Thioredoxin-2 (TXN2) suppresses hydrogen peroxide-activated nuclear factor kappa B signaling via alleviating oxidative stress in bovine adipocytes

During the periparturient period, both oxidative stress and inflammation of adipose tissue are considered high risk factors for metabolic disorder of dairy cows. Oxidative stress can activate transcription factor nuclear factor kappa B (NF-κB ) , which lead to the upregulation of genes involved in inflammatory pathways. Thioredoxin 2 (TXN2) is a mitochondrial protein that regulates cellular redox by suppressing mitochondrial reactive oxygen species (ROS) generation in nonruminant, whereas the function of TXN2 in bovine adipocytes was unclear. Thus, the objective of this study was to evaluate how or by which mechanisms TXN2 regulates oxidative stress and NF-κB signaling pathway in bovine adipocytes. Bovine pre-adipocytes isolated from 5 healthy Holstein cows were differentiated and used for 1) treatment with different concentrations of hydrogen peroxide (H 2 O 2 ; 0, 25, 50, 100, 200 or 400 μ M ) for 2 h; 2) transfection with or without TXN2 small interfering RNA (si-TXN2) for 48 h and then treated with or without 200 μ M H 2 O 2 for 2 h; 3) transfection with scrambled negative control siRNA (si-control) or si-TXN2 for 48 h, and then treatment with or without 10 m M N-acetylcysteine (NAC) for 2 h; 4) transfection with or without TXN2 -overexpressing plasmid for 48 h and then treatment with or without 200 μ M H 2 O 2 for 2 h. High concentrations of H 2 O 2 (200 and 400 μ M ) decreased protein and mRNA abundance of TXN2 , reduced total antioxidant capacity (T-AOC) and adenosine triphosphate (ATP) content in adipocytes. Moreover, 200 and 400 μ M H 2 O 2 reduced protein abundance of inhibitor of kappa B α (IκBα ) , increased phosphorylation of NF-κB and upregulated mRNA abundance of tumor necrosis factor-α ( TNFA ) and interleukin-1B ( IL-1B ), suggesting that H 2 O 2 -induced oxidative stress and activated NF-κB signaling pathway. Silencing of TXN2 increased intracellular ROS content, phosphorylation of NF-κB and mRNA abundance of TNFA and IL-1B , decreased ATP content and protein abundance of IκBα in bovine adipocytes. Knockdown of TXN2 aggravated H 2 O 2 -induced oxidative stress and inflammation. In addition, treatment with antioxidant NAC ameliorated oxidative stress and inhibited NF-κB signaling pathway in adipocytes transfected with si-TXN2. In bovine adipocytes treated with H 2 O 2 , over-expression of TXN2 reduced the content of ROS and elevated the content of ATP and T-AOC. Overexpression of TXN2 alleviated H 2 O 2 -induced inflammatory response in adipocytes, as demonstrated by decreased expression of phosphorylated NF-κB, TNFA, IL-1B , as well as increased expression of IκBα. Furthermore, the protein and mRNA abundance of TXN2 was lower in adipose tissue of dairy cows with clinical ketosis. Overall, our studies contribute to the understanding of the role of TXN2 in adipocyte oxidative stress and inflammatory response


INTRODUCTION
Oxidative stress refers to the imbalance between oxidative and antioxidative processes in cells (Agarwal et al., 2003).In adipocytes, lipolysis generates reactive oxygen species (ROS), including hydroxyl radical, superoxide anion, and hydrogen peroxide (H 2 O 2 ; Rains and Jain, 2011).Of note, in conditions with intense and protracted lipolysis, the production of ROS rapidly depletes antioxidant systems, and oxidative stress develops (Pizzino et al., 2017).During the periparturient period, lipolysis causes a reduction of adipose tissue (AT) mass, which may exceed 30% of the volume (i.e., weight) of adipose depots in dairy cows (Akter et al., 2011).Thus, dairy cows experience oxidative stress of AT around the time of calving, especially in dairy cows with ketosis or fatty liver (Sahoo et al., 2009;Li et al., 2016).
Thioredoxin-2 (TXN2) suppresses hydrogen peroxide-activated nuclear factor kappa B signaling via alleviating oxidative stress in bovine adipocytes Xue Hao,1 Mingchao Liu,2 Xiao Zhang, 1 Hao Yu, 1 Zhiyuan Fang, 1 Xinxing Gao, 1 Meng Chen, 1 Qi Shao, 1 Wenwen Gao, 1 Lin Lei, 1 Yuxiang Song, 1 Xinwei Li, 1 Guowen Liu, 1 and Xiliang Du1* The intracellular ROS are mostly derived from mitochondrial superoxide, which can be converted to H 2 O 2 via superoxide dismutase (McArdle et al., 2004).Key proteins involved in scavenging H 2 O 2 are members of the thioredoxin (TXN) and the glutathione systems (Lu and Holmgren, 2012).Thioredoxin-2 (TXN2), one of the indispensable components of mitochondrialspecific TXN system, is a small mitochondrial redox protein essential for the control of mitochondrial ROS homeostasis (Yoshioka et al., 2006).A previous study demonstrated that heterozygous TXN2-deficient mice exhibit decreased mitochondrial electron transport chain activity and adenosine triphosphate (ATP) production and increased mitochondrial ROS generation in the liver (Pérez et al., 2008).In addition, overexpression of TXN2 decreased ROS level in rat embryonic ventricular myocytes (Li et al., 2017).Kweh et al. (2021) reported that vitamin D increased antioxidant activity in bovine monocytes via enhancing TXN system.Adipocyte-specific TXN2 deletion increased mitochondrial ROS generation in AT and elevated blood levels of free fatty acids in mice (He et al., 2021).However, the role of TXN2 in regulating oxidative stress of bovine adipocyte is not well known.
Studies on nonruminants revealed that localized inflammation plays an important role in coordinating the lipid metabolism and adipokine secretion of adipocytes (Toczylowski et al., 2019;Trayhurn, 2022).Dairy cows experience localized inflammation of AT during the transition period, as evidenced by infiltration of macrophages and upregulation of proinflammatory biomarkers (Vailati-Riboni et al., 2016;Depreester et al., 2018).Furthermore, it was reported that nuclear factor kappa B (NF-κB) signaling was overactivated in AT of ketotic cows (Fan et al., 2021).In rodents, TXN2 deficiency induced macrophage infiltration in AT (He et al., 2021).In contrast, overexpression of TXN2 significantly reduced the production of interleukin-1B (IL-1B) and tumor necrosis factor-α (TNF-α) in murine macrophage (Wang et al., 2020).Because ROS act as central regulators of inflammatory response (Park et al., 2015), we hypothesized that TXN2 affects mitochondrial ROS generation and contributes to inflammatory response of bovine adipocytes.Thus, the objective of this study was to evaluate the effects of oxidative stress inducer H 2 O 2 on TXN2 function and NF-κB inflammatory pathway, as well as the role of TXN2 in regulating oxidative stress and inflammation of bovine adipocytes.

Ethics
The use of animals and experimental procedures were approved by the Ethics Committee on the Use and Care of Animals of Jilin University (Changchun, China, SY202208003).

Isolation of Bovine Pre-adipocytes
All cows received routine physical examinations by the attending veterinarian to ensure there were no clinical diseases, such as hyperketonemia, laminitis, mastitis, hypocalcemia, and endometritis.Five healthy Holstein cows with similar BCS (median = 3.25, range = 3-3.5),DIM (median = 14, range = 10-18) and number of lactations (median = 3, range = 2-4) were enrolled in this study.The values (mean ± SEM) for serum concentrations of free fatty acids, BHB and glucose in cows were 0.43 ± 0.07 mM, 0.69 ± 0.13 mM and 3.75 ± 0.14 mM, respectively.Subcutaneous AT (1 to 2 g) was collected at 14 (±4) d postpartum from the tailhead depot of all cows on the same day through the methods described previously (Xu et al., 2019).The resulting tissue was rinsed in sterile PBS containing penicillin (2,500 U/mL) and streptomycin (2,500 μg/mL).Then, the resulting tissue was cut into small pieces of approximately 1 mm 3 and digested using 50 mL of Dulbecco's modified Eagle medium (DMEM)/F12 digestion solution containing collagenase type I (1 mg/mL; SH30023.01;HyClone, Logan, Utah, USA) at 37°C in a slightly shaking water bath for 1 h.The mixture was removed from the digested tissue fluid through 80-and 40-μm cell strainers and the filtrate was centrifuged at 175 × g at room temperature for 10 min.The residual erythrocytes were removed by adding ammonium-chloride-potassium lysis buffer (C3702; Beyotime Biothechnology Inc., Jiangsu, China) into the precipitate and centrifuging at 175 × g at room temperature for 10 min.The supernatant was discarded, and the pellet was resuspended with basic culture medium (BCM), which was DMEM/F12 with 10% fetal bovine serum and 1% penicillin/streptomycin.After cell counting, the cell suspension was adjusted to a concentration of 1 × 10 4 cells/mL and inoculated in a cell culture flask.The culture was then incubated at 37°C and 5% CO 2 in a cell incubator for 24 h.The medium was replaced to remove nonadherent cells and tissue residues.The BCM was replaced every other day.The pre-adipocytes from different cows were not pooled and all the experiments were repeated in 5 different cows.The minimum and maximum passage numbers of pre-adipocytes were 3 and 8, respectively.

Cell Differentiation
To induce differentiation of pre-adipocytes into mature adipocytes, primary cells were seeded in cell culture plates.The BCM was discarded after cell aggregation reached ~70%.Then, freshly prepared differentiation culture medium (DCM) 1, which was a final concentration of 0.5 mM 3-isobutyl-1-methylxanthine (I-7018; Sigma-Aldrich, MO, USA), 1 μM dexamethasone (D-4902; Sigma-Aldrich), and 1 μg/mL insulin (I-5500; Sigma-Aldrich) in BCM, was used to induce differentiation.After 48 h of culture, DCM1 was discarded and replaced by DCM2, which was a final concentration of 1 μg/mL insulin in BCM, to maintain the differentiation culture.The DCM2 was replaced every other day for 10 d until visible lipid droplets appeared in the cells, a sign that adipocytes were differentiated.The number of mature adipocytes was 4 × 10 5 per well of a 6-well plate.

Cell Treatment
After the adipogenic induction, experiments with differentiated adipocytes were divided into 4 sections: (1) adipocytes were treated with different concentrations of H 2 O 2 (0, 25, 50, 100, 200 or 400 μM; H1009; Sigma-Aldrich) for 2 h; (2) adipocytes were transfected with or without TXN2 small interfering RNA (si-TXN2) for 48 h and then treated with or without 200 μM H 2 O 2 for 2 h; (3) adipocytes were transfected with scrambled negative control siRNA (si-control) or si-TXN2 for 48 h, and then treated with or without 10 mM N-acetylcysteine (NAC; A9165; Sigma-Aldrich) for 2 h; (4) adipocytes were transfected with or without TXN2overexpressing plasmid for 48 h and then treated with or without 200 μM H 2 O 2 for 2 h.Before H 2 O 2 treatment, adipocytes were serum-starved in DMEM/F12 medium for 12 h.

Detection of Total Antioxidant Capacity (T-AOC)
The T-AOC of the adipocytes is defined as the total levels of various antioxidants.In the present study, T-AOC was measured according to the instructions of the T-AOC Assay Kit with a rapid 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) method (S0121; Beyotime Biothechnology Inc.).Briefly, adipocytes were disrupted by using ultrasonication.Then the cell lysate was collected by centrifuging at 4°C, 12,000 × g for 5 min.The protein concentration was determined using the bicinchoninic acid (BCA) Protein Assay Kit (P0012; Beyotime Biothechnology Inc.).Subsequently, 10 μL of each sample were mixed with 170 μL ABTS detection solution, which was prepared according to the manufacturer's protocol.After incubation at room temperature for 6 min, the absorbance at 414 nm was recorded.Trolox was used as a standard antioxidant reagent to generate a standard curve.According the manufacturer's protocol, the T-AOC was calculated and expressed as the trolox-equivalent antioxidant capacity (mmol/g of protein).

Determination of ROS Content
The intracellular ROS concentrations were measured using the peroxide-sensitive fluorescent probe 2′7′-dichlorofluorescein diacetate (S0033S; Beyotime Biothechnology Inc.).The cells were exposed to serumfree medium containing 10 μM DCFH-DA in the dark for 30 min and then washed 3 times with cold PBS.The fluorescence was measured by flow cytometry (BD FACSCalibur, Becton Dickinson, Mountain View, USA).

Adenosine Triphosphate (ATP) Content Determination
The ATP content was determined using an ATP analysis kit (A095; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions.The ATP values were corrected by protein content.Total protein concentration was

RNA Isolation and Quantitative Reverse-Transcription PCR
Total RNA was isolated from adipocytes using RNAiso Plus (9109; TaKaRa Biotechnology Co. Ltd., Dalian, China) according to the manufacturer's instructions.The RNA concentration and quality were measured using a K5500 MicroSpectrophotometer (Beijing Kaiao Technology Development Ltd., Beijing, China) and electrophoresis (1% agarose gels).The OD 260 /OD 280 ratio of the total RNA was determined to be 1.83 to 1.96 and met the specified purity requirements.Then 1 μg of total RNA in each sample was reverse-transcribed to cDNA (TaKaRa Biotechnology Co. Ltd.) according to the supplier's protocol.The SYBR green plus reagent kit (RR420A; TaKaRa Biotechnology Co. Ltd.) was used to prepare a 20 μL mixture and mRNA abundance was detected with a 7500 Real-Time PCR System (Applied Biosystems Inc., Waltham, MA).The reaction conditions were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.Relative transcription of each target gene was normalized against the geometric mean of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB) both of which were stably expressed across treatment groups (Supplemental Figure S1).Relative gene expression was calculated with the 2 -ΔΔCT method.The primer pairs used in this study were designed with Primer Express software 3.0 (Applied Biosystems Inc.) according to bovine reference sequences from the National Center for Biotechnology Information (Supplemental Table S1).

Animal and AT Collection
Detailed information on the healthy and ketotic cow selection and AT collection was reported in our previous study (Fan et al., 2021).

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 8.0 (Graph Pad Software) or SPSS 23.0 software (IBM Corp.).Results are presented as the means ± standard error of the mean (SEM).All data were tested for normality and homoscedasticity using the Shapiro-Wilk and Levene tests, respectively.Linear and quadratic contrasts were conducted to evaluated dosedependent effects.Data were analyzed by t test in cases of 2 comparison groups.One or 2-way ANOVA was performed for multiple comparisons with Bonferroni correction for data meeting homogeneity of variance.P < 0.05 was considered significant and P < 0.01 markedly significant.

Overexpression of TXN2 Alleviates Oxidative Stress and Inflammatory Response in H 2 O 2 -Treated Bovine Adipocytes
The overexpression efficiency of pCMV-FLAG-TXN2 was 192% (P < 0.01, Figure 4A and B).In the absence of H 2 O 2 , overexpression of TXN2 slightly decreased ROS content (P < 0.05, Figure 4C) and marginally increased ATP content (P < 0.05, Figure 4D) and T-AOC (P < 0.05, Figure 4E) in adipocytes.In addition, overexpression of TXN2 did not alter the protein abundance of p-NF-κB (Figure 4F and G) and IκBα (Figure 4F and H) and the mRNA abundance of TNFA (Figure 4I) and IL-1B (Figure 4J).

Expression of TXN2 in AT
Protein abundance of TXN2 in AT used for pre-adipocytes isolation was similar (Figure 5A).The protein (P < 0.01, Figure 5B and C) and mRNA (P < 0.01, Figure 5D) abundance of TXN2 were lower in AT from clinical ketosis cows than in healthy cows.

DISCUSSION
During early lactation, oxidative stress and inflammatory response of AT increase the risk of metabolic diseases of dairy cows (Contreras et al., 2017;Zachut and Contreras, 2022).The main sources of cellular ROS are mitochondria (Piao et al., 2019).Meanwhile, mitochondria are equipped with several antioxidant enzyme systems posed to detoxify ROS (Lismont et al., 2015;Mailloux, 2018).In nonruminants, TXN2 is the main ROS-scavenging enzyme in mitochondria that maintains mitochondrial function (Sugano et al., 2013;Chen et al., 2017).In the present study, decreased TXN2 gene expression was accompanied by ROS overproduction and NF-κB signaling pathway overaction in bovine adipocytes.Moreover, overexpression of TXN2 attenuated oxidative stress and inflammatory response in H 2 O 2 -treated adipocytes, suggesting that TXN2 is a potential target for modulation of redox homeostasis in bovine adipocytes.
In the AT of healthy cows, excessive ROS can be scavenged by various antioxidative defense mechanisms (Yu et al., 2023).However, in dairy cows with prolonged and sustained lipolysis, the production of ROS depletes antioxidant systems, and oxidative stress develops (Mavangira and Sordillo, 2018).As a mitochondrial member of the TXN antioxidant defense system, TXN2 interacts with TXN reductase 2 and peroxiredoxin 3 to scavenge H 2 O 2 and offer protection against oxidative stress in nonruminant (Pérez et al., 2008;He et al., 2021).In the present study, protein and mRNA abundance of TXN2 was lower in AT of dairy cows with clinical ketosis.Moreover, we observed that incubation of bovine adipocytes with 50 and 100 μM H 2 O 2 increased intracellular ROS content and TXN2 expression.In addition, other studies with bovine mammary epithelial cells (Ma et al., 2018) and dairy cow (Xu et al., 2021) or calf adipocytes (Sun et al., 2019) have revealed no deleterious effects on cell viability with 100 μM H 2 O 2 treatment.Although speculative at this time, enhanced TXN2 protein expression could be the adaptation mechanism to combat oxidative stress and maintain cell viability.This idea is partly supported by the observation that enforced TXN2 expression reduced ROS content and enhanced antioxidant capacity of bovine adipocytes treated with high concentration of H 2 O 2 (200 μM).Similarly, Huang et al. (2015) reported that TXN2 inhibited mitochondrial ROS generation to maintain mouse cardiac function.Thus, our results combined with previous study suggest a role for TXN2 in the modulation of ROS homeostasis in ruminants and TXN2 may be a novel target for preventing oxidative stress in adipocytes.
Because mitochondria are both a generator of and target for ROS, oxidative stress is closely linked to mitochondria dysfunction (Silwal et al., 2020).In our study, high concentration of H 2 O 2 (200 and 400 μM) not only increased intracellular ROS content but reduced TXN2 expression, ATP content and antioxidant capacity in bovine adipocytes.Moreover, knockdown of TXN2 induced oxidative stress and decreased ATP content in bovine adipocytes.In line with these observations, deletion of TXN2 in murine adipocytes resulted in severe mitochondrial damage, as evidenced by fragmentation and cristae disruption of mitochondria, as well as decreased mitochondrial DNA copy number and ATP production (He et al., 2021).These studies suggest that dysregulation of TXN2 contributes to development of mitochondrial dysfunction and oxidative stress in adipocytes.Given that oxidative stress of AT is one of the risk factors leading to metabolic dysfunction (Abou-Rjeileh and Contreras, 2021;Zachut and Contreras, 2022), more research is needed to identify It is well established that oxidative stress can cause inflammation in ruminants (Zachut and Contreras, 2022) and nonruminants (Mittal et al., 2014).In humans and rodents, ROS induces the degradation of the inhibitor IκB that holds NF-κB in an inactive state in the cytoplasm, freeing NF-κB to translocate to the nucleus and drive the expression of proinflammatory genes (Morgan and Liu, 2011).We observed that H 2 O 2 activated NF-κB inflammatory signaling pathway in bovine adipocytes, as evidenced by decreased expression of IκBα and increased expression of phosphorylated NF-κB and mRNA expression of TNFA and IL-1B.Thus, the greater phosphorylation of NF-κB along with upregulation of TNFA and IL-1B in AT of ketotic cows might be associated with oxidative stress (Fan et al., 2021).
An activation of NF-κB signaling could induce the upregulation of NLRP3 and IL-1B leading to formation of NLPR3 inflammasome (Bauernfeind et al., 2009).Notably, ROS are shown to be critical mechanism triggering NLRP3 inflammasome formation and activation (Abais et al., 2015).Moreover, in mice, TNF-α and IL-1B produced during activation of the NF-κB and NLRP3 signaling pathways can lead to mitochondria dysfunction and oxidative stress (Zhou et al., 2011;Kastl et al., 2014).This leads to a vicious circle where ROS activates NF-κB pathway and NLRP3 inflammasome, which induces proinflammatory cytokines overproduction and further promotes oxidative stress.Thus, such as vicious circle may promote development of metabolic disorder of AT in dairy cows during early lactation.
In this study, knockdown of TXN2 induced oxidative stress and inflammatory response whereas antioxidant NAC attenuated this effect in bovine adipocytes.Consistently, treatment with mitochondrial ROS scavenger mito-TEMPO abrogated NF-κB and NLRP3 inflammasome activation in TXN2 knockout mouse adipocytes (He et al., 2021;Huang et al., 2022).Thus, current and previous data indicate that ROS overproduction induced by H 2 O 2 treatment or downregulation of TXN2 may contribute to the occurrence of overt inflammatory responses in bovine adipocyte.
Although our previous study demonstrated that the NF-κB signaling pathway was overactivated in adipose tissues of dairy cows with ketosis (Fan et al., 2021), the inflammatory phenotype remained unclear.Furthermore, adipose tissue is heterogeneous; it is comprised of an array of cell types including adipocytes, preadipocytes, immune system cells, endothelial cells and stromal cells.Depreester et al. (2018) reported infiltration of macrophages in adipose tissue was associated with the proinflammatory status of transition dairy cows.Thus, whether TXN2 regulate the inflammatory response of adipocyte in adipose tissue of ketotic cows needs further investigation.
In AT, sustained lipolysis produces excessive ROS and leads to a remodeling process characterized by an inflammatory response (Zachut and Contreras, 2022).In this study, knockdown of TXN2 aggravated while overexpression of TXN2 alleviated H 2 O 2 -induced oxidative stress and inflammatory response in bovine adipo-cytes.However, potential associations between TXN2 function and the resulting effects on adipocyte lipolysis have been neglected in the present study.Furthermore, the total ROS content and T-AOC, not specific mitochondrial-derived ROS and antioxidant capacity, were measured.Thus, based on the present data, we cannot fully evaluate the status of oxidative stress in mitochondrion of adipocyte.Those are limitations in present study.
Hao et al.: TXN2 INHIBITS OXIDATIVE STRESS AND INFLAMMATION liner and quadratic, Supplemental Table Hao et al.: TXN2 INHIBITS OXIDATIVE STRESS AND INFLAMMATION
High concentration of H 2 O 2 inhibited TXN2 expression, induced oxidative stress and activated NF-κB signaling pathway in bovine adipocytes.Knockdown of TXN2 aggravated, whereas overexpression of TXN2 Hao et al.: TXN2 INHIBITS OXIDATIVE STRESS AND INFLAMMATION

Figure 5 .
Figure 5. Expression of thioredoxin-2 (TXN2) in adipose tissue (AT).(A) Representative Western blots of TXN2 in AT from 5 experimental cows.(B) Representative Western blot of TXN2 in AT from healthy cows (n = 6) and cows with clinical ketosis (n = 6).(C) Quantification of protein level of TXN2 in AT. (C) Quantification of protein level of TXN2 in AT from healthy cows (n = 15) and cows with clinical ketosis (n = 15).The data were analyzed using t-tests and expressed as mean ± SEM *P < 0.05, **P < 0.01.