Mao Peng

Bovine mastitis caused by Klebsiella pneumoniae (K. pneumoniae) poses a serious threat to dairy production and milk quality, with mitochondrial dysfunction acting as a core pathological driver. As an important transcription factor regulating mitochondrial function, Forkhead box O 3 (FOXO3) plays a role in mastitis caused by K. pneumoniae infection that has not been deeply studied. This research established in vivo and in vitro mastitis models through K. pneumoniae infection. The results showed that K. pneumoniae upregulates FOXO3 mRNA but reduces its protein abundance. Mechanistically, K. pneumoniae-activated folliculin-interacting protein 1 (FNIP1) inhibits AMP-activated protein kinase (AMPK)-mediated FOXO3 phosphorylation to block its nuclear translocation, while upregulating E3 ligase mouse double minute 2 (MDM2) to promote FOXO3 ubiquitination and degradation. FOXO3 overexpression rescues mitochondrial dysfunction, restores milk synthesis and alleviates inflammation, verifying that FOXO3 activation is a promising strategy for mastitis prevention and control.

Bovine mastitis caused by Klebsiella pneumoniae (K. pneumoniae) severely threatens the dairy industry. The mitochondrial dysfunction plays a pivotal role in the pathogenesis of mastitis caused by K. pneumoniae. Forkhead box O 3 (FOXO3), a key transcription factor known to regulate mitochondrial function, has not been explored in the mastitis induced by K. pneumoniae. This study aims to elucidate the regulatory mechanism of FOXO3 in K. pneumoniae infection-induced mastitis. In vivo and in vitro mastitis models were established using K. pneumoniae infection. K. pneumoniae infection significantly increased FOXO3 mRNA expression but reduced its protein levels in bovine mammary epithelial cells (BMECs) and mammary tissues. Mechanistically, K. pneumoniae activated folliculin-interacting protein 1 (FNIP1) to inhibit AMP-activated protein kinase (AMPK)-mediated FOXO3 phosphorylation, thereby reducing nuclear FOXO3 localization. Concurrently, K. pneumoniae upregulated mouse double minute 2 (MDM2), promoting FOXO3 ubiquitination and degradation via the ubiquitin-proteasome system. Inhibition of FNIP1 attenuated FOXO3 ubiquitination by suppressing MDM2 expression. FOXO3 overexpression ameliorated mitochondrial dysfunction, reduced milk synthesis defects, and alleviated inflammation induced by K. pneumoniae. In summary, these findings demonstrate that K. pneumoniae infection activates FNIP1, which subsequently reduces FOXO3 activity by inhibiting AMPK-mediated phosphorylation and promoting FOXO3 ubiquitination via the E3 ubiquitin ligase MDM2. These dual effects of FNIP1 on FOXO3 contribute to mitochondrial dysfunction, inhibit milk synthesis, and exacerbate inflammation, suggesting that activation of FOXO3 may represent a promising strategy for the prevention and control of K. pneumoniae-induced mastitis.

Klebsiella pneumoniae-induced mastitis poses a severe threat to livestock health with limited therapeutic options. Selenium nanoparticles (SeNP) offer promising biocompatibility and antioxidant potential, but their efficacy and mechanism against bacterial mastitis remain unexplored. This study demonstrated that the oral administration of SeNP (20 μg/kg BW) attenuated K. pneumoniae-induced mastitis in goats. The treatment reduced histopathological damage and neutrophil infiltration. Furthermore, it restored anti-inflammatory IL-10 mRNA expression and suppressed proinflammatory IL-1β and IL-6 mRNA levels. Ultrasonography and Doppler analysis confirmed that mammary hemodynamics and echotextural abnormalities were improved. The SeNP supplementation contributed to increased concentrations of selenium in the serum, enhanced the activity of mammary glutathione, activated the peroxiredoxin 6 (PRDX6)/selenophosphate synthetase 2 (SEPHS2) axis, and upregulated glutathione peroxidase 4 (GPX4) expression. Immunofluorescence revealed that SeNP strengthened the PRDX6-SEPHS2 interaction to improve the activity of GPX4. Crucially, SeNP suppressed ferroptosis by inhibiting nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy, reducing iron overload and lipid peroxidation, and attenuating NCOA4-ferritin heavy chain 1 colocalization. The findings established that SeNP concurrently target inflammatory, antioxidant, hemodynamic, and ferroptotic pathways to prevent bacterial mastitis, suggesting a novel intervention for the prevention and control of mastitis.

Klebsiella pneumoniae (K. pneumoniae), a prominent causative agent of mastitis in dairy cattle, remains enigmatic in its pathogenic mechanisms. This study aimed to reveal the effects of K. pneumoniae on mammary glands via the induction of ferroptosis, as well as the protective role of Ferrostatin-1 (Fer-1) against this pathogen-mediated damage in bovine mammary epithelial cells (BMECs). Holstein cows were used to establish an intramammary infection model of K. pneumoniae. In vitro, primary BMECs were treated with 10 μM Fer-1 and K. pneumoniae alone or in combination. The results showed that mammary glands infected with K. pneumoniae exhibited increased transcriptional levels of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α). Concurrently, significant elevations in iron, 4-hydroxynonenal, and reactive oxygen species (ROS) levels were observed. Conversely, K. pneumoniae infection downregulated nuclear factor erythroid 2-related factor 2 (Nrf2), cystine/glutamate antiporter (xCT), glutathione peroxidase 4 (GPX4), and glutathione levels. Following K. pneumoniae invasion, intracellular Fe²⁺ and lipid ROS accumulated in the BMECs, impeding activation of Nrf2/xCT/GPX4 signal transduction. Additionally, Fer-1 facilitated the nuclear translocation of Nrf2 protein, upregulating the protein levels of Nrf2/xCT/GPX4 while downregulation transcriptional levels of IL-1β, IL-6, IL-8, and TNF-α. In conclusion, Fer-1 alleviates K. pneumoniae-induced inflammatory factor activation and ferroptosis in BMECs via upregulation of the Nrf2/xCT/GPX4 pathway, supporting ferroptosis inhibition holds promise as a feasible therapeutic agent for the control of mastitis.

Klebsiella pneumoniae, a major bovine mastitis pathogen, disrupts mammary gland function through poorly understood mechanisms. This study demonstrates that K. pneumoniae infection triggers ferroptosis in bovine mammary epithelial cells (BMECs) via nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy, leading to lactation impairment. RNA sequencing revealed enrichment in ferroptosis, autophagy, and iron metabolism pathways. Infection time-dependently suppressed glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) expression, causing mitochondrial membrane potential collapse and lipid peroxidation. Crucially, K. pneumoniae activated NCOA4-dependent ferritin degradation, elevating intracellular Fe²⁺. NCOA4 knockdown alleviated ferroptosis, restored GPX4/SLC7A11 expression, reduced the bacterial load, and partially rescued lactation markers (fatty acid synthase, acetyl-CoA carboxylase 1, and β-casein). In vivo, infected mammary glands showed iron overload, 4-hydroxynonenal accumulation, lactation protein suppression, and pathological damage, correlating with NCOA4/ferritin heavy chain 1 upregulation. These findings establish NCOA4-mediated ferritinophagy as a key driver of K. pneumoniae-induced ferroptosis and lactation dysfunction, suggesting novel therapeutic targets for mastitis.

Meloxicam has been identified as an adjuvant therapeutic component in the management of bovine uterine diseases, exhibiting anti-inflammatory and antioxidant effects. However, the mechanisms underlying its antioxidant actions in the context of bovine uterine diseases remain incompletely understood. The objective of this research was to determine whether meloxicam exerts its antioxidant effects through the Nrf2/HO-1 signaling pathway. By employing N-acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), along with inhibitors directed against heme oxygenase-1 (HO-1) or nuclear factor erythroid 2-related factor 2 (Nrf2), we investigated the dynamic changes in oxidative stress markers (ROS and malondialdehyde) and antioxidant indices (comprising catalase, superoxide dismutase, and glutathione), as well as the expression profiles of Nrf2 and inflammation-associated genes and proteins in bovine endometrial epithelial cells (BEECs) subjected to lipopolysaccharide (LPS) stimulation. As a result, meloxicam alleviated the LPS-induced elevation of oxidative stress marker levels and the reduction in antioxidant enzyme activities and antioxidant substance contents in BEECs. Compared to NAC, meloxicam demonstrated superior efficacy in activating the Nrf2 pathway, with the promotion of NRF2 expression (~1.6-fold) and nuclear translocation. The pretreatment of cells with HO-1 or Nrf2 inhibitors markedly attenuated the antioxidant activity of meloxicam. In summary, meloxicam primarily alleviates LPS-induced oxidative stress through the activation of the Nrf2/HO-1 pathway in BEECs.

Background: In dairy cows, the stress-related cortisol level increases the susceptibility to postpartum uterine diseases. Oxidative stress is an important component of the disease process and causes morphological and functional alterations in the bovine endometrium. Selenium (Se) has an antioxidant property, and an appropriate Se supplementation is recommended to enhance bovine disease resistance. Methods and results: Here, we aimed to answer two questions: (1) how does cortisol affect the oxidative status of bovine endometrial cells; and (2) does Se supplementation protect cells from oxidative injury with a high cortisol condition? The oxidative stress of primary bovine endometrial epithelial cells (BEECs) was established by Escherichia coli lipopolysaccharide (LPS) stimulation, as marked by the increased oxidative markers and the suppressions of antioxidant indicators and Nrf2 signaling. In the absence of LPS, cortisol levels of 15 ng/mL showed a more significant antioxidative effect than cortisol levels of 5 and 30 ng/mL. In the presence of LPS, cortisol levels of 15 and 30 ng/mL elicited antioxidation, whereas 5 ng/mL of cortisol did not. Regardless of LPS stimulation, Se pretreatment of 1, 2, and 4 μM protected BEEC from oxidative stress, as evidenced by the decreased oxidative markers, increased antioxidant indices, and the activated Nrf2 signaling. With the presence of 30 ng/mL of cortisol, there was an enhanced Se antioxidant effect, which can be abolished by the block of cortisol receptor. Conclusions: Both cortisol and Se elicited antioxidant properties in BEEC through the Nrf2 pathway. In addition, the Se antioxidation was enhanced by cortisol.

Klebsiella pneumoniae (K. pneumoniae) is one of the pathogens causing clinical mastitis of bovine. Previous studies have demonstrated that mitochondrial damage and dysfunction are important mechanisms of mastitis in dairy cattle. Folliculin interacting protein 1 (FNIP1) is a major metabolic regulator of mitochondrial function with proinflammatory capabilities, but its role in K. pneumoniae-induced mastitis is yet to be elucidated. Thus, the studies were conducted to clarify the role of FNIP1-mediated mitochondrial function in mastitis caused by K. pneumoniae in vivo and in vitro. The experiments verified that K. pneumoniae caused decrease of milk fat and protein synthesis evidently in the mammary glands and bovine mammary epithelial cells (BMECs), accompanied by an imbalance in mitochondrial fission and fusion, increased mitochondrial permeability transition pore opening, decreased membrane potential and ATP content. While the enhancement of mitochondrial function alleviated K. pneumoniae-induced BMECs injury via relieving milk fat and protein dyssynthesis. Notably, transcriptomic analysis revealed that FNIP1 expression was upregulated in BMECs induced by K. pneumoniae. Further investigations revealed FNIP1 silencing improved milk synthesis by alleviating mitochondrial dysfunction caused by K. pneumoniae infection, and further inhibiting the activation of inflammatory factors, which in turn prompted the mammary recovery. In conclusion, K. pneumoniae inhibited mitochondrial function by activating FNIP1, which reducing the synthesis of milk fat and protein, thereby in turn lowers milk quality and induced mastitis. This study showed that FNIP1 has the potential as a novel target for the prevention and control of bovine mastitis.

Carbon fiber‐reinforced resin composites (CFRCs) utilizing vinyl ester (VE) resin as the matrix have found extensive applications in military equipment, transportation, sports and entertainment, wind power generation, and various other fields. However, the inherent inertness of the carbon fiber (CF) surfaces makes it difficult to form covalent bonds between VE resin and CFs, thereby limiting the application of CFs/VE composites. The sizing process is an effective method for the surface modification of CFs that improves the final mechanical properties of CFRCs. In this study, a novel organo‐soluble methacrylamide‐substituted para‐aramid, poly(p‐methacrylamino‐phenylene methacrylamino‐terephthalamide) (mPPTA), characterized by methacrylamide side groups attached to the phenyl moieties of its rigid‐rod‐like backbone, was used as a novel sizing agent for the surface modification of CFs in the fabrication of CFRCs via vacuum‐assisted resin infusion (VARI). CF fabrics were coated with mPPTA through solution impregnation. The CF‐matrix interfacial bonding was evaluated using a transverse fiber bundle tensile test, which indicated a 51.1% enhancement in interfacial bonding strength due to mPPTA. The application of merely 0.281 mg/cm² of mPPTA on each CF sheet resulted in significant improvements in flexural strength, flexural fracture total energy, interlaminar shear strength, and mode II interlaminar fracture toughness of the composites, with increases of 75%, 156.3%, 85.1%, and 90.1%, respectively. These enhancements surpass those reported for CFRCs modified using other sizing agents or matrix reinforcements. This method presents a new opportunity for fabricating of high‐performance VE resin‐based CFRCs using VARI.Highlights: A novel methacrylamide‐substituted rigid‐rod para‐aramid (mPPTA) was synthesized. mPPTA was synthesized by reacting methacryloyl chloride with aminated PPTA. mPPTA serves as a novel sizing agent for producing VE/CF composites through VARI. mPPTA significantly enhances the mechanical properties of the VE/CF composites.

Poly(p‐phenylene terephthamide) (PPTA) is an aromatic para‐polyamide with a rigid rod‐like main chain and superior intrinsic mechanical properties but is insoluble in all organic solvents. It has been demonstrated that introducing amino groups to the phenyl rings on the PPTA main chain yields organic‐soluble aminated para‐polyamide known as poly(p‐aminophenylene aminoterephthalamide) ((NH₂)₂‐PPTA). In this study, an organic‐soluble bismaleimide (BMI)‐grafted (NH₂)₂‐PPTA (BMI‐g‐(NH₂)₂‐PPTA) was synthesized by reacting the amino groups of (NH₂)₂‐PPTA with the double bonds of BMI via Michael addition reaction. BMI‐g‐(NH₂)₂‐PPTA was then employed as a new macromolecular reinforcing agent that significantly enhances the mechanical properties of BMI/2,2′‐diallyl bisphenol A (BD) resin. With the addition of merely 0.5 wt% of BMI‐g‐(NH₂)₂‐PPTA, the strength and toughness of both BD resin and BD/carbon fiber (CF) composite laminates are significantly improved without decreasing the heat resistance of the matrix. The uniform dispersion of BMI‐g‐(NH₂)₂‐PPTA in BD resin and its strong interaction with the matrix result in a more significant enhancement in the mechanical properties compared to directly incorporating (NH₂)₂‐PPTA into the matrix, providing a new and efficient method for enhancing the mechanical properties of BD resin and its CF composites.Highlights: A novel BMI‐grafted aminated para‐polyamide, BMI‐g‐(NH₂)₂‐PPTA, is synthesized. BMI‐g‐(NH₂)₂‐PPTA is organic‐soluble and can disperse uniformly in BD resin. BMI‐g‐(NH₂)₂‐PPTA at 0.5 wt% enhances the strength of BD resin by 50.8%. BMI‐g‐(NH₂)₂‐PPTA is more efficient than (NH₂)₂‐PPTA in strengthening BD resin. Mechanical properties of BD/CF laminates are also enhanced by BMI‐g‐(NH₂)₂‐PPTA.