- Email: [email protected]
Protective effects of polydatin on LPS-induced endometritis in mice
Microbial Pathogenesis 137 (2019) 103720
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Protective effects of polydatin on LPS-induced endometritis in mice a,1
a,1
a,1
Rong Li , T. Maimai , Hongmei Yao Yinan Wangc,∗∗, Guanghong Xiea,∗ a b c
b
a
T
a
, Xueshibojie Liu , Zhaoqi He , Chong Xiao ,
Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province, 130062, China Department of Otolaryngology Head and Neck Surgery, The Second Hospital Affiliated of Jilin University, Changchun, Jilin Province, 130041, China Department of Obstetrics and Gynecology, The Second Hospital Affiliated of Jilin University, Changchun, Jilin Province, 130041, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Polydatin LPS Endometritis NF-κB
Endometritis, a common inflammation of the uterus, often causes severe damage to human and animal reproductive health. Polydatin is a polyphenol extracted from the rhizome of Polygonum cuspidatum that has antiinflammatory and anti-oxidative effects. The purpose of this study was to investigate the underlying protective effects and mechanisms of polydatin against lipopolysaccharide (LPS)-induced endometritis in mice. The mouse model of endometritis was established by injection of LPS through the vagina. The uterine tissues of each group were gathered to analyze histopathological changes, inflammatory cytokine production, and the degree of activation of the NF-κB and Nrf2 signaling pathways. The myeloperoxidase (MPO) activity assay indicated that polydatin treatment significantly alleviated inflammatory cell infiltration in LPS-induced endometritis mice. Furthermore, polydatin treatment remarkably impeded the expression of TNF-α, IL-1β, and IL-6 by ELISA assay. Hematoxylin-eosin staining (H&E) showed that polydatin significantly decreased impairment of the uterus. In addition, polydatin was also found to suppress LPS-induced NF-κB activation in a dose-dependent manner. The expression of Nrf2 and HO-1 was enhanced by polydatin treatment. All the results suggest that polydatin helpfully alleviates LPS-induced endometritis by suppressing the NF-ĸB signaling pathway and activating the Nrf2 signaling pathway.
1. Introduction Endometritis is the inflammation of the uterine lining that is often associated with pelvic pain [1]. There is much relevance between endometritis and postpartum disorders, such as infertility [2,3]. Bacteria are considered the most significant factor in the occurrence of endometritis [4,5]. A large number of neutrophils may infiltrate the uterus when the uterus is stimulated by bacteria. LPS is the major component of bacteria that can induce the activation of inflammatory cells [6]. LPS activates the TLR4 signaling pathway, which subsequently leads to the activation of NF-ĸB and the release of inflammatory cytokines [7], which then leads to injury of the endometrium [8]. Previous studies demonstrated that the TLR4 signaling pathway was activated during the development of endometritis [9]. Inhibition of the TLR4 and NF-ĸB signaling pathways had protective effects against endometritis [10]. Inflammation plays a critical role in the development of endometritis. Normally, a small degree of inflammation can help the host to clear the invading pathogens. However, an excessive
inflammatory response can lead to injury of the endometrium [11]. Previous data indicated that suppression of excessive inflammation could suppress endometritis [12]. Recently, more attention has been given to the effects of herbal medicines on inflammatory diseases, such as endometritis [13]. As a result, it is important for the study to explore the protective response of polydatin on LPS-induced endometritis. Polydatin, a polyphenol extracted from the rhizome of Polygonum cuspidatum, has been discovered to have anti-inflammatory effects [14]. Polydatin can inhibit the inflammatory response in fulminant hepatic failure [15]. It protects against liver injury by attenuating NF-κB activation. Furthermore, polydatin can suppress the LPS-induced inflammatory response in lung injury. However, the effects of polydatin on LPS-induced endometritis have not yet been investigated. In this study, we explored the protective effects of polydatin on LPS-induced endometritis in mice and detected the underlying mechanism.
∗
Corresponding author. Corresponding author. E-mail addresses: [email protected] (Y. Wang), [email protected] (G. Xie). 1 These authors contributed equally to this article. ∗∗
https://doi.org/10.1016/j.micpath.2019.103720 Received 7 March 2019; Received in revised form 3 September 2019; Accepted 4 September 2019 Available online 05 September 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
Microbial Pathogenesis 137 (2019) 103720
R. Li, et al.
2. Materials and methods
rabbit anti-mouse primary antibodies (Cell Signaling Technology, MA, United States) overnight and with goat anti-rabbit secondary antibodies (Cell Signaling Technology, MA, United States). Finally, the bands were exposed using an enhanced chemiluminescence system (Thermo Scientific, United States). The band intensity was quantified with ImageJ software (Amersham Life Science, UK) in a blinded fashion.
2.1. Materials Polydatin (purity: > 98%) was purchased from Chengdu Preferred Biotechnology Co., Ltd. (Chengdu, China). Lipopolysaccharide (LPS) (Escherichia coli O55:B5) was purchased from Sigma (St. Louis, MO, United States). The myeloperoxidase (MPO) determination kit was purchased from the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu Province, China). ELISA kits of TNF-α, IL-1β and IL-6 were purchased from Biolegend (CA, United States). Antibodies against NF-κB p65 and IκBα antibodies were purchased from Cell Signaling Technology (Danvers, MA, United States).
2.7. Statistical analysis The data of this study were analyzed with SPSS software and expressed as means ± SEM. The comparison of the control group and LPS group, as well as the LPS group to the LPS + polydatin (20, 40, 80 mg/kg) groups were measured by using one-way ANOVA followed by Dunn test. A p value < 0.05 was considered to be significant.
2.2. Animals 3. Results Female BALB/c mice (6–8 wk; weight 18–22 g) were purchased from the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China). Mice were housed in a managed environment (24 ± 1 °C) and were given food and clean water. Sixty mice were randomly divided into 5 groups and each group contained twelve mice: Control group, LPS group, LPS + polydatin (20, 40, 80 mg/kg) groups. The mice in the LPS group were injected with 50 ml LPS by vagina (1 mg/ml, dissolved in sterile PBS) [10,16]. Polydatin was given by intraperitoneal injection before LPS injection.
3.1. Effects of polydatin on LPS-induced uterine histopathological changes Histological analysis demonstrated that the mice of the control group showed normal morphology. The LPS group showed severe pathologic changes, such as inflammatory cell infiltration and the endometrial epithelium being hyperemia edema. These histopathological changes of uterus induced by LPS were obviously attenuated by polydatin (Fig. 1).
2.3. Histological analysis
3.2. Effects of polydatin on MPO activity during LPS-induced endometritis
Twenty-four hours after LPS treatment, the uterine tissues were gathered and washed. Afterwards, the tissues were fixed in 10% formalin, desiccated and embedded in paraffin, and chop into 5 μm slices by microtome. Finally, the tissues were stained with hematoxylin and eosin (H&E). The slides were evaluated by three pathologists who were blinded to the treatment.
Compared with MPO activity in the control group, the LPS-treated group had significantly increased MPO activity in the uterine tissues. In addition, polydatin treatment significantly reduced the MPO activity in mice with LPS-induced endometritis (Fig. 2). 3.3. Effects of polydatin on pro-inflammatory cytokine production in uterus of LPS-induced endometritis model mice
2.4. MPO assay Inflammatory cytokines play very significant roles in the process of inflammation. Inflammatory cytokines TNF-α, IL-6, and IL-1β in the LPS group significantly increased than that of the control group. However, treatment of polydatin significantly decreased the levels of inflammatory cytokines (Fig. 3).
The tissues of the uterus were collected. The MPO activity of the tissues was detected by a test kit purchased from the Jiancheng Bioengineering Institute of Nanjing according to the manufacturer's instructions. Briefly, the tissues were weighed and homogenized with PBS (1:9, w/v). Then, 0.9 mL homogenate was mixed with 0.1 mL buffer three and putted in the 37 °C water bath for 15 min. Next, buffer four, TMB Substrate, and buffer six were added and putted in the 60 °C water bath for 10 min. The absorbance was read at 460 nm.
3.4. Effects of polydatin on the activity of the NF-κB signaling pathway in LPS-induced endometritis The NF-κB signaling pathway is involved in the inflammatory response and its phosphorylation level is increased in LPS-induced endometritis. The Western blot results showed that the phosphorylation of NF-κB p65 and IκBα in the LPS group increased significantly. The phosphorylation of NF-κB p65 and IκBα induced by LPS were suppressed by polydatin in a dose-dependent manner (Fig. 4).
2.5. ELISA ELISA was applied to measure the effects of polydatin on LPS-induced inflammatory cytokine production. The standards for the inflammatory cytokines TNF-α, IL-6, and IL-1β were determined by ELISA based on the specifications. The levels of TNF-α, IL-6, and IL-1β were tested in triplicate for in each sample. The absorbance was read at 450 nm. The levels of inflammatory cytokines were measured against the standard curve. The TNF-α inter- and intra-assay coefficient of variation (CV) were 3.6% and 7.9%, respectively. The IL-6 inter- and intra-assay CV were 4.3% and 6.2%, respectively. The IL-1β inter- and intra-assay CV were 3.2% and 9.2%, respectively.
3.5. Effects of polydatin on the activity of the Nrf2 signaling pathway in LPS-induced endometritis In the present study, we investigated the effects of polydatin of Nrf2 and HO-1 expression. Western blot results showed that the expression of Nrf2 and HO-1 in the LPS group increased compared with that of the control group. Polydatin significantly increased the expression of Nrf2 and HO-1 (Fig. 5).
2.6. Western blot analysis
4. Discussion
The uterine tissues were gathered and the tissue proteins were extracted by T-PER (Thermo, USA). This study employed the BCA method to determine the concentrations of protein. The proteins were loaded onto the 12% SPS-PAGE and transferred onto PVDF membranes. The membranes were blocked with fetal bovine serum and probed with
Polydatin is a polyphenol extracted from the rhizome of Polygonum cuspidatum. A previous study confirmed that polydatin had potent antiinflammatory properties [17]. Our study showed that polydatin broadly 2
Microbial Pathogenesis 137 (2019) 103720
R. Li, et al.
Fig. 1. Effects of polydatin on LPS-induced uterus histopathologic changes. The histological changes of uterus were detected by H&E staining. Representative histological changes of kidney obtained from mice of different groups. A: Control group, B: LPS group, C: LPS + polydatin (20 mg/kg) group, D: LPS + polydatin (40 mg/kg) group, E: LPS + polydatin (80 mg/kg) group (Hematoxylin and eosin staining, magnification 200 × ).
Fig. 2. Effects of polydatin on LPS-induced MPO activity. The values presented are mean ± SEM (n = 6 in each group) of three parallel measurements. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
inhibited inflammatory cytokine production and NF-κB activation in LPS-induced endometritis. Polydatin may be beneficial for patients with endometritis and can be used as an anti-inflammatory agent. Inflammation plays a critical role in the development of endometritis. LPS can induce the release of inflammatory cytokines [18]. These inflammatory cytokines can lead to injury of uterus uterine tissue. Thus, inhibition of the production of inflammatory cytokines may be a target for endometritis therapies. The present study showed that polydatin significantly inhibited LPS-induced inflammatory cytokine production. MPO was used as a direct indicator of the neutrophil granulocyte active state in tissues [19]. The results of the study indicated that LPS-induced MPO activity was significantly reduced after treatment with polydatin. These results showed that polydatin had a protective function against LPS-stimulated endometritis. IκBα is known as an inhibitor of NF-κB. However, after activation of NF-κB, IκBα is phosphorylated and then degraded [20,21]. Studies showed that inhibition of NF-κB had protective effects against endometritis. Our results indicated that polydatin inhibited LPS-induced NF-κB activation, which suggests that polydatin protects mice against endometritis by inhibiting NF-κB activation. Nrf2 is the major transcription factor that is necessary for the regulation of oxidative stress. Additionally, Nrf2 is known to be involved in the regulation of the inflammatory response [22]. Previous studies have shown that activation of Nrf2 can inhibit the activation of NF-κB [23]. The activation of Nrf2
Fig. 3. Effects of polydatin on LPS-induced TNF-α, IL-6, and IL-1β production. The values presented are mean ± SEM (n = 6 in each group) of three parallel measurements. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
3
Microbial Pathogenesis 137 (2019) 103720
R. Li, et al.
[2] [3] [4] [5]
[6]
[7] [8]
Fig. 4. Effects of polydatin on LPS-induced NF-κB activation. The values presented are mean ± SEM (n = 6 in each group) of three parallel measurements. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
[9]
[10]
[11]
[12]
[13]
[14]
[15]
Fig. 5. Effects of polydatin on Nrf2 signaling pathway. The values presented are mean ± SEM (n = 6 in each group) of three parallel measurements. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
[16]
has protective effects against endometritis. Therefore, we examined the effects of polydatin on the Nrf2 signaling pathway. The results indicated that polydatin significantly increased the expression of Nrf2 and HO-1. In summary, the data from this study indicate that polydatin exerts an anti-inflammatory effect on LPS-induced endometritis, and its underlying mechanism might inhibit the production of inflammatory cytokines by activating Nrf2 and inhibiting the NF-κB signaling pathway. Therefore, polydatin might be a useful drug for preventing and treating LPS-induced endometritis.
[17]
[18]
[19]
[20]
[21]
Conflicts of interest All authors declare that they have no conflict of interest.
[22]
Acknowledgements
[23]
This work was supported by a grant from the National Natural Science Foundation of China (No. 31672616). References [1] H.C. Wiesenfeld, M.A. Hillier SLKrohn, A.J. Amortegui, R.P. Heine, D.V. Landers,
4
R.L. Sweet, Lower genital tract infection and endometritis: insight into subclinical pelvic inflammatory disease, Obstet. Gynecol. 100 (2002) 456–463. I.M. Sheldon, E.J. Williams, A.N.A. Miller, D.M. Nash, H. Shan, Uterine diseases in cattle after parturition, Vet. J. 176 (2008) 115–121. M.L. Turner, G.D. Healey, I.M. Sheldon, Immunity and inflammation in the uterus, Reprod. Domest. Anim. 47 (2012) 402–409. D.E. Soper, Bacterial vaginosis and postoperative infections, Am. J. Obstet. Gynecol. 169 (1993) 467–469. W.W. Andrews, J.C. Hauth, S.P. Cliver, M.G. Conner, R.L. Goldenberg, A.R. Goepfert, Association of asymptomatic bacterial vaginosis with endometrial microbial colonization and plasma cell endometritis in nonpregnant women, Am. J. Obstet. Gynecol. 195 (2006) 1611–1616. A.C. Asbury, K.T. Schultz, P.H. Klesius, G.W. Foster, S.M. Washburn, Factors affecting phagocytosis of bacteria by neutrophils in the mare's uterus, J. Reprod. Fertil. Suppl. 32 (1982) 151. D. Heumann, T. Roger, Initial responses to endotoxins and Gram-negative bacteria, Clin. Chim. Acta 323 (2002) 59–72. R. Roberto, G. Francesca, P. Beth, K. Juan Pedro, Inflammation in pregnancy: its roles in reproductive physiology, obstetrical complications, and fetal injury, Nutr. Rev. 65 (2010) S194–S202. J.F. Ju, L.P. Li, J.Y. Xie, Y. Wu, X. Wu, W.H. Li, Toll-like receptor-4 pathway is required for the pathogenesis of human chronic endometritis, Exp. Ther. Med. 8 (2014) 1896–1900. M. Zhou, Y. Yi, L. Hong, Oridonin ameliorates lipopolysaccharide-induced endometritis in mice via inhibition of the TLR-4/NF-kappaBpathway, Inflammation 42 (2019) 81–90. A. Augoulea, A. Alexandrou, M. Creatsa, N. Vrachnis, I. Lambrinoudaki, Pathogenesis of endometriosis: the role of genetics, inflammation and oxidative stress, Arch. Gynecol. Obstet. 286 (2012) 99–103. N.,. Rana, D.P. Braun, R.,. House, H.,. Gebel, C.,. Rotman, W.P. Dmowski, Basal and stimulated secretion of cytokines by peritoneal macrophages in women with endometriosis, Fertil. Steril. 65 (1996) 925–930. H. Mikamo, K. Kawazoe, Y. Sato, Y. Hayasaki, K. Izumi, T. Tamaya, Effects of crude herbal ingredients on serum levels of inflammatory cytokines in a rat uterine endometritis model, Curr. Ther. Res. Clin. Exp. 60 (1999) 105–110. T. Lou, W.J. Jiang, D.H. Xu, T. Chen, Y.L. Fu, Inhibitory effects of polydatin on lipopolysaccharide-stimulated RAW 264.7 cells, Inflammation 38 (2015) 1213–1220. M.J. Wu, X. Gong, R. Jiang, L. Zhang, X.H. Li, J.Y. Wan, Polydatin protects against lipopolysaccharide-induced fulminant hepatic failure in D-galactosamine-sensitized mice, Int. J. Immunopathol. Pharmacol. 25 (2012) 923. X. Hu, D. Li, J. Wang, J. Guo, Y. Li, Y. Cao, et al., Melatonin inhibits endoplasmic reticulum stress-associated TXNIP/NLRP3 inflammasome activation in lipopolysaccharide-induced endometritis in mice, Int. Immunopharmacol. 64 (2018) 101–109. J. Qi, Y. Min, G. Qianqian, W. Ciman, W. Huimin, M. Shanshan, et al., Protective effects of polydatin on lipopolysaccharide-induced acute lung injury through TLR4MyD88-NF-κB pathway, Int. Immunopharmacol. 29 (2015) 370–376. L.O. Brandenburg, M. Kipp, R. Lucius, T. Pufe, C.J. Wruck, Sulforaphane suppresses LPS-induced inflammation in primary rat microglia, Inflamm. Res. 59 (2010) 443–450. I.V. Gorudko, D.V. Grigorieva, A.V. Sokolov, E.V. Shamova, V.A. Kostevich, I.V. Kudryavtsev, et al., Neutrophil activation IN response to monomeric myeloperoxidase, Biochem. Cell Biol. 96 (2018) 592–601. Y. Fu, L. Bo, X. Feng, F. Li, D. Liang, Z. Liu, et al., The effect of magnolol on the tolllike receptor 4/nuclear factor kappa B signaling pathway in lipopolysaccharideinduced acute lung injury in mice, Eur. J. Pharmacol. 689 (2012) 255–261. J.J. Wang, C.M. Guo, Z.K. Wei, X.X. He, J.H. Kou, E.S. Zhou, et al., Morin suppresses inflammatory cytokine expression by downregulation of nuclear factor-kappa B and mitogen-activated protein kinase (MAPK) signaling pathways in lipopolysaccharide-stimulated primary bovine mammary epithelial cells, J. Dairy Sci. 99 (2016) 3016–3022. J. Ye, H. Piao, J. Jiang, G. Jin, M. Zheng, J. Yang, et al., Polydatin inhibits mast cellmediated allergic inflammation by targeting PI3K/Akt, MAPK, NF-κB and Nrf2/HO1 pathways, Sci. Rep. 7 (2017) 11895. C. Antonio, M.M. Zaira, Y. Jianping, L.B. Isabel, Transcription factors NRF2 and NFκB are coordinated effectors of the Rho family, GTP-binding protein RAC1 during inflammation, J. Biol. Chem. 289 (2014) 15244–15258.
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Protective effects of polydatin on LPS-induced endometritis in mice a,1
a,1
a,1
Rong Li , T. Maimai , Hongmei Yao Yinan Wangc,∗∗, Guanghong Xiea,∗ a b c
b
a
T
a
, Xueshibojie Liu , Zhaoqi He , Chong Xiao ,
Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province, 130062, China Department of Otolaryngology Head and Neck Surgery, The Second Hospital Affiliated of Jilin University, Changchun, Jilin Province, 130041, China Department of Obstetrics and Gynecology, The Second Hospital Affiliated of Jilin University, Changchun, Jilin Province, 130041, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Polydatin LPS Endometritis NF-κB
Endometritis, a common inflammation of the uterus, often causes severe damage to human and animal reproductive health. Polydatin is a polyphenol extracted from the rhizome of Polygonum cuspidatum that has antiinflammatory and anti-oxidative effects. The purpose of this study was to investigate the underlying protective effects and mechanisms of polydatin against lipopolysaccharide (LPS)-induced endometritis in mice. The mouse model of endometritis was established by injection of LPS through the vagina. The uterine tissues of each group were gathered to analyze histopathological changes, inflammatory cytokine production, and the degree of activation of the NF-κB and Nrf2 signaling pathways. The myeloperoxidase (MPO) activity assay indicated that polydatin treatment significantly alleviated inflammatory cell infiltration in LPS-induced endometritis mice. Furthermore, polydatin treatment remarkably impeded the expression of TNF-α, IL-1β, and IL-6 by ELISA assay. Hematoxylin-eosin staining (H&E) showed that polydatin significantly decreased impairment of the uterus. In addition, polydatin was also found to suppress LPS-induced NF-κB activation in a dose-dependent manner. The expression of Nrf2 and HO-1 was enhanced by polydatin treatment. All the results suggest that polydatin helpfully alleviates LPS-induced endometritis by suppressing the NF-ĸB signaling pathway and activating the Nrf2 signaling pathway.
1. Introduction Endometritis is the inflammation of the uterine lining that is often associated with pelvic pain [1]. There is much relevance between endometritis and postpartum disorders, such as infertility [2,3]. Bacteria are considered the most significant factor in the occurrence of endometritis [4,5]. A large number of neutrophils may infiltrate the uterus when the uterus is stimulated by bacteria. LPS is the major component of bacteria that can induce the activation of inflammatory cells [6]. LPS activates the TLR4 signaling pathway, which subsequently leads to the activation of NF-ĸB and the release of inflammatory cytokines [7], which then leads to injury of the endometrium [8]. Previous studies demonstrated that the TLR4 signaling pathway was activated during the development of endometritis [9]. Inhibition of the TLR4 and NF-ĸB signaling pathways had protective effects against endometritis [10]. Inflammation plays a critical role in the development of endometritis. Normally, a small degree of inflammation can help the host to clear the invading pathogens. However, an excessive
inflammatory response can lead to injury of the endometrium [11]. Previous data indicated that suppression of excessive inflammation could suppress endometritis [12]. Recently, more attention has been given to the effects of herbal medicines on inflammatory diseases, such as endometritis [13]. As a result, it is important for the study to explore the protective response of polydatin on LPS-induced endometritis. Polydatin, a polyphenol extracted from the rhizome of Polygonum cuspidatum, has been discovered to have anti-inflammatory effects [14]. Polydatin can inhibit the inflammatory response in fulminant hepatic failure [15]. It protects against liver injury by attenuating NF-κB activation. Furthermore, polydatin can suppress the LPS-induced inflammatory response in lung injury. However, the effects of polydatin on LPS-induced endometritis have not yet been investigated. In this study, we explored the protective effects of polydatin on LPS-induced endometritis in mice and detected the underlying mechanism.
∗
Corresponding author. Corresponding author. E-mail addresses: [email protected] (Y. Wang), [email protected] (G. Xie). 1 These authors contributed equally to this article. ∗∗
https://doi.org/10.1016/j.micpath.2019.103720 Received 7 March 2019; Received in revised form 3 September 2019; Accepted 4 September 2019 Available online 05 September 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
Microbial Pathogenesis 137 (2019) 103720
R. Li, et al.
2. Materials and methods
rabbit anti-mouse primary antibodies (Cell Signaling Technology, MA, United States) overnight and with goat anti-rabbit secondary antibodies (Cell Signaling Technology, MA, United States). Finally, the bands were exposed using an enhanced chemiluminescence system (Thermo Scientific, United States). The band intensity was quantified with ImageJ software (Amersham Life Science, UK) in a blinded fashion.
2.1. Materials Polydatin (purity: > 98%) was purchased from Chengdu Preferred Biotechnology Co., Ltd. (Chengdu, China). Lipopolysaccharide (LPS) (Escherichia coli O55:B5) was purchased from Sigma (St. Louis, MO, United States). The myeloperoxidase (MPO) determination kit was purchased from the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu Province, China). ELISA kits of TNF-α, IL-1β and IL-6 were purchased from Biolegend (CA, United States). Antibodies against NF-κB p65 and IκBα antibodies were purchased from Cell Signaling Technology (Danvers, MA, United States).
2.7. Statistical analysis The data of this study were analyzed with SPSS software and expressed as means ± SEM. The comparison of the control group and LPS group, as well as the LPS group to the LPS + polydatin (20, 40, 80 mg/kg) groups were measured by using one-way ANOVA followed by Dunn test. A p value < 0.05 was considered to be significant.
2.2. Animals 3. Results Female BALB/c mice (6–8 wk; weight 18–22 g) were purchased from the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China). Mice were housed in a managed environment (24 ± 1 °C) and were given food and clean water. Sixty mice were randomly divided into 5 groups and each group contained twelve mice: Control group, LPS group, LPS + polydatin (20, 40, 80 mg/kg) groups. The mice in the LPS group were injected with 50 ml LPS by vagina (1 mg/ml, dissolved in sterile PBS) [10,16]. Polydatin was given by intraperitoneal injection before LPS injection.
3.1. Effects of polydatin on LPS-induced uterine histopathological changes Histological analysis demonstrated that the mice of the control group showed normal morphology. The LPS group showed severe pathologic changes, such as inflammatory cell infiltration and the endometrial epithelium being hyperemia edema. These histopathological changes of uterus induced by LPS were obviously attenuated by polydatin (Fig. 1).
2.3. Histological analysis
3.2. Effects of polydatin on MPO activity during LPS-induced endometritis
Twenty-four hours after LPS treatment, the uterine tissues were gathered and washed. Afterwards, the tissues were fixed in 10% formalin, desiccated and embedded in paraffin, and chop into 5 μm slices by microtome. Finally, the tissues were stained with hematoxylin and eosin (H&E). The slides were evaluated by three pathologists who were blinded to the treatment.
Compared with MPO activity in the control group, the LPS-treated group had significantly increased MPO activity in the uterine tissues. In addition, polydatin treatment significantly reduced the MPO activity in mice with LPS-induced endometritis (Fig. 2). 3.3. Effects of polydatin on pro-inflammatory cytokine production in uterus of LPS-induced endometritis model mice
2.4. MPO assay Inflammatory cytokines play very significant roles in the process of inflammation. Inflammatory cytokines TNF-α, IL-6, and IL-1β in the LPS group significantly increased than that of the control group. However, treatment of polydatin significantly decreased the levels of inflammatory cytokines (Fig. 3).
The tissues of the uterus were collected. The MPO activity of the tissues was detected by a test kit purchased from the Jiancheng Bioengineering Institute of Nanjing according to the manufacturer's instructions. Briefly, the tissues were weighed and homogenized with PBS (1:9, w/v). Then, 0.9 mL homogenate was mixed with 0.1 mL buffer three and putted in the 37 °C water bath for 15 min. Next, buffer four, TMB Substrate, and buffer six were added and putted in the 60 °C water bath for 10 min. The absorbance was read at 460 nm.
3.4. Effects of polydatin on the activity of the NF-κB signaling pathway in LPS-induced endometritis The NF-κB signaling pathway is involved in the inflammatory response and its phosphorylation level is increased in LPS-induced endometritis. The Western blot results showed that the phosphorylation of NF-κB p65 and IκBα in the LPS group increased significantly. The phosphorylation of NF-κB p65 and IκBα induced by LPS were suppressed by polydatin in a dose-dependent manner (Fig. 4).
2.5. ELISA ELISA was applied to measure the effects of polydatin on LPS-induced inflammatory cytokine production. The standards for the inflammatory cytokines TNF-α, IL-6, and IL-1β were determined by ELISA based on the specifications. The levels of TNF-α, IL-6, and IL-1β were tested in triplicate for in each sample. The absorbance was read at 450 nm. The levels of inflammatory cytokines were measured against the standard curve. The TNF-α inter- and intra-assay coefficient of variation (CV) were 3.6% and 7.9%, respectively. The IL-6 inter- and intra-assay CV were 4.3% and 6.2%, respectively. The IL-1β inter- and intra-assay CV were 3.2% and 9.2%, respectively.
3.5. Effects of polydatin on the activity of the Nrf2 signaling pathway in LPS-induced endometritis In the present study, we investigated the effects of polydatin of Nrf2 and HO-1 expression. Western blot results showed that the expression of Nrf2 and HO-1 in the LPS group increased compared with that of the control group. Polydatin significantly increased the expression of Nrf2 and HO-1 (Fig. 5).
2.6. Western blot analysis
4. Discussion
The uterine tissues were gathered and the tissue proteins were extracted by T-PER (Thermo, USA). This study employed the BCA method to determine the concentrations of protein. The proteins were loaded onto the 12% SPS-PAGE and transferred onto PVDF membranes. The membranes were blocked with fetal bovine serum and probed with
Polydatin is a polyphenol extracted from the rhizome of Polygonum cuspidatum. A previous study confirmed that polydatin had potent antiinflammatory properties [17]. Our study showed that polydatin broadly 2
Microbial Pathogenesis 137 (2019) 103720
R. Li, et al.
Fig. 1. Effects of polydatin on LPS-induced uterus histopathologic changes. The histological changes of uterus were detected by H&E staining. Representative histological changes of kidney obtained from mice of different groups. A: Control group, B: LPS group, C: LPS + polydatin (20 mg/kg) group, D: LPS + polydatin (40 mg/kg) group, E: LPS + polydatin (80 mg/kg) group (Hematoxylin and eosin staining, magnification 200 × ).
Fig. 2. Effects of polydatin on LPS-induced MPO activity. The values presented are mean ± SEM (n = 6 in each group) of three parallel measurements. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
inhibited inflammatory cytokine production and NF-κB activation in LPS-induced endometritis. Polydatin may be beneficial for patients with endometritis and can be used as an anti-inflammatory agent. Inflammation plays a critical role in the development of endometritis. LPS can induce the release of inflammatory cytokines [18]. These inflammatory cytokines can lead to injury of uterus uterine tissue. Thus, inhibition of the production of inflammatory cytokines may be a target for endometritis therapies. The present study showed that polydatin significantly inhibited LPS-induced inflammatory cytokine production. MPO was used as a direct indicator of the neutrophil granulocyte active state in tissues [19]. The results of the study indicated that LPS-induced MPO activity was significantly reduced after treatment with polydatin. These results showed that polydatin had a protective function against LPS-stimulated endometritis. IκBα is known as an inhibitor of NF-κB. However, after activation of NF-κB, IκBα is phosphorylated and then degraded [20,21]. Studies showed that inhibition of NF-κB had protective effects against endometritis. Our results indicated that polydatin inhibited LPS-induced NF-κB activation, which suggests that polydatin protects mice against endometritis by inhibiting NF-κB activation. Nrf2 is the major transcription factor that is necessary for the regulation of oxidative stress. Additionally, Nrf2 is known to be involved in the regulation of the inflammatory response [22]. Previous studies have shown that activation of Nrf2 can inhibit the activation of NF-κB [23]. The activation of Nrf2
Fig. 3. Effects of polydatin on LPS-induced TNF-α, IL-6, and IL-1β production. The values presented are mean ± SEM (n = 6 in each group) of three parallel measurements. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
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[2] [3] [4] [5]
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[7] [8]
Fig. 4. Effects of polydatin on LPS-induced NF-κB activation. The values presented are mean ± SEM (n = 6 in each group) of three parallel measurements. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
[9]
[10]
[11]
[12]
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Fig. 5. Effects of polydatin on Nrf2 signaling pathway. The values presented are mean ± SEM (n = 6 in each group) of three parallel measurements. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
[16]
has protective effects against endometritis. Therefore, we examined the effects of polydatin on the Nrf2 signaling pathway. The results indicated that polydatin significantly increased the expression of Nrf2 and HO-1. In summary, the data from this study indicate that polydatin exerts an anti-inflammatory effect on LPS-induced endometritis, and its underlying mechanism might inhibit the production of inflammatory cytokines by activating Nrf2 and inhibiting the NF-κB signaling pathway. Therefore, polydatin might be a useful drug for preventing and treating LPS-induced endometritis.
[17]
[18]
[19]
[20]
[21]
Conflicts of interest All authors declare that they have no conflict of interest.
[22]
Acknowledgements
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