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Alpinetin ameliorates inflammatory response in LPS-induced endometritis in mice
International Immunopharmacology 62 (2018) 309–312
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Alpinetin ameliorates inflammatory response in LPS-induced endometritis in mice Yuanjiao Lianga, Duanrong Zhub a b
⁎,1
T
, Tao Shena,1, Qi Minga, Guoqing Hana, Yan Zhanga, Jinlan Lianga,
Reproductive Medicine Center, Zhongda Hospital, Southeast University, Jiangsu, Nanjing 210009, China The Department of Gynecology and Obstetrics, Jinling Hospital, Medical School of Nanjing University, Jiangsu, Nanjing 210002, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Alpinetin LPS PPAR-γ Endometritis
Alpinetin has been reported to have anti-inflammatory effects. However, whether alpinetin has anti-inflammatory effects in LPS-induced endometritis has not been thoroughly elucidated to date. The aim of this study was to investigate the protective effects of alpinetin on LPS-induced endometritis in mice. A mouse model of endometritis was induced by LPS and alpinetin was given 1 h before LPS treatment. According to the results, alpinetin protected mice against LPS-induced endometritis by attenuating uterine histological changes and myeloperoxidase (MPO) activity. The LPS-induced inflammatory response was inhibited by alpinetin as confirmed by the inhibition of TNF-α, IL-1ß, and IL-6 production. Furthermore, LPS-induced TLR4 expression and NF-κB activation were significantly suppressed by alpinetin. In addition, the expression of PPAR-γ was dosedependently increased by the treatment of alpinetin. Taken together, the results of this study showed that alpinetin had protective effects against LPS-induced endometritis in mice, and the beneficial effects were occurred through the activation of PPAR-γ and inhibition of the TLR4 signaling pathway.
1. Introduction Endometritis, the inflammation of the uterine lining, often causes infertility in women [1]. Previous studies have shown a relationship between endometritis and infertility [2, 3]. Endometritis can be caused by many factors and bacteria is one of the most important factors leading to endometritis [4, 5]. Among these bacteria, Gram-negative bacteria, such as Escherichia coli and Fusobacterium necrophorum, have been known as the major bacteria that lead to endometritis [6]. LPS is the major activator of inflammatory cells [7]. During the development of endometritis, LPS can induce the activation of neutrophils and lead to the release of inflammatory cytokines [8]. Recent studies have suggested that suppression of these inflammatory cytokines has potential therapeutic effects for endometritis [9]. Antibiotics are widely used for the treatment of endometritis. The use of antibiotics is likely to cause bacterial resistance. Therefore, new agents to treat endometritis are urgently needed. Alpinetin has been known to have anti-inflammatory effects. Alpinetin was found to suppress LPS-induced inflammatory cytokine production in RAW264.7 cells [10]. Additionally, alpinetin can attenuate the production of
inflammatory mediators in LPS-stimulated THP-1 cells [11]. In vivo, alpinetin had protective effects on LPS-induced mastitis in mice [12]. Alpinetin can also attenuate DSS-induced acute colitis in mice by suppressing the TLR4 signaling pathway [13]. In addition, alpinetin can inhibit LPS-induced acute kidney injury in mice [14]. However, whether alpinetin can protect against LPS-induced endometritis is unknown. This study is aimed at determining the protective effects of alpinetin on LPS-induced endometritis in vivo. 2. Materials and methods 2.1. Reagents Alpinetin (purity > 98%) was purchased from the National Institutes for Food and Drug Control (Beijing, China). The PPAR-γ antibody was purchased from Santa Cruz (TX, USA), and the TLR4 and NF-κB signaling pathway antibodies were purchased from Biolegend (CA, USA). LPS was purchased from Sigma (St. Louis, MO, USA). ELISA kits were obtained from R&D systems (Minneapolis, MN, USA).
⁎
Corresponding author. E-mail address: [email protected] (Y. Liang). 1 Contributed equally to this article. https://doi.org/10.1016/j.intimp.2018.07.010 Received 15 March 2018; Received in revised form 7 July 2018; Accepted 10 July 2018 1567-5769/ © 2018 Elsevier B.V. All rights reserved.
International Immunopharmacology 62 (2018) 309–312
Y. Liang et al.
Fig. 1. Alpinetin attenuates LPS-induced uterine histopathological changes (magnification 200×). A: Control group, B: LPS group, C–E: LPS + alpinetin (10, 20, 40 mg/kg) groups.
Fig. 2. Alpinetin inhibits LPS-induced MPO activity. The data of this study are presented as mean ± SEM of three parallel measurements. p# < 0.01 vs. control group, p** < 0.01 vs. LPS group.
2.2. Animals Female C57BL/6 mice, weighing 18–22 g, were purchased from Southeast University. The experiment was approved by the Ethical Committee of Southeast University. 2.3. Experimental groups Sixty mice were randomly divided into five groups: Control group, LPS group, and LPS + alpinetin (10, 20, 40 mg/kg) groups. LPS-induced endometritis was induced by giving 50 μL of LPS (1 mg/mL) to each side of the mouse uterus through the vagina using a 100 μl syringe (patent no.: 201410391820.8). Alpinetin was given intraperitoneally 1 h before LPS treatment. 24 h later, the mice were sacrificed and the uterine tissues were collected for subsequent experiments. 2.4. Histological analysis The uterine tissues were collected and washed. The tissues were then fixed in 10% formalin, dehydrated, and embedded in paraffin. The uterine tissues were cut into 5 μm sections using a microtome and stained with hematoxylin and eosin (H&E).
Fig. 3. Effects of alpinetin on LPS-induced TNF-α, IL-6, and IL-1β production. The data of this study are presented as mean ± SEM of three parallel measurements. p# < 0.01 vs. control group, p** < 0.01 vs. LPS group. 310
International Immunopharmacology 62 (2018) 309–312
Y. Liang et al.
2.5. ELISA assay
3.2. Effects of alpinetin on LPS-induced MPO activity
The production of TNF-α, IL-1ß, and IL-6 in uterine tissues was quantified in duplicate by ELISA kits (Biolegend, CA, USA) according to the manufacturer's instructions.
MPO, a biomarker of neutrophils, was detected in this study. The results showed that a significant increase was observed in MPO activity after LPS treatment. In contrast, compared with the LPS group, alpinetin significantly inhibited LPS-induced MPO activity (Fig. 2).
2.6. Western blot analysis
3.3. Effects of alpinetin on LPS-induced TNF-α, IL-6, and IL-1β production
The uterine tissues were collected, frozen, and homogenized using the T-PER Tissue Protein Extraction Reagent (Thermo, USA). The BCA method was used to measure the concentration of protein. The samples were loaded onto 12% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with primary antibodies and secondary antibodies. Finally, the bands were revealed by an enhanced ECL chemiluminescence reagent (Thermo, MA, USA).
Inflammatory cytokines are involved in the pathological progression of endometritis. In this study, TNF-α, IL-6, and IL-1β production were detected by ELISA. As shown in Fig. 3, a significant increase was observed in TNF-α, IL-6, and IL-1β production after LPS treatment. In contrast, treatment of alpinetin significantly inhibited LPS-induced inflammatory cytokine production.
2.7. Statistical analysis
3.4. Effects of alpinetin on LPS-induced TLR4 expression and NF-κB activation
The values were analyzed with SPSS software and expressed as means ± SEM. The differences among groups were measured using oneway ANOVA followed by Dunn's test. A p value < 0.05 was considered to be significant.
As shown in Fig. 4, a significant increase was observed in the TLR4 expression, as well as the phosphorylation levels of NF-κB and IκBα after LPS treatment. In contrast, compared with LPS group, alpinetin significantly attenuated LPS-induced TLR4 expression and phosphorylation levels of NF-κB and IκBα (Fig. 4).
3. Results 3.5. Effects of alpinetin on PPAR-γ expression 3.1. Effects of alpinetin on LPS-induced uterine histopathological changes Finally, we detected the expression of PPAR-γ by Western blot analysis. The results in Fig. 4 showed a significant decrease in the expression of PPAR-γ after LPS treatment. In contrast, compared with the LPS group, alpinetin significantly increased the expression of PPAR-γ (Fig. 5).
The protective effects of alpinetin on uterine histopathological changes were detected. The results showed that the uterine tissues of the control group showed normal structure. As shown in Fig. 1B, significant uterine histopathological changes including the shedding of epithelial cells and the infiltration of inflammatory cells were observed in the LPS-treated group (Fig. 1B). However, LPS-induced histopathological changes were attenuated by the treatment of alpinetin (Fig. 1C–E).
4. Discussion In this study, we found that alpinetin protected against LPS-induced
Fig. 4. Effects of alpinetin on LPS-induced TLR4 expression and NF-κB activation. The data of this study are presented as mean ± SEM of three parallel measurements. p# < 0.01 vs. control group, p** < 0.01 vs. LPS group.
311
International Immunopharmacology 62 (2018) 309–312
Y. Liang et al.
References [1] E. Cicinelli, D. De Ziegler, R. Nicoletti, R. Tinelli, N. Saliani, L. Resta, et al., Poor reliability of vaginal and endocervical cultures for evaluating microbiology of endometrial cavity in women with chronic endometritis, Gynecol. Obstet. Investig. 68 (2009) 108–115. [2] B. Czernobilsky, Endometritis and infertility, Fertil. Steril. 30 (1978) 119–130. [3] F. Polisseni, E.A. Bambirra, A.F. Camargos, Detection of chronic endometritis by diagnostic hysteroscopy in asymptomatic infertile patients, Gynecol. Obstet. Investig. 55 (2003) 205–210. [4] D.E. Soper, Bacterial vaginosis and postoperative infections, Am. J. Obstet. Gynecol. 169 (1993) 467–469. [5] 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. [6] A. Sens, W. Heuwieser, Presence of Escherichia coli, Trueperella pyogenes, alpha-hemolytic streptococci, and coagulase-negative staphylococci and prevalence of subclinical endometritis, J. Dairy Sci. 96 (2013) 6347–6354. [7] D. Heumann, T. Roger, Initial responses to endotoxins and Gram-negative bacteria, Clin. Chim. Acta 323 (2002) 59–72 International Journal of Clinical Chemistry. [8] W. Li, K. Fu, X. Lv, Y. Wang, J. Wang, H. Li, et al., Lactoferrin suppresses lipopolysaccharide-induced endometritis in mice via down-regulation of the NF-kappaB pathway, Int. Immunopharmacol. 28 (2015) 695–699. [9] 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. [10] M. Huo, N. Chen, G. Chi, X. Yuan, S. Guan, H. Li, et al., Traditional medicine alpinetin inhibits the inflammatory response in raw 264.7 cells and mouse models, Int. Immunopharmacol. 12 (2012) 241–248. [11] K. Hu, Y. Yang, Q. Tu, Y. Luo, R. Ma, Alpinetin inhibits LPS-induced inflammatory mediator response by activating PPAR-gamma in THP-1-derived macrophages, Eur. J. Pharmacol. 721 (2013) 96–102. [12] H. Chen, X. Mo, J. Yu, Z. Huang, Alpinetin attenuates inflammatory responses by interfering toll-like receptor 4/nuclear factor kappa B signaling pathway in lipopolysaccharide-induced mastitis in mice, Int. Immunopharmacol. 17 (2013) 26–32. [13] X. He, Z. Wei, J. Wang, J. Kou, W. Liu, Y. Fu, et al., Alpinetin attenuates inflammatory responses by suppressing TLR4 and NLRP3 signaling pathways in DSSinduced acute colitis, Sci. Rep. 6 (2016) 28370. [14] Y. Huang, L.S. Zhou, L. Yan, J. Ren, D.X. Zhou, S.S. Li, Alpinetin inhibits lipopolysaccharide-induced acute kidney injury in mice, Int. Immunopharmacol. 28 (2015) 1003–1008. [15] J.F. Peipert, R.B. Ness, J. Blume, D.E. Soper, R. Holley, H. Randall, et al., Clinical predictors of endometritis in women with symptoms and signs of pelvic inflammatory disease, Am. J. Obstet. Gynecol. 184 (2001) 856–863 (discussion 63-4). [16] X. Lv, K. Fu, W. Li, Y. Wang, J. Wang, H. Li, et al., TIIA attenuates LPS-induced mouse endometritis by suppressing the NF-kappaB signaling pathway, Can. J. Physiol. Pharmacol. 93 (2015) 967–971. [17] J.R. Challis, C.J. Lockwood, L. Myatt, J.E. Norman, J.F. Strauss III, F. Petraglia, Inflammation and pregnancy, Reprod. Sci. 16 (2009) 206–215. [18] M.D. Mueller, D.I. Lebovic, E. Garrett, R.N. Taylor, Neutrophils infiltrating the endometrium express vascular endothelial growth factor: potential role in endometrial angiogenesis, Fertil. Steril. 74 (2000) 107–112. [19] J. Wang, C. Xiao, Z. Wei, Y. Wang, X. Zhang, Y. Fu, Activation of liver X receptors inhibit LPS-induced inflammatory response in primary bovine mammary epithelial cells, Vet. Immunol. Immunopathol. 197 (2018) 87–92. [20] P.P. Tak, G.S. Firestein, NF-kappaB: a key role in inflammatory diseases, J. Clin. Invest. 107 (2001) 7–11. [21] J. Wang, C. Guo, Z. Wei, X. He, J. Kou, E. Zhou, et al., Morin suppresses inflammatory cytokine expression by downregulation of nuclear factor-kappaB and mitogen-activated protein kinase (MAPK) signaling pathways in lipopolysaccharide-stimulated primary bovine mammary epithelial cells, J. Dairy Sci. 99 (2016) 3016–3022. [22] R. Gonzalez-Ramos, A. Van Langendonckt, S. Defrere, J.C. Lousse, S. Colette, L. Devoto, et al., Involvement of the nuclear factor-kappaB pathway in the pathogenesis of endometriosis, Fertil. Steril. 94 (2010) 1985–1994. [23] S.J. Bensinger, P. Tontonoz, Integration of metabolism and inflammation by lipidactivated nuclear receptors, Nature 454 (2008) 470–477. [24] C.G. Su, X. Wen, S.T. Bailey, W. Jiang, S.M. Rangwala, S.A. Keilbaugh, et al., A novel therapy for colitis utilizing PPAR-gamma ligands to inhibit the epithelial inflammatory response, J. Clin. Invest. 104 (1999) 383–389. [25] T.H. Huang, A.W. Teoh, B.L. Lin, D.S. Lin, B. Roufogalis, The role of herbal PPAR modulators in the treatment of cardiometabolic syndrome, Pharmacol. Res. 60 (2009) 195–206. [26] A. Jacob, R. Wu, M. Zhou, P. Wang, Mechanism of the anti-inflammatory effect of curcumin: PPAR-gamma activation, PPAR Res. 2007 (2007) 89369. [27] S. Appel, V. Mirakaj, A. Bringmann, M.M. Weck, F. Grunebach, P. Brossart, PPARgamma agonists inhibit toll-like receptor-mediated activation of dendritic cells via the MAP kinase and NF-kappaB pathways, Blood 106 (2005) 3888–3894.
Fig. 5. Effects of alpinetin on PPAR-γ expression. The data of this study are presented as mean ± SEM of three parallel measurements. p# < 0.01 vs. control group, p** < 0.01 vs. LPS group.
endometritis by inhibiting uterine histopathological changes and inflammatory cytokine production. We also found that alpinetin suppressed the LPS-induced inflammatory response by activating PPAR-γ. Inflammation plays a critical role in the development of endometritis [15]. A significant elevation of TNF-α, IL-1ß, and IL-6 in uterine tissues was observed in the LPS-induced endometritis model [16]. These inflammatory cytokines could induce other cytokines and result in epithelial cell necrosis. Furthermore, these cytokines could lead to the accumulation of neutrophils in the uterus [17]. The overactivation of neutrophils may result in injury of the endometrial epithelium [18]. We found that alpinetin inhibited LPS-induced neutrophils infiltration. Furthermore, alpinetin could inhibit LPS-induced inflammatory cytokines production. In the following studies, we investigated the anti-inflammatory mechanism of alpinetin. Inflammatory cytokines are known to be regulated by NF-κB [19]. NF-κB is known as the target of many inflammatory diseases [20, 21]. Inhibition of NF-κB activation had protective effects against endometritis [22]. In this study, alpinetin exhibited anti-inflammatory effects by inhibiting NF-κB activation. PPAR-γ is an important nuclear receptor that regulates lipid metabolism [23]. It also plays critical roles in the regulation of the inflammatory response [24]. Furthermore, many herbal compounds have been known to activate PPAR-γ and exhibited their anti-inflammatory effects through activating PPAR-γ [25, 26]. In addition, PPAR-γ agonists can inhibit NF-κB activation [27]. Therefore, PPAR-γ expression was measured in this study. We found that alpinetin increased the expression of PPAR-γ. These results indicated that alpinetin exhibited anti-inflammatory effects by activating PPAR-γ. In conclusion, our study provided evidence that alpinetin could protect LPS-induced endometritis by inhibiting the inflammatory response. The mechanism was through activation of PPAR-γ, which subsequently attenuated the inflammatory response induced by LPS. Alpinetin might be a useful agent for treating endometritis.
Conflict of interest All authors declare that they have no conflict of interest.
312
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Alpinetin ameliorates inflammatory response in LPS-induced endometritis in mice Yuanjiao Lianga, Duanrong Zhub a b
⁎,1
T
, Tao Shena,1, Qi Minga, Guoqing Hana, Yan Zhanga, Jinlan Lianga,
Reproductive Medicine Center, Zhongda Hospital, Southeast University, Jiangsu, Nanjing 210009, China The Department of Gynecology and Obstetrics, Jinling Hospital, Medical School of Nanjing University, Jiangsu, Nanjing 210002, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Alpinetin LPS PPAR-γ Endometritis
Alpinetin has been reported to have anti-inflammatory effects. However, whether alpinetin has anti-inflammatory effects in LPS-induced endometritis has not been thoroughly elucidated to date. The aim of this study was to investigate the protective effects of alpinetin on LPS-induced endometritis in mice. A mouse model of endometritis was induced by LPS and alpinetin was given 1 h before LPS treatment. According to the results, alpinetin protected mice against LPS-induced endometritis by attenuating uterine histological changes and myeloperoxidase (MPO) activity. The LPS-induced inflammatory response was inhibited by alpinetin as confirmed by the inhibition of TNF-α, IL-1ß, and IL-6 production. Furthermore, LPS-induced TLR4 expression and NF-κB activation were significantly suppressed by alpinetin. In addition, the expression of PPAR-γ was dosedependently increased by the treatment of alpinetin. Taken together, the results of this study showed that alpinetin had protective effects against LPS-induced endometritis in mice, and the beneficial effects were occurred through the activation of PPAR-γ and inhibition of the TLR4 signaling pathway.
1. Introduction Endometritis, the inflammation of the uterine lining, often causes infertility in women [1]. Previous studies have shown a relationship between endometritis and infertility [2, 3]. Endometritis can be caused by many factors and bacteria is one of the most important factors leading to endometritis [4, 5]. Among these bacteria, Gram-negative bacteria, such as Escherichia coli and Fusobacterium necrophorum, have been known as the major bacteria that lead to endometritis [6]. LPS is the major activator of inflammatory cells [7]. During the development of endometritis, LPS can induce the activation of neutrophils and lead to the release of inflammatory cytokines [8]. Recent studies have suggested that suppression of these inflammatory cytokines has potential therapeutic effects for endometritis [9]. Antibiotics are widely used for the treatment of endometritis. The use of antibiotics is likely to cause bacterial resistance. Therefore, new agents to treat endometritis are urgently needed. Alpinetin has been known to have anti-inflammatory effects. Alpinetin was found to suppress LPS-induced inflammatory cytokine production in RAW264.7 cells [10]. Additionally, alpinetin can attenuate the production of
inflammatory mediators in LPS-stimulated THP-1 cells [11]. In vivo, alpinetin had protective effects on LPS-induced mastitis in mice [12]. Alpinetin can also attenuate DSS-induced acute colitis in mice by suppressing the TLR4 signaling pathway [13]. In addition, alpinetin can inhibit LPS-induced acute kidney injury in mice [14]. However, whether alpinetin can protect against LPS-induced endometritis is unknown. This study is aimed at determining the protective effects of alpinetin on LPS-induced endometritis in vivo. 2. Materials and methods 2.1. Reagents Alpinetin (purity > 98%) was purchased from the National Institutes for Food and Drug Control (Beijing, China). The PPAR-γ antibody was purchased from Santa Cruz (TX, USA), and the TLR4 and NF-κB signaling pathway antibodies were purchased from Biolegend (CA, USA). LPS was purchased from Sigma (St. Louis, MO, USA). ELISA kits were obtained from R&D systems (Minneapolis, MN, USA).
⁎
Corresponding author. E-mail address: [email protected] (Y. Liang). 1 Contributed equally to this article. https://doi.org/10.1016/j.intimp.2018.07.010 Received 15 March 2018; Received in revised form 7 July 2018; Accepted 10 July 2018 1567-5769/ © 2018 Elsevier B.V. All rights reserved.
International Immunopharmacology 62 (2018) 309–312
Y. Liang et al.
Fig. 1. Alpinetin attenuates LPS-induced uterine histopathological changes (magnification 200×). A: Control group, B: LPS group, C–E: LPS + alpinetin (10, 20, 40 mg/kg) groups.
Fig. 2. Alpinetin inhibits LPS-induced MPO activity. The data of this study are presented as mean ± SEM of three parallel measurements. p# < 0.01 vs. control group, p** < 0.01 vs. LPS group.
2.2. Animals Female C57BL/6 mice, weighing 18–22 g, were purchased from Southeast University. The experiment was approved by the Ethical Committee of Southeast University. 2.3. Experimental groups Sixty mice were randomly divided into five groups: Control group, LPS group, and LPS + alpinetin (10, 20, 40 mg/kg) groups. LPS-induced endometritis was induced by giving 50 μL of LPS (1 mg/mL) to each side of the mouse uterus through the vagina using a 100 μl syringe (patent no.: 201410391820.8). Alpinetin was given intraperitoneally 1 h before LPS treatment. 24 h later, the mice were sacrificed and the uterine tissues were collected for subsequent experiments. 2.4. Histological analysis The uterine tissues were collected and washed. The tissues were then fixed in 10% formalin, dehydrated, and embedded in paraffin. The uterine tissues were cut into 5 μm sections using a microtome and stained with hematoxylin and eosin (H&E).
Fig. 3. Effects of alpinetin on LPS-induced TNF-α, IL-6, and IL-1β production. The data of this study are presented as mean ± SEM of three parallel measurements. p# < 0.01 vs. control group, p** < 0.01 vs. LPS group. 310
International Immunopharmacology 62 (2018) 309–312
Y. Liang et al.
2.5. ELISA assay
3.2. Effects of alpinetin on LPS-induced MPO activity
The production of TNF-α, IL-1ß, and IL-6 in uterine tissues was quantified in duplicate by ELISA kits (Biolegend, CA, USA) according to the manufacturer's instructions.
MPO, a biomarker of neutrophils, was detected in this study. The results showed that a significant increase was observed in MPO activity after LPS treatment. In contrast, compared with the LPS group, alpinetin significantly inhibited LPS-induced MPO activity (Fig. 2).
2.6. Western blot analysis
3.3. Effects of alpinetin on LPS-induced TNF-α, IL-6, and IL-1β production
The uterine tissues were collected, frozen, and homogenized using the T-PER Tissue Protein Extraction Reagent (Thermo, USA). The BCA method was used to measure the concentration of protein. The samples were loaded onto 12% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with primary antibodies and secondary antibodies. Finally, the bands were revealed by an enhanced ECL chemiluminescence reagent (Thermo, MA, USA).
Inflammatory cytokines are involved in the pathological progression of endometritis. In this study, TNF-α, IL-6, and IL-1β production were detected by ELISA. As shown in Fig. 3, a significant increase was observed in TNF-α, IL-6, and IL-1β production after LPS treatment. In contrast, treatment of alpinetin significantly inhibited LPS-induced inflammatory cytokine production.
2.7. Statistical analysis
3.4. Effects of alpinetin on LPS-induced TLR4 expression and NF-κB activation
The values were analyzed with SPSS software and expressed as means ± SEM. The differences among groups were measured using oneway ANOVA followed by Dunn's test. A p value < 0.05 was considered to be significant.
As shown in Fig. 4, a significant increase was observed in the TLR4 expression, as well as the phosphorylation levels of NF-κB and IκBα after LPS treatment. In contrast, compared with LPS group, alpinetin significantly attenuated LPS-induced TLR4 expression and phosphorylation levels of NF-κB and IκBα (Fig. 4).
3. Results 3.5. Effects of alpinetin on PPAR-γ expression 3.1. Effects of alpinetin on LPS-induced uterine histopathological changes Finally, we detected the expression of PPAR-γ by Western blot analysis. The results in Fig. 4 showed a significant decrease in the expression of PPAR-γ after LPS treatment. In contrast, compared with the LPS group, alpinetin significantly increased the expression of PPAR-γ (Fig. 5).
The protective effects of alpinetin on uterine histopathological changes were detected. The results showed that the uterine tissues of the control group showed normal structure. As shown in Fig. 1B, significant uterine histopathological changes including the shedding of epithelial cells and the infiltration of inflammatory cells were observed in the LPS-treated group (Fig. 1B). However, LPS-induced histopathological changes were attenuated by the treatment of alpinetin (Fig. 1C–E).
4. Discussion In this study, we found that alpinetin protected against LPS-induced
Fig. 4. Effects of alpinetin on LPS-induced TLR4 expression and NF-κB activation. The data of this study are presented as mean ± SEM of three parallel measurements. p# < 0.01 vs. control group, p** < 0.01 vs. LPS group.
311
International Immunopharmacology 62 (2018) 309–312
Y. Liang et al.
References [1] E. Cicinelli, D. De Ziegler, R. Nicoletti, R. Tinelli, N. Saliani, L. Resta, et al., Poor reliability of vaginal and endocervical cultures for evaluating microbiology of endometrial cavity in women with chronic endometritis, Gynecol. Obstet. Investig. 68 (2009) 108–115. [2] B. Czernobilsky, Endometritis and infertility, Fertil. Steril. 30 (1978) 119–130. [3] F. Polisseni, E.A. Bambirra, A.F. Camargos, Detection of chronic endometritis by diagnostic hysteroscopy in asymptomatic infertile patients, Gynecol. Obstet. Investig. 55 (2003) 205–210. [4] D.E. Soper, Bacterial vaginosis and postoperative infections, Am. J. Obstet. Gynecol. 169 (1993) 467–469. [5] 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. [6] A. Sens, W. Heuwieser, Presence of Escherichia coli, Trueperella pyogenes, alpha-hemolytic streptococci, and coagulase-negative staphylococci and prevalence of subclinical endometritis, J. Dairy Sci. 96 (2013) 6347–6354. [7] D. Heumann, T. Roger, Initial responses to endotoxins and Gram-negative bacteria, Clin. Chim. Acta 323 (2002) 59–72 International Journal of Clinical Chemistry. [8] W. Li, K. Fu, X. Lv, Y. Wang, J. Wang, H. Li, et al., Lactoferrin suppresses lipopolysaccharide-induced endometritis in mice via down-regulation of the NF-kappaB pathway, Int. Immunopharmacol. 28 (2015) 695–699. [9] 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. [10] M. Huo, N. Chen, G. Chi, X. Yuan, S. Guan, H. Li, et al., Traditional medicine alpinetin inhibits the inflammatory response in raw 264.7 cells and mouse models, Int. Immunopharmacol. 12 (2012) 241–248. [11] K. Hu, Y. Yang, Q. Tu, Y. Luo, R. Ma, Alpinetin inhibits LPS-induced inflammatory mediator response by activating PPAR-gamma in THP-1-derived macrophages, Eur. J. Pharmacol. 721 (2013) 96–102. [12] H. Chen, X. Mo, J. Yu, Z. Huang, Alpinetin attenuates inflammatory responses by interfering toll-like receptor 4/nuclear factor kappa B signaling pathway in lipopolysaccharide-induced mastitis in mice, Int. Immunopharmacol. 17 (2013) 26–32. [13] X. He, Z. Wei, J. Wang, J. Kou, W. Liu, Y. Fu, et al., Alpinetin attenuates inflammatory responses by suppressing TLR4 and NLRP3 signaling pathways in DSSinduced acute colitis, Sci. Rep. 6 (2016) 28370. [14] Y. Huang, L.S. Zhou, L. Yan, J. Ren, D.X. Zhou, S.S. Li, Alpinetin inhibits lipopolysaccharide-induced acute kidney injury in mice, Int. Immunopharmacol. 28 (2015) 1003–1008. [15] J.F. Peipert, R.B. Ness, J. Blume, D.E. Soper, R. Holley, H. Randall, et al., Clinical predictors of endometritis in women with symptoms and signs of pelvic inflammatory disease, Am. J. Obstet. Gynecol. 184 (2001) 856–863 (discussion 63-4). [16] X. Lv, K. Fu, W. Li, Y. Wang, J. Wang, H. Li, et al., TIIA attenuates LPS-induced mouse endometritis by suppressing the NF-kappaB signaling pathway, Can. J. Physiol. Pharmacol. 93 (2015) 967–971. [17] J.R. Challis, C.J. Lockwood, L. Myatt, J.E. Norman, J.F. Strauss III, F. Petraglia, Inflammation and pregnancy, Reprod. Sci. 16 (2009) 206–215. [18] M.D. Mueller, D.I. Lebovic, E. Garrett, R.N. Taylor, Neutrophils infiltrating the endometrium express vascular endothelial growth factor: potential role in endometrial angiogenesis, Fertil. Steril. 74 (2000) 107–112. [19] J. Wang, C. Xiao, Z. Wei, Y. Wang, X. Zhang, Y. Fu, Activation of liver X receptors inhibit LPS-induced inflammatory response in primary bovine mammary epithelial cells, Vet. Immunol. Immunopathol. 197 (2018) 87–92. [20] P.P. Tak, G.S. 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Fig. 5. Effects of alpinetin on PPAR-γ expression. The data of this study are presented as mean ± SEM of three parallel measurements. p# < 0.01 vs. control group, p** < 0.01 vs. LPS group.
endometritis by inhibiting uterine histopathological changes and inflammatory cytokine production. We also found that alpinetin suppressed the LPS-induced inflammatory response by activating PPAR-γ. Inflammation plays a critical role in the development of endometritis [15]. A significant elevation of TNF-α, IL-1ß, and IL-6 in uterine tissues was observed in the LPS-induced endometritis model [16]. These inflammatory cytokines could induce other cytokines and result in epithelial cell necrosis. Furthermore, these cytokines could lead to the accumulation of neutrophils in the uterus [17]. The overactivation of neutrophils may result in injury of the endometrial epithelium [18]. We found that alpinetin inhibited LPS-induced neutrophils infiltration. Furthermore, alpinetin could inhibit LPS-induced inflammatory cytokines production. In the following studies, we investigated the anti-inflammatory mechanism of alpinetin. Inflammatory cytokines are known to be regulated by NF-κB [19]. NF-κB is known as the target of many inflammatory diseases [20, 21]. Inhibition of NF-κB activation had protective effects against endometritis [22]. In this study, alpinetin exhibited anti-inflammatory effects by inhibiting NF-κB activation. PPAR-γ is an important nuclear receptor that regulates lipid metabolism [23]. It also plays critical roles in the regulation of the inflammatory response [24]. Furthermore, many herbal compounds have been known to activate PPAR-γ and exhibited their anti-inflammatory effects through activating PPAR-γ [25, 26]. In addition, PPAR-γ agonists can inhibit NF-κB activation [27]. Therefore, PPAR-γ expression was measured in this study. We found that alpinetin increased the expression of PPAR-γ. These results indicated that alpinetin exhibited anti-inflammatory effects by activating PPAR-γ. In conclusion, our study provided evidence that alpinetin could protect LPS-induced endometritis by inhibiting the inflammatory response. The mechanism was through activation of PPAR-γ, which subsequently attenuated the inflammatory response induced by LPS. Alpinetin might be a useful agent for treating endometritis.
Conflict of interest All authors declare that they have no conflict of interest.
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