasis of mitochondria [31]. Therefore, targeting autophagy during MG infection will provide novel insights for therapeutic targets.
Recently, a large number of studies demonstrated the anti-inflammatory, anti-cancer, anti-viral properties of baicalin, a flavonoid compound found in the roots of Scutellaria baicalensis [32]. Our previous studies also investigated the protective effects of baicalin against MG infection in lungs [33], thymus [34], spleen [35] and bursa of fabricius in chickens [36]. However, the complete molecular mechanisms of baicalin are still unknown in the MG-infected HD11 cells. Therefore, in the present study, the preventive effects of baicalin have been studied in vitro on MG-infected HD11 cells with the aim to identify molecular targets that might be helpful for the prevention of MG infection. The effect on MG-induced oxidative stress and inflammation-related signaling pathways including TLR-2-NF-κB, NLRP3 inflammasome and autophagy pathway were evaluated that might contribute to the therapeutic effects of baicalin. The study could provide an essential regulatory loop through which baicalin orchestrates a reparative anti-inflammatory response and prevents excessive MG-induced oxidative and inflammatory damage.
Section snippets
Ethical statement
All the experiments were conducted under the approval of Laboratory Animal Ethics Committee of Northeast Agricultural University (Heilongjiang province, China) in accordance with Laboratory Animal-Guideline for ethical review of animal welfare (GB/T 35892–2018, National Standards of the People’s Republic of China).
Culture of bacteria
MG strain (Rlow) were cultured as mentioned in a previous published study [37]. In brief, 0.1% Nicotinamide adenine dinucleotide (NAD), 10% freshly prepared yeast extract, and 0.05%
Effects of MG infection and baicalin on HD11 cell’s viability
Cell’s viability was determined by cell counting kit (CCK-8) assay as shown in Fig. 1A. The effect of different multiplicity of infection (MOI) of MG ranged from low to high (0–1000 MOI) was tested on the cell’s viability of HD11 cells at different time points. MG infection decreased the cell’s viability in a dose and time-dependent manner. Significant (p < 0.01) cell death was observed at a dose greater than 400 MOI at 4 h and 6 h, and at a dose greater than 200 MOI at 8 h compared to the
Discussion
Previous studies reported that oxidative stress damage was caused by the imbalance of exogenous or endogenous oxidants and antioxidant system [30]. Cellular redox signals can be mediated by certain active oxides. The oxidative stress response can be associated with the antioxidant system, increased production of oxides, and apoptosis of cells or even death [29], [31]. Enzymes including GSH-PX, MDA, and CAT are the key to balance the body's oxidation and anti-oxidation effects [38], [39]. MG
Conclusion
In conclusion, the data showed that baicalin could protect from MG-induced inflammation and oxidative stress by activation of autophagy and suppression of TLR-2-NF-κB pathway and NLRP3-inflammasome. These results provide evidence for supporting the application of autophagy promoter in the treatment MG infection. Furthermore, the mechanism of baicalin has been clarified and laid a foundation and reference for the applications and comparative studies of baicalin for the prevention and treatment
CRediT authorship contribution statement
Muhammad Ishfaq: Writing – review & editing. Zhiyong Wu: Data curation, Formal analysis. Jian Wang: Methodology, Validation. Rui Li: Methodology, Software. Chunli Chen: Resources, Supervision, Validation. Jichang Li: Conceptualization, Methodology, Project administration, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
All the authors greatly acknowledge the funding support from the National Natural Science Foundation of China (31772801, 31973005). The authors also acknowledge the kind support from “Professor Guangxing Li” of the department of Basic Veterinary Medicine, Northeast Agricultural University (Harbin, China) by providing chicken like macrophages (HD11 cells).
Ethical statement
All the experiments were conducted under the approval of Laboratory Animal Ethics Committee of Northeast Agricultural
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