Research Article
Progesterone inhibits lipopolysaccharide-induced oxidative stress through Nrf2/Keap1 pathway in bovine endometrial epithelial cells
https://doi.org/10.21203/rs.3.rs-1021821/v2
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progesterone
oxidative stress
lipopolysaccharide
bovine endometrial epithelial cells
Nrf2/Keap1
In modern dairy industry, endometritis is one of the common postpartum diseases of dairy cows, and can lead to infertility and even death. The disease is mainly caused by the invasion of pathogens such as Escherichia coli (E. coli) into the uterus (Wagener et al., 2017). Lipopolysaccharide (LPS) is a component of the outer membrane of E. coli, and has been used as a non-specific immune stimulant to induce inflammatory response (Cui et al., 2020). LPS has been observed to increase the level of reactive oxygen species (ROS) in bovine endometrial cells and induce cellular oxidative damage. LPS-induced release of inflammatory cytokines, such as interleukin 6 and tumor necrosis factor α, was involved in the pathogenesis of oxidative damage (Gugliandolo et al., 2020).
Oxidative stress refers to the imbalance between oxidation and antioxidation in bodies. When the cells cannot remove excess free radicals in time, the accumulation of oxidation intermediates aggravates tissue damage (Dandekar et al., 2015). ROS is a marker product of cell oxidative damage. ROS caused oxidative damage of Marc-145 cells (monkey embryonic kidney epithelial cells) by increasing inducible nitric oxide synthase and upregulating apoptotic genes (Liu et al., 2019). ROS attacks the phospholipid layer of biofilm to produce malondialdehyde (MDA) and inhibits the activity of antioxidant enzymes such as glutathione peroxidase (GSH-PX) (Kwiecien et al., 2014). LPS has been shown to upregulate ROS level and MDA content, and decrease the activities of superoxide dismutase (SOD) and GSH-PX in bovine mammary epithelial cells (Liu et al., 2021).The elevated levels of ROS and peroxiredoxin-1 have also been observed in LPS-treated bovine endometrial epithelial cells (Guo et al., 2019; Piras et al., 2017).
Progesterone can maintain pregnancy, thicken the endometrium, and inhibit the maternal immune rejection to fetus and plays an important role in maintaining sexual cycle and pregnancy (Standeven et al., 2020). Progesterone elicits an immunosuppressive function, and the pathogenesis of endometritis can be related to progesterone. Normally the progesterone level keeps low during early postpartum period. However, an early ovulation and the subsequent formation of corpus luteum, which generates progesterone, makes the animal more susceptible to uterine infection when the uterine involution is incomplete (Lewis, 2004; Opsomer et al., 2000). Progesterone has been observed to inhibit inflammation (Cui et al., 2020; Cui et al., 2022), but its effect on oxidative stress has not been reported in bovine endometrial cells. Progesterone has been found to play a neuroprotective role on the developing brain by reducing lipid peroxidation and fighting free radicals (Theis and Theiss, 2019), and has been reported to reduce oxidative damage and apoptosis of nerve cells through progesterone receptor membrane component 1 in mice (Guennoun, 2020).
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a newfound cellular antioxidant regulator, and is negatively regulated by the kelch like ECH associated protein 1 (Keap1) (Bellezza et al., 2018). The Nrf2/Keap1 pathway mediates the expression of a series of antioxidant enzymes and signal proteins and plays an important role in regulating drug metabolism, antioxidant defense, and oxidant signal. Nrf2 knockout mice were more susceptible to diseases related to oxidative pathology (Qiu et al., 2020). Activation of Nrf2/Keap1 pathway has been found to alleviate oxidative damage of RAW264.7 cells (mouse leukemic monocyte/macrophage cell line) (Lin et al., 2019), and has been demonstrated to participate in the diverse neuroprotective effect of progesterone on traumatic brain injury, so as to reduce brain edema, apoptosis and inflammatory response (Zhang et al., 2017). These reports indicated that progesterone alleviated oxidative damage by regulating Nrf2/Keap1 pathway. However, the mechanism of progesterone on LPS-induced oxidative damage of bovine endometrium is not clear.
In this study, the effect of progesterone on E. coli LPS-induced oxidative damage of primary bovine endometrial epithelial cells (BEEC) was determined by observing the changes in oxidative damage markers and antioxidative enzyme activities. The underlying mechanism was explored by detecting the related genes and proteins of Nrf2/Keap1 pathway, and by the use of progesterone receptor (PGR) antagonist mifepristone (RU486).
All experimental procedures were approved by the Animal Care and Use Committee of Yangzhou University (NSFC2020-SYXY24). The primary bovine endometrial epithelial cells were isolated and cultured as described from a previous report (Dong et al., 2018). The uterus of healthy nonpregnant cows was collected aseptically from a local slaughterhouse. The uterus was collected from the cows on days 1 to 4 of the estrous cycle because peripheral plasma progesterone concentration in these cows were basal. The determination of stage of the estrous cycle was based on a published literature (Cronin et al., 2012). It was necessary to ensure that the uterus was free of infection and disease. The uterine horns of healthy cows were collected and placed in an ice box at 4°C and were brought back to the laboratory. The uterine surface was disinfected with iodophor and 75% alcohol. Under sterile conditions, the uterine horn was cut into small pieces of 3 to 4 cm, and was rinsed repeatedly with phosphate-buffered saline (PBS, pH values from 7.2 to 7.4) containing 500 U/mL penicillin and 500 U/mL streptomycin. The uterine horn was cut longitudinally to expose endometrial tissue. The endometrial tissue was put into 0.1% streptoproteinase (P5147, sigma, USA) and then diluted in DMEM/F-12 (D8900, sigma, USA) at 4°C for 16 h. We use a sterile scalpel to scrape the surface of endometrium to collect cells on a clean bench. The obtained cell suspension was centrifuged at 100×g for 5 min and was washed with PBS for three times. The cells were resuspended in DMEM/F-12 containing 15% fetal bovine serum (Gibco, USA) and were cultured at 37°C with 5% CO2. The medium was changed after 24 to 48 h for routine culture. The BEEC could be obtained after around 3 days. The epithelial cell populations were determined to be more than 95% in purity. The medium was changed every 1 to 2 days until the cells reached about 90% confluence.
The E. coli LPS (L6529), progesterone (P0130), and RU486 (M8046) were purchased from Sigma-Aldrich. LPS lyophilized powder was dissolved in DMEM/F12 at a concentration of 1 mg/mL as stock solutions at -20°C. LPS was further diluted to 1 µg/mL by DMEM/F12 during experiment. Progesterone was dissolved in ethanol, and was then configured to a storage concentration of 100 µg/mL using DMEM/F12. Under physiological conditions, the concentration of progesterone in the serum of dairy cattle ranges from 1.03 to 5.1 ng/mL (Hasler et al., 1980), so the final progesterone concentration in the current study was 1, 3, and 5 ng/mL. RU486 is an antagonist of the classical PGR. The 50% inhibition concentration of RU486 as a PGR inhibitor was 0.2 nM (Jiang et al., 2006). RU486 was dissolved in ethanol and was stored at -20°C with the concentration of 118.5 µg/mL, and was further diluted to 35 ng/mL with DMEM/F12 during experiment. To determine the effect of progesterone on the LPS-induced oxidative damage of BEEC, the cells were treated with LPS (1 µg/mL), or cotreated with LPS and progesterone (1, 3, and 5 ng/mL). RU486 was used to reveal the underlying mechanism of progesterone on oxidative damage, and the cells were cotreated with LPS, progesterone (3 ng/mL), and RU486 (35 ng/mL). To detect the oxidative markers, the cells were collected 12 h post treatment. For analysis of gene expression, the cells were collected at 6 and 12 h. The changes in key protein levels of Nrf2/Keap1 signaling pathway were detected at 90 min.
ROS level was detected by ROS assay kit (S0033M, Beyotime, Beijing, China) using the fluorescent probe DCFH-DA combined with flow cytometry (FACS Calibur, BD Biosciences). The cells (90% confluence) were plated into a 60 mm dish at a density of 5×106 cells per dish and were treated according to the experiment design. Then the cells were collected and washed with PBS for three times, followed by the addition of the probe containing DCFH-DA (10 µM). The cells were incubated at 37°C for 30 min. Then the cells were washed three times with serum-free cell culture medium. Flow cytometry was performed using 488 nm excitation wavelength and 525 nm emission wavelength. The result was quantified by the FlowJo software V10.0 (Ashland, KY, USA). The ROS level was presented as the ratio of the mean fluorescence intensity in the experimental group relative to that in the control group.
The cells with 90% confluence were plated into a 60 mm dish at a density of 5×106 cells per dish. After treatment for 12 h, the collected cells were lysed by ultrasound on ice and then centrifuged at 6000×g for 10 min at 4°C. The supernatant was collected for oxidative damage labeling and antioxidant enzyme activity analysis. The detection assay kits for MDA (A003-4-1), SOD (A001-3-2), catalase (CAT, A007-1-1), and total antioxidant capacity (T-AOC, A015-2-1) were purchased from Nanjing Jiancheng Bioengineering Institute (China).
The cells were inoculated in a 6-well plate (90% confluent) at a density of 2×105 cells per well and were treated as described. Total RNA was extracted using Trizol reagent (ET111, Tran, China). The quantity and purity of RNA was detected by a Nanodrop 2000 spectrophotometer (Thermo, USA). The RNA with absorption ratios (A260/A280) between 1.8 and 2.1 was used for subsequent experiments. The total RNA was converted into cDNA using the primescript RT Regent kit gDNA eraser (DRR047A, Takara, Japan). Quantitative PCR was used to detect the mRNA expression according to the instruction of SYBR Premix Ex Taq™ II (RR820A, TaKaRa, Japan) on the CFX 96 Real-Time PCR Detection System (BIO-RAD, USA). The SYBR Premix, primers of target gene, and template DNA were added into the amplification mixtures with the total volume of 20 μL. The following cycling conditions were performed: 95°C for 2 min; 95°C for 5 s and 60°C for 34 s, 40 cycles; 95°C for 15 s; 60°C for 5 s; 60°C to 95°C, 0.5°C gradient heating. The 2-△△Ct method was carried out to measure the relative abundance of mRNA transcripts. A single product was amplified by each primer pair. The products were purified and sequenced (TsingKe Biotech, Beijing, China), and then the sequence results were analysed through BLAST (http://blast.ncbi.nlm.nih.gov/blast.cg) and compared to GenBank database. The sequences of primers were shown in Table 1.
The cells with 90% fusion were plated into a 60 mm dish at a density of 5×106 cells per dish. The total protein was extracted using the RIPA lysis buffer (P0013B, Beyotime, China) containing phenylmethanesulfonyl fluoride (ST506, Beyotime, China). The nuclear protein was extracted by a nuclear protein and cytoplasmic protein extraction kit (P0027, Beyotime, China). Both the total and nuclear proteins were quantified by a bicinchoninic acid protein assay kit (P0010, Beyotime, China). The protein (20-30 μg) was separated on 10% SDS polyacrylamide gel after electrophoresis, and was transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was incubated in 5% skimmed milk for 1.5 h at room temperature. Then the membrane was washed with TBST (0.05% Tween-20 Tris-HCl buffer) for six times with each 5 min. Then the PVDF membranes were incubated with corresponding primary antibodies specific for NRF2 (#12721, Cell Signaling Technology, USA), KEAP1 (ab227828, Abcam, UK), heme oxygenase 1 (HO-1, ab52947, Abcam, UK), NAD(P)H quinone dehydrogenase 1 (NQO1, ab80588, Abcam, UK), LaminB1 (#13435, Cell Signaling Technology, USA), and β-actin (#4970, Cell Signaling Technology, USA) at 4°C overnight. These antibodies were diluted to 1:1000 with 5% bovine serum albumin. Then the PVDF membrane was incubated with the secondary antibody (diluted to 1:2000 with 5% skimmed milk) (#7074, Cell Signaling Technology, USA) for 1.5 h at room temperature. The protein bands were detected using a chemiluminescence assay. The antigen-antibody complexes were visualized on horseradish peroxidase substrate (Millipore, Billerica, MA, USA) by a ChemiScope 5300Pro CCD camera (Clinx Science Instruments, Shanghai, China). The band intensity was quantified by the Quantity One software (Bio-Rad, California, USA).
The cells were inoculated into a 24-well plate（40% confluence) at a density of 2×103 cells per well and were treated according to the experiment design. Then the cells were covered with 4% formaldehyde to a depth of 2 to 3 mm. The cells were fixed at room temperature for 15 min and was washed with PBS three times with each 5 min. The cell membrane was penetrated with 0.4% Triton X-100 (ST797, Beyotime, China) for 15 min. After washing with PBS, the cells were blocked in blocking buffer (5% bovine serum albumin) for 60 min at room temperature. The primary antibody for NRF2 (dilution ratio 1:200) was prepared in 5% bovine serum albumin. The cells were incubated with the primary antibody at 4°C overnight and were washed with PBS three times with each 5 min. The fluorescein coupled secondary antibody (A0423, Beyotime, China) was diluted with antibody dilution buffer, and the cells were incubated in a dark room for 1.5 h at room temperature. The nuclei were stained with DAPI (C1005, Beyotime, China). The fluorescence microscope (TCS Sp8, Leica, Germany) was used for observation. The signal intensity of nuclear NRF2 was quantified using the Image J software (National Institutes of Health, USA).
All data were analyzed using the SPSS-Statistics 21.0 software (IBM, NY, USA). Statistically significant differences were calculated by one-way ANOVA, followed by Dunnett’s test. The data were presented as means ± standard error of the means (SEM). A two-sided P < 0.05 was considered statistically significant.
We first observed the oxidative status of BEEC treated with progesterone alone. As shown in Figure 1, progesterone of 1, 3, and 5 ng/mL caused no change (P > 0.05) in the levels of ROS, MDA, and T-AOC, and the activities of SOD and CAT. In addition, the PGR inhibitor RU486 did not affect (P > 0.05) these oxidative indicators either.
Then LPS was used to induce oxidative stress of BEEC. As depicted in Figure2, LPS evoked the production (P < 0.05) of ROS and MDA, and the reduced (P < 0.05) level of T-AOC and the activities of SOD and CAT. Progesterone reduced (P < 0.05) the levels of ROS and MDA, and upregulated T-AOC and the activities of SOD and CAT in LPS-stimulated BEEC. It should be noted that, compared with the cells treated with LPS alone, no difference (P > 0.05) was found in MDA and T-AOC in cells cotreated with LPS and 5 ng/mL progesterone. These results suggested that progesterone exerted antioxidative effect in BEEC with oxidative stress, and this effect was more prominent in progesterone treatment of 1 and 3 ng/mL than that of 5 ng/mL.
The relative abundance of genes related to Nrf2/Keap1 pathway was determined by quantitative PCR. As depicted in Fig 3 A-D, LPS resulted in a general decrease (P < 0.05) in the expression of NRF2, NQO1, heme oxygenase 1 (HMOX1), and an increase (P < 0.05) in KEAP1. Compared with the cells in LPS group, the expression of NRF2, NQO1, and HMOX1 were generally higher (P < 0.05) in cells cotreated with LPS and progesterone (1 and 3 ng/mL), but not the cells cotreated with LPS and 5 ng/mL P4.
The changes in the protein levels of NRF2, KEAP1, NQO1, and HO-1 were detected using western blot (Fig 3 E-H). Similar to the mRNA results, LPS treatment caused decreased (P < 0.05) protein levels of total NRF2 and downstream HO-1 and NQO1, and increased (P < 0.05) KEAP1 level. The cotreatment of LPS and progesterone (1 and 3 ng/mL) generally upregulated the level of total NRF2, nuclear NRF2, NQO1, and HO-1, and downregulated KEAP1 level in comparison with LPS treatment. Taken together, these results suggested that progesterone activated Nrf2/Keap1 pathway in BEEC with oxidative stress.
To verify whether the antioxidant effect of progesterone is mediated by PGR, the BEEC was treated with LPS, progesterone, and PGR antagonist RU486. As depicted in Fig 4, cotreatment of LPS, progesterone (3 ng/mL), and RU486 upregulated (P < 0.05) the levels of ROS and MDA, and downregulated (P < 0.05) the activities of SOD and CAT and T-AOC as compared with the cells cotreated with LPS and progesterone. In addition, the cotreatment of LPS and RU486 downregulated (P < 0.05) the levels of ROS and MDA as compared with the LPS group.
The influence of RU486 on Nrf2/Keap1 pathway was shown in Fig 5. Compared with the cells cotreated with LPS and progesterone, there were decreases (P < 0.05) in the relative abundances of NRF2, HMOX1, and NQO1 genes, as well as the protein levels of total and nuclear NRF2, HO1, and NQO1 after the addition of RU486. Moreover, there was an increase (P < 0.05) in the gene expression of NRF2, NQO1, and HMOX1 in RU486 and LPS cotreatment group as compared with the LPS group.
The Nrf2 translocation result (Fig 6A), as observed by immunofluorescence, was similar to the changes of the nuclear NFR2 level. Co-treatment with progesterone (3 ng/mL) and LPS showed higher NRF2 level in the nucleus as compared with the LPS group. The addition of RU486 reduced the amount of nuclear NRF2 in BEEC cotreated with LPS and progesterone (Fig 6B). In summary, these results revealed that RU486 reversed the activation of Nrf2/Keap1 pathway and the antioxidant capacity of BEEC elicited by progesterone.
According to our study, progesterone inhibited the production of ROS, and promoted the activities of antioxidant enzymes and Nrf2/Keap1 pathway to prevent BEEC from LPS-induced oxidative stress. This antioxidant property of progesterone was most prominent at the concentration of 3 ng/mL and can be reversed by PGR antagonist RU486.
Excessive ROS accumulation is one of the important signs of oxidative injury. LPS stimulates ROS production and inhibits antioxidant enzyme activity in BEEC and bovine mammary epithelial cells (Gugliandolo et al., 2020; Liu et al., 2021). Our data were consistent with these studies, verifying the oxidative damage of BEEC caused by LPS. The antioxidant enzyme system is the first line of defense against damage. Stimulation with 1 or 10 μg/mL LPS for 9 h inhibited the SOD activity and T-AOC in bovine mammary epithelial cells (Li et al., 2021). BEEC challenged with 100 μg/mL LPS exhibited a decreased SOD activity (Fu et al., 2021b). Similarly, our study found that 1 μg/mL LPS decreased the activities of SOD and CAT, and T-AOC in BEEC at 12 h. However, it has been reported by Huang et al. (2019) that glutathione, a classical antioxidant, increased in bovine mammary epithelial cells after 0.5 μg/mL LPS stimulation for 12 h. Piras et al. (2017) observed the induction of SOD protein in BEEC treated with 16 μg/mL LPS for 72 h. The contradictory results from literature were probably related to the difference in treatment dosage and time.
Progesterone has been reported to diminish retinal MDA concentration in retinitis pigmentosa of mice (150 mg/kg, oral administration) (Benlloch-Navarro et al., 2019), and has been observed to protect against oxidative stress in nerve cells and in experimental colitis of rats (Karatepe et al., 2012; Wei and Xiao, 2013). Consistently, we found the reduced production of ROS and MDA after progesterone treatment in LPS-stimulated BEEC. In order to study the relationship between ROS clearance and progesterone antioxidant capacity, we detected the activities of some enzymes in the antioxidant enzyme system. Alese et al. (2021) demonstrated that progesterone enhanced the activities of SOD, CAT, and glutathione in rat hepatic tissue with oxidative injury caused by cadmium. Matsuoka et al. (2010) reported that progesterone increased SOD protein level in human endometrial stromal cells through Wnt5a pathway. Similarly, our results showed elevated activities of SOD, CAT, and T-AOC after the addition of progesterone, suggesting that progesterone relieved the cellular oxidative injury of BEEC by enhancing antioxidant enzyme activity.
Nrf2/Keap1 signaling is a key pathway regulating the expression of antioxidant enzymes and protect cells from oxidation-induced cytotoxicity. Under physiological conditions, Nrf2 is anchored in the cytoplasm through Keap1 dependent ubiquitination proteasome degradation to maintain antioxidant and cytoprotective enzymes at a basal level (Lu et al., 2016). The phosphorylation of oxidants prompts Keap1 to release Nrf2, and finally Nrf2 enters the nucleus (Li et al., 2012). Nrf2 regulates the antioxidant defense system through a variety of mechanisms, including the regeneration of oxidative cofactors and proteins, the synthesis of reducing factors, the increase in redox transports, and the induction of stress response proteins. As a stress response protein, HO-1 enhances antioxidant activity and maintains redox homeostasis. NQO1 is an important reductant that regulates transcription through anti-oxidative response element (ARE) (McDonnell et al., 2017). The combination of activated Nrf2 and ARE promotes antioxidant gene expression, and further enhances antioxidant enzyme activity (Bey et al., 2013). The status of Nrf2 pathway in response to LPS challenge varied in different studies. Treatment of 1 μg/mL LPS for 12 h showed no influence on cytosolic NRF2 level, but upregulated nuclear NRF2 level in immortalized goat endometrial epithelial cells (Bao et al., 2021). The BEEC subjected 100 μg/mL LPS for 24 h showed higher level of total NRF2 and KEAP1, alone with an increased or unchanged gene expression of HO1 and NQO1 (Fu et al., 2021a; Fu et al., 2021b). Stimulation with 1 μg/mL LPS for 24 h caused reduced protein levels of total NRF2 and HO1 in RAW264.7 cells (Yu et al., 2021). Here we detected the decreased levels of HO-1, NQO1, and total NRF2, and increased level of KEAP1 in BEEC after 1 μg/mL LPS stimulation for 12 h. The down-regulation of NRF2, HMOX1, and NQO1 genes in LPS group coincided with the Western blot result, together suggesting an inhibited status of Nrf2 pathway in BEEC. Chiou et al. (2016) detected the time course change in the NRF2 protein level in RAW264.7 cells stimulated with 100 ng/mL LPS, and observed that the cytosolic NRF2 increased at 1 h post-treatment and decreased thereafter, and that the nuclear NRF2 started to decrease until 2 h and began returning to control level after 3 h of treatment. The inconsistent result among these studies could be related to the detection time and LPS concentration.
Antioxidative effect of progesterone is associated with Nrf2 pathway. It has been reported that progesterone treatment (16 and 32 mg/kg per day) for 14 days increased the amount of NRF2 and HO-1 proteins in rats with traumatic brain injury (Ghadiri et al., 2019). Progesterone decreased the apoptosis index and the release of inflammatory cytokines in C57 mouse model with traumatic brain injury, and such effect of progesterone was absent in Nrf2-knockout mice, indicating the involvement of Nrf2/ARE signaling (Zhang et al., 2017). Progesterone (3.14 ng/mL) has been observed to induce HO-1 protein and mRNA expression in the human myometrium (Acevedo and Ahmed, 1998). In agreement with these reports, we demonstrated that progesterone of 1 and 3 ng/mL activated Nrf2/Keap1 pathway in BEEC with oxidative stress. Progesterone effect is mainly mediated by PGR. RU486 has high affinity on PGR and exhibits potent antagonistic activity after binding to PGR. As expected, the use of RU486 reversed the antioxidant effect of progesterone. In addition, we noticed that RU486 attenuated LPS-induced ROS and MDA level, suggesting an antioxidant property of RU486 per se. This antioxidant effect of RU486 has been suggested previously and is unrelated to its antiprogesterone effects (Parthasarathy et al., 1994).
In our study, 5 ng/mL progesterone showed less antioxidative effect in BEEC as compared with 1 and 3 ng/mL progesterone. Our results showed that 5 ng/mL progesterone decreased the level of ROS, and increased the activities of CAT and SOD, but showed no significant effect on the levels of MDA and T-AOC, and the Nrf2 pathway. High level progesterone (10-4 M and 10-5 M) has been suggested to inhibit the expression of progesterone receptor B (Rekawiecki et al., 2015). We speculated that 5 ng/mL progesterone may inhibit the PGR expression and thus the antioxidant effect is not obvious. As a result, however, 5 ng/mL progesterone did not affect PGR expression. It has been reported that the anti-inflammatory effect of 5 ng/mL progesterone showed no obvious difference from that of 1 and 3 ng/mL progesterone in BEEC, indicating that the antioxidant effect of 5 ng/mL progesterone was not related to PGR (Cui et al., 2020). Further research is needed to clarify the underlying mechanism.
Progesterone of 1 and 3 ng/mL inhibited the LPS-induced oxidative damage in primary bovine endometrial epithelial cells through activating Nrf2/Keap1 signaling pathway. This antioxidative effect was mediated by progesterone receptor.
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LC: conceptualization, funding acquisition, supervision, and writing - review & editing. JG: methodology, data curation, formal analysis. QZ: methodology, data curation, and formal analysis. JZ, CY, KL, LG: data curation. JD: funding acquisition. JL (Jun Li): supervision. WH: funding acquisition. JL (Jianji Li): conceptualization, funding acquisition and supervision. All authors contributed to the article and approved the final version of the article.
This work was supported by the National Natural Science Foundation of China (NO: 32072937, 31802253, 32102735); the China Postdoctoral Science Foundation (NO: 2018M632398); the earmarked fund for Jiangsu Agricultural Industry Technology System (JATS[2023]499); 333 High-level Talent Training Project of Jiangsu Province, China; the Natural Science Foundation of Jiangsu Province (NO: BK20210808); The Postgraduate Training Innovation Program of Jiangsu Province (KYCX21_3279); the Postgraduate Research &Practice Innovation Program of Jiangsu Province (Yangzhou University) (SJCX21_1642); the 111 Project (D18007); and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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