Baicalin ameliorates Mycoplasma gallisepticum-induced inflammatory injury via inhibiting STIM1-regulated ceramide accumulation in DF-1 cells

Abstract

Mycoplasma gallisepticum (MG) is dependent on its host for many nutrients due to the loss of many important metabolic pathways. Ceramide is a sphingolipid that regulates multiple cellular processes in eukaryotic cell. Several studies highlighted the crucial role of ceramide on the pathogenesis of various pathogens. This study aimed to determine whether ceramide plays a crucial role in the pathogenesis of MG. Based on an MG infection model in DF-1 cells, the results revealed that MG infection induced ceramide accumulation in DF-1 cells. Inhibiting the de novo synthesis of ceramide significantly inhibited MG proliferation and inflammatory injury caused by MG in DF-1 cells. Meanwhile, MG infection led to endoplasmic reticulum stress, and pharmacologic inhibition of endoplasmic reticulum stress prevented ceramide accumulation and MG proliferation in DF-1 cells, alleviating the inflammatory injury caused by MG. In addition, MG infection significantly promoted expression level of stromal interaction molecule 1 (STIM1), thus induced calcium overload and oxidative stress. Furthermore, inhibition of STIM1 expression partially restored calcium homeostasis and mitigated oxidative stress, thus alleviated endoplasmic reticulum stress. Importantly, the inflammatory injury caused by MG were partially ameliorated by baicalin treatment (20 µg/mL) through downregulating STIM1 expression. In summary, these results suggests that ceramide accumulation through the de novo pathway plays an important role to promote MG proliferation and baicalin can alleviate MG infection induced inflammatory injury via regulating STIM1-related oxidative stress, endoplasmic reticulum stress and ceramide accumulation in DF-1 cells.

INTRODUCTION

The Mycoplasma gallisepticum (MG) is one of the most important avian Mycoplasma species in the poultry industry (Mahdizadeh et al., 2021). It is well documented that MG causes chronic respiratory disease in chickens that contribute to reduced weight gain of broilers and egg production of layers (Ishfaq et al., 2020). MG-infected chickens are also more susceptible to other infectious diseases as a result of immune dysregulation (Wang et al., 2021b). MG is a continuing problem in poultry industry and efforts to curb MG infection including maintaining flocks free of infection, vaccines, and antibiotics (Ishfaq et al., 2020; Yadav et al., 2021). The most effective strategy for control MG infection is maintenance of MG-free flocks by adhering to strict biosecurity protocols and all-in all-out management system. However, this strategy is not suitable for all chicken farms, especially in developing countries, because it will increase the cost burden. In chicken farms where it is not feasible to maintain MG-free flocks, vaccinations and antibiotics are effective measures to control MG infection (Ishfaq et al., 2020; Yadav et al., 2021). Several vaccines such as F strain, strain 6/85 and ts-11 have been shown to good efficacy in prevention of MG infection (Ishfaq et al., 2020), however, the high cost and individual chicken administration limit widespread use of these vaccines. Administration of tetracyclines or macrolides antibiotics in the feed or water are low-cost and effective approaches to control MG infection in chicken farms (Yadav et al., 2021). However, long time use of antibiotics in poultry industry have contributed to increasing antibiotic resistance (Gautier-Bouchardon, 2018; Yadav et al., 2021). Consequently, it is imperative to explore the pathogenesis of MG infection in order to develop new interventions to enhance existing measures to control this infection.

Mycoplasma are the smallest prokaryote microorganisms that are genome-reduced bacteria with a parasitic lifestyle, resulting in a dependence on the hosts for metabolic requirement (Ishfaq et al., 2019b; Mahdizadeh et al., 2021). Sphingolipids metabolites are one of the major classes of host metabolites and key constituents of the plasma membranes (Leier et al., 2020). Ceramide, a kind of sphingolipid metabolite, has emerged as a crucial mediator in the development of many infectious diseases (Soudani et al., 2019; Leier et al., 2020). A study found that H1N1 virus infection significantly increased ceramide levels in human lung epithelial cells, and that ceramide accumulation functions as an antiviral agent against H1N1 virus infection (Soudani et al., 2019). However, Zika virus infection caused ceramide metabolic reprogramming and ceramide accumulation that promoted to Zika virus replication and infection (Leier et al., 2020). It is indicated that ceramide may play an important role during pathogenic microbial infection, but the specific role varies with different types of infection. De novo biosynthesis of ceramide mainly takes place in the endoplasmic reticulum (Leier et al., 2020). Therefore, endoplasmic reticulum malfunction may contribute to abnormal ceramide biosynthesis. Cellular calcium homeostasis is closely related to endoplasmic reticulum function (Liang et al., 2022). The level of intracellular Ca²⁺ is mainly regulated by the store-operated Ca²⁺ entry (SOCE) moiety ORAI calcium release-activated calcium modulator 1 (ORAI1) located in plasma membrane (Liang et al., 2022). The SOCE opens upon binding to its regulator stromal interaction molecule 1 (STIM1), which senses Ca²⁺ levels of endoplasmic reticulum (Zhang et al., 2020; Liang et al., 2022). Previous studies have shown that STIM1 deficiency or overexpression caused calcium homeostasis imbalance that enhanced endoplasmic reticulum stress (Liang et al., 2022). In addition, beyond a certain threshold, intracellular Ca²⁺ changes affected mitochondrial malfunction and oxidative stress, thus induced endoplasmic reticulum stress (Zhang et al., 2020). Therefore, understanding the effects of ceramide metabolism on MG pathogenesis may provide crucial insights into the development of control strategies against MG infection.

There is increasing evidence that the antimicrobial and anti-inflammatory properties of baicalin, an active ingredient of Scutellaria baicalensis (Hu et al., 2022). Our recently studies also confirmed that baicalin alleviated MG infection induced lung injury (Ishfaq et al., 2019b; Wang et al., 2021a). However, the exact molecular mechanism of baicalin against MG infection is still unclear. It has been reported that baicalin could exert an anti-infectious role by improving lipid metabolism of host (Wang et al., 2020). Consequently, this study explored whether baicalin's anti-MG effect was related to ceramide metabolism.

MATERIALS AND METHODS

MG Culture

MG strain (R_low) was purchased from China Veterinary Culture Collection Center. MG were grown at 37 °C in 5% CO₂ in Mycoplasma basal medium (BasalMedia, Shanghai, China) supplemented with 20% fetal bovine serum (Gibco Hyclone, Shanghai, China), 0.1% Nicotinamide adenine dinucleotide (Solarbio, Beijing, China) and 0.05% penicillins (Beyotime, Shanghai, China). MG were prepared for subculture every 3 to 5 d, the passage 3 used in the subsequent experiments.

DF-1 Cells Culture

The chicken fibroblast cell line DF-1 cells (ATCC CRL-12203) were grown at 37°C in 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) (Procell, Wuhan, China) supplemented with 10% fetal bovine serum (Gibco Hyclone, Shanghai, China), 1% penicillins and streptomycin (Beyotime, Shanghai, China) and 2 mM L-glutamine (Beyotime, Shanghai, China). The details of MG, baicalin (Macklin, Shanghai, China) and inhibitors were administered at amounts/concentration and times stated in figure legends.

Cell Viability Evaluation

The cells were inoculated in 6-well plate (1 × 10⁶ cells/well) and treated with MG (multiplicity of infection (MOI) = 0, 25, 50, 75 and 100) for 6, 12, and 24 h at 37°C, respectively. The medium was discarded, and the cells were co-cultured with 5 mg/mL MTT for 4 h. DMSO was used to dissolve formazan, then the absorbance value was detected at 490 nm in a microplate reader (Molecular Devices, Sunnyvale, CA).

Ceramide Quantification

Inhibitors including Myriocin (Myr, IM3130, Solarbio, Beijing, China), GSK2606414 (GSK, IG1330, Solarbio, Beijing, China) and N-acetylcysteine (NAC, C8460, Solarbio, Beijing, China) were used to inhibited ceramide synthesis, endoplasmic reticulum stress and oxidative stress, respectively. The DF-1 cells were inoculated in 6-well plate (1 × 10⁶ cells/well) and pretreated with inhibitors (0.1 µM Myr, 2 µM GSK or 10 mM NAC) or baicalin (20 µg/mL) for 2 h. Then the cells were infected with MG (MOI = 0, 25, 50, 75, and 100) for 6, 12, and 24 h at 37°C, respectively. The cells were prepared to a suspended condition by using a cell scraper, and the cell pellets were obtained by centrifugation (1,000 rpm for 3 min). Ceramide levels were evaluated according to a diacylglycerol kinase assay as described previously (Dbaibo et al., 1998; Soudani et al., 2019). Briefly, cell pellets were re-suspended in 1 mL of 8:4:3 chloroform: methanol: water. The mixture was mixed fully, then chilled in an ice-bath for 5 min. The samples were placed at 4 °C for 2 h. The lower organic layer was discarded, the lipids layer was obtained. Lipids were incubated in 20 µL of β-octylglucoside/dioleoyl-PG micelles (7.5%), 70 µL of the reaction mixture (1 mM dithiothreitol, 5 µg diacylglycerol kinase) and 10 µL of ATP mixture (2.5 mM ATP and 0.13 µCi/µL adenosine 5′-triphosphate [γ-^32P]ATP) for 30 min at 25°C. After several steps of adding organic solvents and water followed by centrifugation, the lower phase was discarded and dried by speed vacuum. The dried lipids and standards were re-suspended in 50 µL of 9:1 chloroform-methanol, and the lipids were separated using thin-layer chromatographic plates. Radioactive ceramide species were detected using X-ray films by autoradiography. The area of interest was separated, and the radioactivity was determined by liquid scintillation. Ceramide levels were detected according to the standard curve.

MG Proliferation Assessment

The amounts of MG were measured by color changing units method as described previously (Luo et al., 2020). The DF-1 cells were inoculated in 6-well plate (1 × 10⁶ cells/well) and pretreated with inhibitors (0.1 µM Myr, 2 µM GSK or 10 mM NAC) or baicalin (20 µg/mL, Macklin, Shanghai, China) for 2 h. Then the cells were infected with MG (MOI = 100) for 24 h at 37°C. The medium and lysed cells were obtained, and 10-fold gradient diluted into fifteen tubes that containing the above Mycoplasma medium. All tubes were placed at 37 °C in 5% CO₂ for at least 1 wk to detect the amounts of MG.

Western Blot Analysis

The cells were lysed by lysis buffer (Boster, Wuhan, China) containing 1 mM protease inhibitor PMSF (Boster, Wuhan, China). To obtain total protein, the lysate was centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant was the total protein. The total protein was denatured and then separated by SDS-PAGE (8–15%). The separated protein was transferred to a nitrocellulose membrane (Univ-bio, Shanghai, China). The membrane was blocked with a protein free quick blocking solution (Beyotime, Shanghai, China) for 15 min and incubated with specific primary antibodies for 3 h at room temperature. The membrane was washed for 3 times with Tris-buffered saline with 1% Tween 20 (TBST) (Beyotime, Shanghai, China) and incubated with secondary goat anti-rabbit IgG (1:5000, BA1056, Boster, Wuhan, China) for 1 h at room temperature. The membrane was washed again for 3 times with TBST, and the blot was detected by enhanced chemiluminescence solution (Meilune, Dalian, China) and analyzed by Image J (version 1.4.3.67) software. The primary antibodies used are as follows: β-actin (1:5000, bsm-33036M, Bioss, Beijing, China), GRP78 (1:1000, bs-1219R, Bioss, Beijing, China), CHOP (1:500, bs-20669R, Bioss, Beijing, China), STIM1 (1:500, A9764, Abclonal, Wuhan, China), ORAI1 (1:500, A7412, Abclonal, Wuhan, China).

Detection of Proinflammatory Cytokines

The cells were lysed by lysis buffer containing 1 mM protease inhibitor PMSF, then the lysate was centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant was used to detect the levels of TNF-α, IL-1β and IL-6 by using commercial ELISA kits (Saipei Bio, Wuhan, China) according to the manufacturer's instructions.

Determination of Oxidative Stress Markers and ATPase Activities

Cell suspension was collected and examined for malonaldehyde (MDA) level and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH), Mg²⁺-ATPase, Na⁺-K⁺-ATPase, Ca²⁺-Mg²⁺-ATPase and Ca²⁺ATPase by commercial ELISA kits according to the manufacturer's instructions (Jiancheng, Nanjing, China).

Determination of Reactive Oxygen Species

Reactive oxygen species (ROS) levels were measured by using a commercial kit (Beyotime, Shanghai, China). Briefly, cells were incubated with fluorescence probe DCFH-DA for at least 20 min at 37 °C. The cells were washed 3 times with serum-free cell culture medium to fully remove the extracellular DCFH-DA. Cell suspension was collected and examined for ROS by a fluorescence spectrophotometer (Thermo Scientific, Shanghai, China).

Measurement of Intracellular Ca²⁺ Concentration

Intracellular Ca²⁺ concentration was detected as previously described (Yan et al., 2022). Briefly, cells were incubated with calcium indicator fura-2/AM (Beyotime, Shanghai, China) for 1 h at 37 °C in the dark. The cells were washed 3 times and re-suspended in calcium-free Hank's Balanced Salt Solution. The levels of fluorescence of fura-2 were detected by a fluorescence spectrophotometer with the excitation wavelength at 340 and 380 nm, and emission fluorescence at 510 nm.

The siRNA Interference Assay

The siRNAs against STIM1 gene (si-STIM1) were purchased from GenePharma Co. Ltd (Shanghai, China). The siRNA sequences for inhibition of STIM1 expression included siRNA-STIM1#1 (sense, 5′-CCAGGUUAGCGGUGAACAATT-3′), siRNA-STIM1#2 (sense, 5′-GCAACACUCUGUUUGGAACTT-3′). The sense sequence of negative control siRNA (siRNA-NC) was 5′-UUCUCCGAACGUGUCACGUTT-3′. The siRNA with the highest silence efficiency was chosen for subsequent experiments. The siRNAs were transfected into DF-1 cells using RNAiMAX (Invitrogen, Shanghai, China) according to the manufacturer's recommended procedures. After 48 h, cells were treated with MG (MOI = 100) for 24 h at 37°C, then the cells were collected for assessment of oxidative stress, endoplasmic reticulum stress, ceramide quantification, MG proliferation and proinflammatory cytokines production.

Statistical Analysis

The data were represented as mean ± SD and analyzed using GraphPad Prism 8.0 (GraphPad Software). Multiple-group comparisons were analyzed with one-way analysis of variance (with post-hoc Sidak's test). P value < 0.05 was deemed as statistically significant.

RESULTS

MG Infection Induces Ceramide Accumulation in DF-1 Cells

MG infection reduced cell viability in a time- and dose-dependent manner (Figure 1A). A dose of 100 MOI at 6 h significantly reduced cell viability, and at a dose of 50 MOI at 24 h compared to the control group (Figure 1A, all P < 0.01). Next, the effects of MG infection on ceramide accumulation in DF-1 cells were evaluated. DF-1 cells were infected with 100 MOI of MG, and the cells were collected at 6, 12, and 24 h post infection. At each time point, the fold change of ceramide levels in MG-infected group were computed relative to the time matching uninfected controls. The results showed that a significant increase in ceramide levels were found at 6 h post MG infection, and continued to elevate until 24 h (Figure 1B, all P < 0.01). In addition, DF-1 cells were infected with 50, 75, and 100 MOI of MG, and the cells were collected at 24 h post infection. The results showed that MG infection induced ceramide accumulation in DF-1 cells in a dose-dependent manner (Figure 1C, all P < 0.01).

Figure 1.

Effects of MG infection on cell viability and ceramide levels in DF-1 cells. (A) Cell viability was evaluated by MTT assay. The chicken fibroblast cell line DF-1 cells were incubated with different multiplicity of infection (MOI) (0–100) for different time points (6, 12, and 24 h). (B–C) MG infection caused ceramide accumulation in DF-1 cells. DF-1 cells were challenged with 100 MOI MG for 6, 12, and 24 h, or 50, 75, and 100 MOI MG for 24 h, then ceramide levels were detected by diacylglycerol kinase (DGK) assay. For (B–C), for each condition, ceramide level was normalized to total phosphate, and the ceramide fold change was calculated with respect to time matching uninfected controls. ^⁎⁎ indicates P < 0.01. Abbreviation: MG, Mycoplasma gallisepticum.

Inhibition of the De Novo Ceramide Pathway Inhibits MG Proliferation and MG Infection Induced Inflammatory Injury

To address the role of ceramide in MG infection, a small-molecule inhibitor of serine palmitoyltransferase (SPT) called Myr was used to block the de novo synthesis of ceramide. The inhibitor did not directly affect MG proliferation and cell viability (Supplementary Figures 1A and 1B). Myr treatment effectively reduced ceramide levels in DF-1 cells (Figure 2A, all P < 0.01). In addition, compared to the MG infection group, Myr treatment inhibited MG proliferation in DF-1 cells (Figure 2B, P < 0.01). Furthermore, Myr treatment significantly alleviated MG infection caused inflammatory injury as reflected by decreased production of proinflammatory cytokines TNF-α, IL-1β and IL-6 (Figure 2C, all P < 0.01) compared to the MG infection group. Meanwhile, DF-1 cells were treated with exogenous C6-ceramide for 24 h, the results showed that exogenous C6-ceramide significantly promoted ceramide accumulation, MG proliferation and inflammatory injury caused by MG (Supplementary Figure 2). These results indicated that blockage of the de novo synthesis of ceramide inhibited MG proliferation and inflammatory injury caused by MG.

Figure 2.

Effects of ceramide accumulation on MG proliferation and inflammatory injury caused by MG. (A) Myriocin (Myr) treatment inhibited ceramide accumulation in DF-1 cells. (B) Blockage of ceramide accumulation by Myr inhibited MG proliferation in DF-1 cells. (C) Blockage of ceramide accumulation by Myr reduced MG infection induced proinflammatory cytokines production in DF-1 cells. For (A–C), DF-1 cells were treated with 0.1 µM Myr or vehicle for 2 h, then the cells were infected with 100 MOI MG for 24 h, then the indicated indices were detected. ^⁎⁎ indicates P < 0.01. Abbreviation: MG, Mycoplasma gallisepticum.

Inhibition of Endoplasmic Reticulum Stress Inhibits Ceramide Accumulation and Inflammatory Injury Caused by MG

Endoplasmic reticulum is the major site of ceramide synthesis. To address MG infection how affected ceramide synthesis, the effects of endoplasmic reticulum stress on ceramide accumulation were evaluated during MG infection. The results showed that MG infection triggered endoplasmic reticulum stress in DF-1 cells as reflected by significantly elevated expression levels of endoplasmic reticulum stress markers GRP78 and CHOP (Figure 3A, all P < 0.01). Next, an inhibitor GSK was used to block endoplasmic reticulum stress (Figure 3B, all P < 0.01). The inhibitor did not directly affect MG proliferation and cell viability (Supplementary Figures 3A and 3B). Compared to MG infection group, inhibition of endoplasmic reticulum stress reduced ceramide levels and inhibited MG proliferation in DF-1 cells (Figures 3C and 3D, all P < 0.01). Furthermore, compared to the MG infection group, inhibition of endoplasmic reticulum stress significantly ameliorated MG infection induced inflammatory injury as reflected by decreased production of proinflammatory cytokines TNF-α, IL-1β, and IL-6 (Figure 3E, all P < 0.01). These results indicated that inhibition of endoplasmic reticulum stress inhibited ceramide accumulation and MG proliferation, and alleviated inflammatory injury caused by MG.

Figure 3.

Effects of endoplasmic reticulum stress on ceramide accumulation, MG proliferation and inflammatory injury caused by MG in DF-1 cells. (A) MG infection triggered endoplasmic reticulum stress in DF-1 cells. (B) The inhibitor GSK2606414 (GSK) inhibited endoplasmic reticulum stress activation in DF-1 cells. (C) Inhibition of endoplasmic reticulum stress by GSK inhibited ceramide accumulation in DF-1 cells. (D) Inhibition of endoplasmic reticulum stress by GSK inhibited MG proliferation in DF-1 cells. (E) Inhibition of endoplasmic reticulum stress by GSK reduced MG infection induced proinflammatory cytokines production in DF-1 cells. For (A), DF-1 cells were challenged with 100 MOI MG for 12 and 24 h, and the indicated indices were detected. For (B–E), DF-1 cells were treated with 2 µM GSK or vehicle for 2 h. Then the cells were infected with 100 MOI MG for 24 h, and the indicated indices were detected. ^⁎⁎ indicates P < 0.01. Abbreviation: MG, Mycoplasma gallisepticum.

Downregulated STIM1 Inhibits the Endoplasmic Reticulum Stress Caused by MG

STIM1 and ORAI1 take part in the maintenance of calcium homeostasis and have been shown to play important roles in activating endoplasmic reticulum stress. The results showed that, compared to the control group, the expression levels of STIM1 and ORAI1 were significantly increased in the MG infection group (Figure 4A, all P < 0.01). In addition, MG infection significantly increased intracellular Ca²⁺ levels compared to the control group (Figure 4B, all P < 0.01). To further explore the role of STIM1 in MG infection induced endoplasmic reticulum stress and ceramide accumulation, the STIM1 expression was inhibited by siRNA (Figure 4C). Compared to MG infection group, inhibition of STIM1 expression significantly reduced ORAI1 expression and intracellular Ca²⁺ levels, and alleviated endoplasmic reticulum stress as reflected by reduced expression levels of GRP78 and CHOP (Figures 4D and 4E, all P < 0.01). In addition, inhibition of STIM1 expression significantly decreased ceramide levels and inhibited MG proliferation in DF-1 cells compared to the MG infection group (Figures 4F and 4G, all P < 0.01). Furthermore, compared to MG infection group, inhibition of STIM1 expression significantly ameliorated MG infection induced inflammatory injury as reflected by decreased production of proinflammatory cytokines TNF-α, IL-1β, and IL-6 (Figure 4H, all P < 0.01). These results indicated that inhibition of STIM1 expression inhibited calcium homeostasis imbalance, endoplasmic reticulum stress, ceramide accumulation and MG proliferation, and alleviated inflammatory injury caused by MG.

Figure 4.

Effects of inhibition of STIM1 expression on endoplasmic reticulum stress, ceramide accumulation, MG proliferation and inflammatory injury caused by MG in DF-1 cells. (A) MG infection increased expression levels of STIM1 and ORAI1 in DF-1 cells. (B) MG infection increased intracellular calcium levels in DF-1 cells. (C) Inhibition of STIM1 expression by siRNA. (D) Inhibition of STIM1 improved MG-induced calcium homeostasis imbalance. (E) Inhibition of STIM1 inhibited MG infection-mediated endoplasmic reticulum stress and abnormal ORAI1 expression. (F) Inhibition of STIM1 inhibited ceramide accumulation in DF-1 cells. (G) Inhibition of STIM1 inhibited MG proliferation in DF-1 cells. (H) Inhibition of STIM1 reduced MG infection-mediated proinflammatory cytokines production in DF-1 cells. For (A–B), DF-1 cells were challenged with 100 MOI MG for 12 and 24 h, and indices were detected. For (D–H), DF-1 cells were treated with siRNA or vehicle for 48 h, then the cells were infected with 100 MOI MG for 24 h, and the indicated indices were detected. ^⁎⁎ indicates P < 0.01. Abbreviations: MG, Mycoplasma gallisepticum; ORAI1, ORAI calcium release-activated calcium modulator 1; STIM1, stromal interaction molecule 1.

Downregulated STIM1 Inhibits Oxidative Stress-related Endoplasmic Reticulum Stress Caused by MG

It has been noted that GSH and SOD activities significantly decreased, and MDA and ROS levels increased markedly in MG infection group compared to the control group (Figure 5A, all P < 0.01). In addition, compared to control group, Na⁺-K⁺-ATPase, Mg²⁺-ATPase, Ca²⁺ATPase and Ca²⁺-Mg²⁺-ATPase activities were significantly impaired in the MG infection group (Figure 5B, all P < 0.01). To address the role of oxidative stress in MG infection induced endoplasmic reticulum stress and ceramide accumulation, an oxidative stress inhibitor NAC was used. Compared to MG infection group, DF-1 cells treated with the oxidative stress inhibitor NAC had significantly lower expression levels of the endoplasmic reticulum stress-associated proteins GRP78 and CHOP (Figure 5C, all P < 0.01). In addition, compared to MG infection group, NAC treatment significantly inhibited ceramide accumulation and MG proliferation in DF-1 cells (Figures 5D and 5E, all P < 0.01). NAC did not directly affect MG proliferation and cell viability (Supplementary Figures 4A and 4B). Furthermore, compared to MG infection group, NAC treatment significantly ameliorated MG infection induced inflammatory injury as reflected by decreased production of proinflammatory cytokines TNF-α, IL-1β and IL-6 (Figure 5F, all P < 0.01). NAC treatment did not affect STIM1 and ORAI1 expression levels caused by MG (Figure 6A). While, inhibition of STIM1 expression significantly ameliorated MG infection induced oxidative stress as reflected by increased activities of GSH, SOD, and ATPase, and decreased production of MDA and ROS (Figures 6B and 6C, all P < 0.01). These results indicated that inhibition of STIM1 expression alleviated oxidative stress, thus inhibited endoplasmic reticulum stress, ceramide accumulation, MG proliferation, and alleviated inflammatory injury caused by MG.

Figure 5.

Effects of oxidative stress on endoplasmic reticulum stress, ceramide accumulation, MG proliferation and inflammatory injury caused by MG in DF-1 cells. (A) MG infection caused oxidative stress in DF-1 cells. (B) MG infection impaired ATPase activities in DF-1 cells. (C) Inhibition of oxidative stress by N-acetylcysteine (NAC) inhibited MG infection induced endoplasmic reticulum stress. (D) Inhibition of oxidative stress by NAC inhibited ceramide accumulation in DF-1 cells. (E) Inhibition of oxidative stress by NAC inhibited MG proliferation in DF-1 cells. (F) Inhibition of oxidative stress by NAC reduced MG infection induced proinflammatory cytokines production in DF-1 cells. For (A–B), DF-1 cells were challenged with 100 MOI MG for 12 and 24 h, and the indicated indices were detected. For (C–F), DF-1 cells were treated with 10 mM NAC for 2 h. The cells were infected with 100 MOI MG for 24 h, and the indicated indices were detected. ^⁎⁎ indicates P < 0.01. Abbreviation: MG, Mycoplasma gallisepticum.

Figure 6.

Effects of inhibition of STIM1 expression on oxidative stress caused by MG in DF-1 cells. (A) 10 mM NAC did not affect STIM1 and ORAI1 expression. (B) Inhibition of STIM1 reduced oxidative stress caused by MG in DF-1 cells. (C) Inhibition of STIM1 enhanced ATPase activities in MG-infected DF-1 cells. For (A), DF-1 cells were treated with 10 mM NAC for 2 h, then the cells were infected with 100 MOI MG for 24 h, the indicated indices were detected. For (B–C), DF-1 cells were treated with siRNA or vehicle for 48 h. The cells were infected with 100 MOI MG for 24 h, and the indicated indices were detected. ^⁎⁎ indicates P < 0.01. Abbreviations: MG, Mycoplasma gallisepticum; ORAI1, ORAI calcium release-activated calcium modulator 1; STIM1, stromal interaction molecule 1.

Baicalin Ameliorates MG-induced Inflammatory Injury through Inhibiting STIM1 Expression

The effect of different concentrations of baicalin on cell viability was evaluated by MTT assay. It has been noted that a concentration ranged from 0 to 20 µg/mL baicalin treatment for 24 h did not affect cell viability. While higher concentrations of baicalin significantly reduced cell viability (Supplementary Figure 5A). Thus, baicalin concentrations of 20 µg/mL for 24 h was chosen for the following experiments. Compared to MG infection group, baicalin treatment significantly inhibited STIM1 and ORAI1 expression levels, and decreased intracellular Ca²⁺ levels (Figures 7A and 7B, all P < 0.01). In addition, compared to MG infection group, baicalin treatment significantly inhibited oxidative stress and endoplasmic reticulum stress reflected by increased activities of GSH, SOD and ATPase, decreased production of MDA and ROS, and decreased expression levels of endoplasmic reticulum stress-associated proteins GRP78 and CHOP (Figures 7C–7E, all P < 0.01). 20 µg/mL baicalin treatment did not directly affect MG proliferation (Supplementary Figure 5B). Compared to MG infection group, baicalin treatment significantly inhibited ceramide accumulation and MG proliferation in DF-1 cells (Figures 7F and 7G, all P < 0.01). Furthermore, baicalin treatment significantly alleviated MG infection caused inflammatory injury as reflected by decreased production of proinflammatory cytokines TNF-α, IL-1β and IL-6 (Figure 7H, all P < 0.01). These results indicated that the protective effects of baicalin on MG infection caused inflammatory injury by inhibiting STIM1-related calcium homeostasis imbalance, oxidative stress, endoplasmic reticulum stress, ceramide accumulation and MG proliferation.

Figure 7.

The protective effects of baicalin on MG infection-induced inflammatory injury. (A) Baicalin inhibited the abnormal expression of STIM1 and ORAI1 in MG-infected DF-1 cells. (B) Baicalin reduced intracellular calcium levels in MG-infected DF-1 cells. (C) Baicalin alleviated oxidative stress in MG-infected DF-1 cells. (D) Baicalin partially restored ATPase activities in MG-infected DF-1 cells. (E) Baicalin inhibited MG infection induced endoplasmic reticulum stress. (F) Baicalin inhibited ceramide accumulation in MG-infected DF-1 cells. (G) Baicalin inhibited MG proliferation in DF-1 cells. (H) Baicalin reduced MG infection-induced proinflammatory cytokines production in DF-1 cells. For (A–H), DF-1 cells were treated with 0 or 20 µg/mL baicalin before 2 h of MG infection at 100 MOI and cultured for 24 h, then the indicated indices were detected. ^⁎⁎ indicates P < 0.01. Abbreviations: MG, Mycoplasma gallisepticum; ORAI1, ORAI calcium release-activated calcium modulator 1; STIM1, stromal interaction molecule 1.

DISCUSSION

Ceramide is an important bioactive lipid in sphingolipid metabolism (Soudani et al., 2019). Ceramide is mainly regulated at the cellular level by its metabolic pathway of de novo synthesis (Soudani et al., 2019; Leier et al., 2020). The de novo synthesis starts in endoplasmic reticulum with the condensation of serine and palmitoyl-coenzyme A, a rate-limiting step catalyzed by SPT (Soudani et al., 2019; Leier et al., 2020). Accumulating evidence suggests that ceramide is not only responsible for maintaining membrane composition and fluidity, but also involved in host-pathogen interactions (Finnegan et al., 2004; Martín-Acebes et al., 2011; Soudani et al., 2019; Leier et al., 2020; Zheng et al., 2022). For instance, treating cells with pharmacologic agents such as fenretinide or exogenous addition of ceramide that enhanced ceramide accumulation resulted in inhibition of HIV-1 infection in TZM-bl cells (Finnegan et al., 2004). The anti-HIV effects of accumulated ceramide was partially mediated the perturbation of host membrane structure leading to rearranging HIV receptors (Finnegan et al., 2004). On the contrary, West Nile virus and Zika virus triggered and utilized the de novo biosynthesis of ceramide that contributed to virus replication and infection (Martín-Acebes et al., 2011; Leier et al., 2020). Ceramide can promote or suppress pathogenic microorganisms infection suggesting that regulation of ceramide metabolism may be a potential and promising target to defense pathogenic microorganisms. In the present study, the results indicated that increased ceramide accumulation in response to MG challenge in a dose- and time-dependent manner. In addition, inhibiting the rate-limiting enzyme SPT in the de novo biosynthesis of ceramide suppressed MG proliferation and alleviated MG-induced inflammatory injury in DF-1 cells. This is the first study to demonstrated that ceramide plays a vital role during MG infection. The mechanism by which ceramide promotes MG proliferation remains unclear. It has been reported that cholesterol is a crucial lipid affecting the growth of Mycoplasma hyopneumoniae (Liu et al., 2017). The growth-enhancing effect of cholesterol was related to an up-regulation of M. hyopneumoniae genes responsible for DNA and protein synthesis (Liu et al., 2017). In addition, previous studies indicated that bacterial pathogens hijacked host lipid metabolism and utilized host lipid as carbon source that contribute to rapid proliferation of pathogens (Chatterjee et al., 2021). It remains to be determined whether MG can utilize host ceramide for the purpose of promoting its own proliferation. In addition, MG is a prokaryote and does not possess organelles capable of synthesizing sphingolipids (Chatterjee et al., 2021). The MG genome encodes numerous nutrient transport systems that help MG acquire lipids and other nutrients from the host (Chatterjee et al., 2021; Mahdizadeh et al., 2021). Further studies are required in order to determine which transport system is employed by MG in order to obtain ceramide from the host.

The endoplasmic reticulum is an important intracellular organelle that is responsible for synthesis and folding of secretory and membrane proteins in eukaryotic cells (Pan et al., 2020; Wu et al., 2021). In addition, endoplasmic reticulum acts as a main site of sphingolipids synthesis (Leier et al., 2020). Many factors, such as pathogen infection, tumor and inflammation, cause overload of endoplasmic reticulum function, referred to as endoplasmic reticulum stress (Pan et al., 2020; Wu et al., 2021). Previous study indicated that endoplasmic reticulum stress is a critical event during Mycoplasma bovis infection (Wu et al., 2021). However, the effect of MG infections on endoplasmic reticulum function is rarely reported. In this study, our data showed that the endoplasmic reticulum stress markers GRP78 and CHOP were significantly upregulated in the MG-infected DF-1 cells. In addition, pharmacologic inhibition of endoplasmic reticulum stress impaired MG proliferation and alleviated MG-mediated inflammatory injury. Based on these results, it appears that endoplasmic reticulum stress plays an important role during infection with MG. It has been reported that prolonged endoplasmic reticulum stress instigated ceramide metabolic disorder (Liang et al., 2022). In this study, our data confirmed that pharmacologic inhibition of endoplasmic reticulum stress alleviated MG infection-mediated ceramide metabolic disorder. The present study provides evidence that endoplasmic reticulum stress-regulated ceramide metabolism could be involved in MG-induced inflammatory injury in DF-1 cells. Previous studies demonstrated that endoplasmic reticulum is the primary calcium store in eukaryotic cells (Biczo et al., 2018; Zhang et al., 2020). Imbalanced endoplasmic reticulum-calcium homeostasis accompanied by intracellular calcium overload can cause overload of endoplasmic reticulum function, which results in endoplasmic reticulum stress (Liang et al., 2022; Zhang et al., 2020). As the major constitutes of SOCE, STIM1 and ORAI1 are involved in the maintenance of calcium homeostasis and have been confirmed to play crucial roles in activating endoplasmic reticulum stress (Zhang et al., 2020; Liang et al., 2022). It has been reported that STIM1 overexpression induced disturbance of calcium homeostasis and thus exacerbated induction of endoplasmic reticulum stress (Zhang et al., 2020; Liang et al., 2022). In the present study, MG challenge induced a significant increase in the expression of STIM1 and ORAI1. In addition, the cellular ceramide level and the expression levels of endoplasmic reticulum stress markers were significantly reduced in STIM1 silenced group indicating that STIM1-regulated endoplasmic reticulum stress activation may be the major mechanism by which MG promotes ceramide metabolic disorder and inflammatory injury. In the present study, MG infection induced STIM1-regulated oxidative stress in DF-1 cell. Furthermore, pharmacologic inhibition of oxidative stress ameliorated endoplasmic reticulum stress and ceramide accumulation during MG infection. Cellular calcium homeostasis imbalance can increase metabolic rate of mitochondria, resulting in excessive ROS production, oxidative stress and mitochondrial dysfunction (Biczo et al., 2018). Mitochondrial dysfunction caused decreased levels of ATP that compromised unfolded protein response and caused overload of endoplasmic reticulum function, resulting in endoplasmic reticulum stress (Raffaello et al., 2016; Biczo et al., 2018).

Baicalin, the major flavonoid compound isolated from S. baicalensis, has been found to show multiple pharmacologic properties, including anti-infective, anti-inflammatory, and anti-oxidant activities (Wang et al., 2020; Wang et al., 2021a; Hu et al., 2022). Our recent studies demonstrated that oral administration of baicalin alleviated MG infection induced inflammatory injury of lung, spleen and bursa of fabricius (Ishfaq et al., 2019a,2021; Wang et al., 2021a). However, the anti-MG effects of baicalin are in notable contrast to its poor bioavailability. Previous studies confirmed that oral administrations of 400 mg/kg baicalin alleviated MG-induced lung injury, whereas baicalin concentration in the lung tissues was much lower than the minimum inhibitory concentration of baicalin against MG (Bao et al., 2021). These results suggests that the anti-MG effects of baicalin do not depend on the direct killing of MG. It has been reported that baicalin can regulate lipid metabolism, oxidative stress, calcium homeostasis and endoplasmic reticulum stress of host (Cao et al., 2018; Ishfaq et al., 2019a; Wang et al., 2020; Yu et al., 2020). For example, according to a network pharmacology study, S. baicalensis may exert its beneficial effects by regulating the cholesterol biosynthesis and sphingolipid metabolism pathways of host, and baicalin, a major ingredient of S. baicalensis, may regulate lipid metabolism by inhibiting activities of key enzymes such as squalene monooxygenase and lanosterol synthase (Ge et al., 2021). In addition, baicalin can alleviate oxidative stress and endoplasmic reticulum stress via Nrf2 signaling pathway activation (Lin et al., 2014). Moreover, as disclosed in this context, MG could utilize the host ceramide to promote its own growth that related to oxidative stress, calcium homeostasis and endoplasmic reticulum stress of host. Hence, we investigated whether baicalin can exert anti-MG effects by affecting host lipid metabolism, oxidative stress, and endoplasmic reticulum function. In the present study, the results showed that baicalin significantly inhibited STIM1-regulated calcium homeostasis imbalance, oxidative stress, endoplasmic reticulum stress and ceramide accumulation, and thus inhibited inflammatory injury caused by MG in DF-1 cells. The data provided evidence that the protective role of baicalin against MG infection is partially attributed to its ability to inhibit STIM1 expression and improve ceramide metabolism. The molecular docking of baicalin and STIM1 showed very low fitness score and interaction (data not shown), this indicated that baicalin may not influence STIM1 expression directly. It has been reported that, based on the random transposon mutagenesis technology, several important proteins in M. bovis were identified that were involved in the metabolic interaction between M. bovis and host (Zhu et al., 2020). If these key proteins were mutated, the M. bovis would not growth and survive in the host (Zhu et al., 2020). We speculated that MG may enhance STIM1 expression by secreting some proteins that interact with STIM1 in the host. Baicalin has been found to exert its antibacterial effect not by killing bacteria directly, but by inhibiting bacterial virulence proteins. For example, baicalin alleviated Staphylococcus aureus infection induced lung injury via inhibition of the cytolytic activity of α-hemolysin (Qiu et al., 2012). It is speculated that baicalin may inhibit the expression of STIM1 by blocking the interaction between MG key proteins and STIM1, and then affect sphingolipid metabolism. However, further studies are needed to clarify which MG proteins were involved in the interaction with STIM1, and how baicalin affects STIM1 expression.

CONCLUSIONS

In conclusion, MG infection induced ceramide accumulation and thus promoted MG proliferation and caused inflammatory injury in DF-1 cells. Furthermore, the antagonistic effects of baicalin on MG infection-mediated inflammatory injury include inhibition of STIM1-regulated calcium homeostasis imbalance, oxidative stress, endoplasmic reticulum stress, ceramide accumulation, and MG proliferation (Figure 8).

Figure 8.

Schematic diagram of the protective effects of baicalin against MG-induced inflammatory injury. MG infection induced increased expression levels of STIM1 and ORAI1 that contributed to increased Ca²⁺ levels and induced Ca²⁺ overload. Ca²⁺ overload triggered oxidative stress and endoplasmic reticulum stress, and thus promoted ceramide accumulation. Furthermore, ceramide accumulation enhanced MG proliferation and inflammatory injury caused by MG in DF-1 cells. While baicalin suppressed MG proliferation and mitigated MG infection caused inflammatory injury via inhibiting STIM1-related oxidative stress, endoplasmic reticulum stress and abnormal ceramide accumulation. Abbreviations: MG, Mycoplasma gallisepticum; ORAI1, ORAI calcium release-activated calcium modulator 1; STIM1, stromal interaction molecule 1.