Vitexin Mitigates Staphylococcus aureus-Induced Mastitis via Regulation of ROS/ER Stress/NF- κ B/MAPK Pathway

Abstract

Mastitis, caused by a variety of pathogenic microorganisms, seriously threatens the safety and economic benefits of the dairy industry. Vitexin, a flavone glucoside found in many plant species, has been widely reported to have antioxidant, anti-inflammatory, antiviral, anticancer, neuroprotective, and cardioprotective effects. However, few studies have explored the effect of vitexin on mastitis. This study is aimed at exploring whether the antioxidant and anti-inflammatory functions of vitexin can improve Staphylococcus aureus-induced mastitis and its possible molecular mechanism. The expression profiles of S. aureus-infected bovine mammary epithelial cells and gland tissues from the GEO data set (GSE94056 and GSE139612) were analyzed and found that DEGs were mainly involved in immune signaling pathways, apoptosis, and ER stress through GO and KEGG enrichment. Vitexin blocked the production of ROS and increased the activity of antioxidant enzymes (SOD, GSH-PX, and CAT) via activation of PPARγ in vivo and in vitro. In addition, vitexin reduced the production of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and inhibited apoptosis in MAC-T cells and mouse mammary tissues infected with Staphylococcus aureus. Moreover, vitexin decreased the expression of PDI, Ero1-Lα, p-IRE1α, PERK, p-eIF2α, and CHOP protein but increased BiP in both mammary gland cells and tissues challenged by S. aureus. Western blot results also found that the phosphorylation levels of JNK, ERK, p38, and p65 were reduced in vitexin-treated tissues and cells. Vitexin inhibited the production of ROS through promoting PPARγ, increased the activity of antioxidant enzymes, and reduced inflammatory cytokines and apoptosis by alleviating ER stress and inactivation MAPKs and NF-κB signaling pathway. Vitexin maybe have great potential to be a preventive and therapeutic agent for mastitis.

Figure 1

DEG analysis of S. aureus-induced mastitis from GEO database. (a) The Venn diagram displayed the distribution of DEGs in two GEO datasets (GSE94056 and GSE139612). (b) KEGG enriched these common DEG pathways. (c–e) GO enrichment analysis on 283 DEGs, including biological process, cellular components, and molecular functions. (f) The interaction between 283 DEGs was analyzed through the STRING database. (g) Emphasized the interaction relationship with CHOP- (DDIT3-) related DEGs.

Figure 2

Network pharmacology analysis of vitexin. (a) The chemical structural formula of the searched molecule. (b) The target classes of vitexin. (c) The potential targets of vitexin were predicted on the SwissTarget Prediction. (d) The common target genes of vitexin and S. aureus-induced mastitis.

Figure 3

The purified substance vitexin had no effect on the viability of MAC-T cells. (a) The chemical structure of vitexin. (b) HPLC for purity inspection of vitexin. (c) CCK-8 assessed the effect of vitexin on the viability of MAC-T cells.

Figure 4

Vitexin ameliorated cell damage on S. aureus challenged-MAC-T cells. (a) MAC-T cells were stimulated with S. aureus at MOI of 100 for 6 h. And then, different concentrations of vitexin (10, 20, and 40 μM) were added to coincubate for 24 h. Flow cytometry was used to evaluate the effect of vitexin on the apoptosis of MAC-T cells stimulated by S. aureus. The horizontal axis represented Annexin V-FITC, and the vertical axis expressed PI. (b) Flow cytometry results were statistically analyzed for apoptosis rate by the GraphPad Prism 9.00 software. (c–e) The cells were dealt with S. aureus and/or vitexin; the relative expression of TNF-α, IL-1β, and IL-6 mRNA was examined by RT-qPCR. GAPDH served as an internal reference gene. (f–h) The proinflammatory factors produced were tested by ELISA. Data were presented as means ± SEM of three independent experiments. #p < 0.01 vs. the control group. ^∗p < 0.05 vs. the S. aureus group; ^∗∗p < 0.01 vs. the S. aureus group.

Figure 5

Vitexin inhibited ROS production on S. aureus stimulated-MAC-T cells. (a) The cells were stimulated with S. aureus for 6 h and then treated with different concentrations of vitexin (10, 20, and 40 μM) for 24 h. ROS red fluorescence was displayed with DHE probe. (b) ROS levels were quantified by the ImageJ software. Relative ROS levels were expressed as red fluorescence intensity/cell number. (c) The level of T-AOC. (d) The activity of SOD. (e) The activity of GSH-PX. (f) The activity of CAT. (g) The concentration of MDA. (h) Western blot detection of the expression level of PPARγ. (i) PPARγ gray values were measured by the ImageJ software. (j) MAC-T cells were pretreated with GW9662 (10 μM) or GW1929 (10 μM) for 30 min to downregulate or upregulate the expression of PPARγ, respectively, then stimulated with S. aureus to mimic inflammatory stimulation, and finally treated with vitexin. ROS was detected with DHE probe. Data was expressed as means ± SEM of three independent experiments. #p < 0.01 vs. the control group. ^∗p < 0.05 vs. the S. aureus group; ^∗∗p < 0.01 vs. the S. aureus group.

Figure 6

Vitexin suppressed the activation of MAPK and NF-κB signaling pathways by blocking the ER stress caused by S. aureus on MAC-T cells. (a) The protein levels of PDI, BiP, Ero1-Lα, p-IRE1α, PERK, p-eIF2α, and CHOP were detected by Western blot on MAC-T cells. (c) The expression levels of JNK, ERK, p38, and p65 proteins in MAC-T cells were determined by Western blot. (b, d) Protein gray values were measured by the ImageJ software. Data was expressed as means ± SEM of three independent experiments. #p < 0.01 vs. the control group. ^∗p < 0.05 vs. the S. aureus group; ^∗∗p < 0.01 vs. the S. aureus group.

Figure 7

Vitexin improved S. aureus-induced mastitis in mice. All mouse mammary gland tissues were stimulated with S. aureus for 24 h, then intraperitoneally injected with different concentrations of vitexin (15, 30, and 60 mg/kg) three times. (a) Histopathological analysis of mammary gland tissues with H&E staining. Left images scale bar = 100 μm, right large images scale bar = 50 μm. (b) The activity of MPO. (c) The expression of TNF-α, IL-1β, and IL-6 mRNA in vivo was measured by RT-qPCR. GAPDH was used as an endogenous control. (d) The proinflammation cytokines TNF-α, IL-1β, and IL-6 in mammary gland tissues were detected by ELISA. Data was expressed as means ± SEM of three independent experiments. #p < 0.01 vs. the control group. ^∗p < 0.05 vs. the S. aureus group; ∗∗p < 0.01 vs. the S. aureus group.

Figure 8

Vitexin reduced ROS production in mammary tissues of mice infected with S. aureus. (a) The level of tissue ROS. (b) The level of T-AOC. (c) The activity of SOD. (d) The activity of GSH-PX. (e) The activity of CAT. (f) The concentration of MDA. (g) PPARγ protein was examined with Western blot. (h) PPARγ gray values were calculated by the ImageJ software. Data was expressed as means ± SEM of three independent experiments. #p < 0.01 vs. the control group. ^∗p < 0.05 vs. the S. aureus group; ^∗∗p < 0.01 vs. the S. aureus group.

Figure 9

Vitexin relieved ER stress to inactivate MAPK and NF-κB signaling pathways in S. aureus-induced mastitis on mice. (a) The protein levels of PDI, BiP, Ero1-Lα, p-IRE1α, PERK, p-eIF2α, and CHOP in mammary gland tissues. (c) The expression levels of JNK, ERK, p38, and p65 proteins in mammary gland tissues. (b, d) Protein gray values were measured by the ImageJ software. Data was expressed as means ± SEM of three independent experiments. #p < 0.01 vs. the control group. ^∗p < 0.05 vs. the S. aureus group; ^∗∗p < 0.01 vs. the S. aureus group.

Figure 10

Schematic diagram of the therapeutic effect of vitexin on Staphylococcus aureus-induced mastitis. Vitexin inhibited the production of ROS by promoting PPARγ activity, increased the activity of antioxidant enzymes, and reduced inflammatory cytokines and apoptosis by alleviating ER stress and inactivation MAPKs and NF-κB signaling pathways.