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Abstract The juice leaked from broken phloem of Paulownia elongate S.Y. Hu has been used to cure acute inflammation resulted from stung injury by poisonous insects for a long time in China, but its potential mechanism remains unclear. The present study was designed to evaluate anti-inflammatory activity and mechanism of total flavonoids from the phloem of P. elongate S.Y. Hu in RAW264.7 cells. Lipopolysaccharide (LPS)-induced nitric oxide (NO) was measured by Griess and mRNA of pro-inflammatory mediators was analyzed by q-PCR. Cell viability was measured using cell counting kit (CCK)-8 assay. The protein level was analyzed by Western blot. The results showed that the total flavonoids of P. elongate significantly inhibited the production of the pro-inflammatory mediators such as NO, interleukin (IL)-1β and interleukin (IL)-6 in LPS-stimulated RAW264.7 cells. Total flavonoids of P. elongate exerted potential anti-inflammatory activity through the regulation of several signaling pathways. Total flavonoids of P. elongate inhibited JAK/STAT by blocking JAK2 and STAT3 phosphorylation levels. This is the first report on anti-inflammatory activity of total flavonoids from the phloem of P. elongate, which suggests that the total flavonoids of P. elongate may have great potential for the development of anti-inflammatory drug to treat inflammatory disorders.
Key words Paulownia elongate; Total flavonoids; Anti-inflammatory activity; Mechanisms
Received: March 23, 2021 Accepted: May 6, 2021
Supported by Henan Provincial Department of Science & Technology (172102310165); Henan Provincial Department of Education (21B350001).
Liyan LI (1974-), female, P. R. China, associate professor, devoted to research about anti-tumor and anti-UV radiation.
*Corresponding author.
Inflammation is a complex physiological process triggered by harmful stimuli such as infection injury. The immune system will notice and remove the harmful stimuli and heal damages［1］. Several types of immune cells play a role in maintaining homeostasis by regulating the levels of inflammatory mediators. Macrophages are the most important inflammatory regulatory cells, which execute antigen presentation, phagocytosis and immune regulation functions through secreting various inflammatory cytokines and growth factors. In particular, lipopolysaccharide (LPS) stimulation on macrophages initiates a series of downstream signaling cascades, which eventually lead to an increase in inflammatory mediator levels, such as nitric oxide (NO), inducible NO synthase (iNOS) and pro-inflammatory cytokines, interleukin (IL)-1β, IL-6 and so on［2］. Statistical analysis
Values were expressed as mean±SEM. The analysis of Variance (ANOVA) and Tukey test were used to assess biological activity data, with P<0.05 established as statistically significant.
Results and Discussion
Cytotoxicity assay
Prior to the evaluation of anti-inflammatory effect of total flavonoids from P. elongate in LPS-activated RAW 264.7 macrophage, the cytotoxic concentration of the extract was determined using CCK-8 assay. As shown in Fig. 1, total flavonoids of P. elongate showed almost no cytotoxic effect against RAW 264.7 macrophages at concentration of less 100 μg/ml. Therefore, 50 μg/ml of total flavonoids of P. elongate were chosen for the maximum test concentration.
Liyan LI et al. Anti-inflammatory Activity and Mechanism of Total Flavonoids from the Phloem of Paulownia elongate S.Y. Hu in LPS-stimulated RAW264.7 Macrophages
Effect of total flavonoids of P. elongate on NO production in LPS-stimulated RAW264.7 cells
After the treatment with total flavonoids of P. elongate followed stimulation of LPS, NO concentration in the cultured medium was determined here. For control group, the basal concentration of NO in RAW264.7 macrophages was 0.095 μM, but this value was significantly increased to 4.96 μM after the treatment with 0.5 μg/ml of LPS, indicating inflammatory response in RAW264.7 macrophages. However, NO concentration by LPS treatment was significantly decreased by pretreatment with total flavonoids of P. elongate in a dose-dependent manner and it was1.5, 2.08 and 3.27 μM, respectively, and which was 0.35 μM in aspirin group (Fig. 2).
Effect of total flavonoids of P. elongate on inflammatory factors
We also examined whether total flavonoids of P. elongate could regulate production of pro-inflammatory cytokines by ELISA assay. As shown in Fig. 3a-c, LPS treatment significantly increased the expression level of IL-6 and IL-1β compared with those of the nontreatment group, which was consistent with the results of mRNA expression (Fig. 3b and Fig. 3c) and protein level (Fig. 3a). While, the pretreatment of total flavonoids of P. elongate significantly inhibited the expression of proinflammatory cytokines in a dose-dependent manner. The present results indicate that total flavonoids of P. elongate may be beneficial for preventing inflammatory disorder.
Western blot analysis
To determine whether the blockade of JAK/STAT signaling activation plays a role in mediating the anti-inflammatory effect of total flavonoids of P. elongate, we investigated the effects of total flavonoids of P. elongate on LPS-stimulated activation of p-JAK2 (Tyr1007/1008) and p-STAT3 (Tyr705) in macrophages by western blot. As illustrated in Fig. 4a, total flavonoids of P. elongate attenuated the phosphorylation of p-JAK2 induced by LPS (1 μg/ml) stimulation for 4 h in a dose-dependent manner. Subsequently, total flavonoids of P. elongate were also shown to attenuate LPS-induced STAT3 phosphorylation levels in a concentration-dependent manner (Fig. 4b). It suggested that total flavonoids of P. elongate prevented the JAK/STAT signaling pathway in LPS-treated inflammatory condition. Discussion
The juice leaked from the broken phloem of Paulownia elongate has been used to cure acute inflammation resulted from stung injury by poisonous insects in folk of China for longtime. However, the mechanism through which the effective constituent exerts anti-inflammatory activity is not elucidated. Flavonoids in plant always show anti-inflammatory activity. In this study, therefore, we investigated the anti-inflammatory activity and mechanism of total flavonoids from phloem of P. elongate in LPS-stimulated murine macrophages, RAW264.7 cells.
The process of inflammation response include extensive leukocytes infiltration, secretion of inflammatory mediators, and abnormal activation of signaling pathways regulating inflammation, which may result in the development and ongoing of various diseases［7］. Macrophages are crucial cells that play a variety of roles in the inflammatory process, through produce a variety of proinflammatory mediators such as NO, IL-1β and IL-6 in response to LPS stimuli. Thus, we evaluated whether flavonoids of P. elongate exerts its anti-inflammatory properties by inhibiting other inflammatory mediators and cytokines, as well as NO, and investigate the signaling pathways involved in its mechanism in RAW 264.7 cells stimulated by LPS, which is widely used for searching proinflammatory agents.
In this study, we observed that total flavonoids of P. elongate significantly lowered LPS-induced NO levels, a key biomarker of oxidative stress in inflammatory reactions, in RAW264.7 cells［8］. Our study also indicate that total flavonoids of P. elongate displays great potency in deceasing LPS-stimulated proinflammatory cytokines IL-1β and IL-6 in RAW264.7 cells without exhibiting cytotoxicity. This suggested that it may be a promising candidate as a novel anti-inflammatory drug.
Furtherly, we investigated the possible mechanism of the anti-inflammatory effect of total flavonoids of P. elongate in RAW264.7 cells.
The transcriptional activation of JAK2/STAT3 signal pathway
has been reported to be essential in mediating inflammatory diseases［9-11］. The transcriptional activity of STATs could be activated by upstream inflammatory cytokines, and activated STATs protein mediates a positive feedback loop and results in the overproduction of inflammatory mediators and proinflammatory cytokines［12-13］. We observed a decreasing of phosphorylated JAK2 and phosphorylated STAT3 proteins expression level in LPS-activated macrophages following pretreatment with flavonoids of P. elongate, which in turn might inhibit the proinflammatory factors, such as IL-1β and IL-6. Conclusions
Our study demonstrated that total flavonoids of P. elongate significantly inhibited the production of the pro-inflammatory mediators such as NO, IL-1β and IL-6 in LPS-stimulated RAW264.7 cells. Total flavonoids of P. elongate exerted potential anti-inflammatory activity through the regulation of several signaling pathways. Total flavonoids of P. elongate inhibited JAK/STAT by blocking JAK2 and STAT3 phosphorylation levels. These findings suggest that flavonoids of P. elongate may have great potential for the development of anti-inflammatory drug to treat inflammatory disorders. In further, the experiments in vivo need to be done to further identify to supply a probability of flavonoids of P. elongate to develop as an anti-inflammatory drug.
References
［1］ CHEN L, DENG H, CUI H, et al. Inflammatory responses and inflammation-associated diseases in organs［J］. Oncotarget, 2017, 9(6): 7204-7218.
［2］ FANG H, PENGAL RA, CAO X, et al. Lipopolysaccharide-induced macrophage inflammatory response is regulated by SHIP［J］. J. Immunol. (Baltimore, Md.: 1950), 2004, 173(1): 360-366.
［3］ KHANSARI N, SHAKIBA Y, MAHMOUDI M. Chronic inflammation and oxidative stress as a major cause of agerelated diseases and cancer［J］. Recent Patents on Inflammation & Allergy Drug Discovery, 2009, 3(1): 73-80.
［4］ LIU BX, DONG GP, LIU HY, et al. Traditional application and modern research and development of skin phytomedicine in dermatosis treatment［J］. China Journal of Traditional Chinese Medicine and Pharmacy, 2018, 33(12): 5654-5659.
［5］ SITTISART P, CHITSOMBOON P, KAMINSKI NE. Pseuderanthemum palatiferum leaf extract inhibits the proinflammatory cytokines, TNF-α and IL-6 expression in LPS-activated macrophages［J］. Food Chem. Toxicol., 2016(97): 11-22.
［6］ OH Y, AHN CB, YOON NY, et al. Protective effect of enzymatic hydrolysates from seahorse (Hippocampus abdominalis) against H2O2-mediated human umbilical vein endothelial cell injury［J］. Biomed. Pharmacother, 2018(108): 103-110.
［7］ LEE DH, SHIN JS, KANG SY, et al. Iridoids from the roots of Patrinia scabra and their inhibitory potential on LPS-induced nitric oxide production［J］. J. Nat. Prod., 2018, 81(6): 1468-1473.
［8］ XUAN YT, GUO Y, ZHU Y, et al. Mechanism of cyclooxygenase-2 upregulation in late preconditioning［J］. J. Mol. Cell. Cardiol., 2003, 35(5): 525-537.
［9］ YU X, KENNEDY RH, LIU SJ. JAK2/STAT3, not ERK1/2, mediates interleukin-6-induced activation of inducible nitric-oxide synthase and decrease in contractility of adult ventricular myocytes［J］. J. Biol. Chem., 2003, 278(18): 16304-16309.
［10］ GORINA R, FONT-NIEVES M, MRQUEZ-KISINOUSKY L, et al. Astrocyte TLR4 activation induces a proinflammatory environment through the interplay between MyD88-dependent NFκB signaling, MAPK, and Jak1/Stat1 pathways［J］. Glia, 2011, 59(2): 242-255.
［11］ BOLLI R, DAWN B, XUAN YT. Role of the JAK–STAT pathway in protection against myocardial ischemia/reperfusion injury［J］. Trends Cardiovas. Med., 2003, 13(2): 72-79.
［12］ MORI T, MIYAMOTO T, YOSHIDA H, et al. IL-1β and TNFα-initiated IL-6-STAT3 pathway is critical in mediating inflammatory cytokines and RANKL expression in inflammatory arthritis［J］. Int. Immunol., 2011, 23(11): 701-712.
Key words Paulownia elongate; Total flavonoids; Anti-inflammatory activity; Mechanisms
Received: March 23, 2021 Accepted: May 6, 2021
Supported by Henan Provincial Department of Science & Technology (172102310165); Henan Provincial Department of Education (21B350001).
Liyan LI (1974-), female, P. R. China, associate professor, devoted to research about anti-tumor and anti-UV radiation.
*Corresponding author.
Inflammation is a complex physiological process triggered by harmful stimuli such as infection injury. The immune system will notice and remove the harmful stimuli and heal damages［1］. Several types of immune cells play a role in maintaining homeostasis by regulating the levels of inflammatory mediators. Macrophages are the most important inflammatory regulatory cells, which execute antigen presentation, phagocytosis and immune regulation functions through secreting various inflammatory cytokines and growth factors. In particular, lipopolysaccharide (LPS) stimulation on macrophages initiates a series of downstream signaling cascades, which eventually lead to an increase in inflammatory mediator levels, such as nitric oxide (NO), inducible NO synthase (iNOS) and pro-inflammatory cytokines, interleukin (IL)-1β, IL-6 and so on［2］. Statistical analysis
Values were expressed as mean±SEM. The analysis of Variance (ANOVA) and Tukey test were used to assess biological activity data, with P<0.05 established as statistically significant.
Results and Discussion
Cytotoxicity assay
Prior to the evaluation of anti-inflammatory effect of total flavonoids from P. elongate in LPS-activated RAW 264.7 macrophage, the cytotoxic concentration of the extract was determined using CCK-8 assay. As shown in Fig. 1, total flavonoids of P. elongate showed almost no cytotoxic effect against RAW 264.7 macrophages at concentration of less 100 μg/ml. Therefore, 50 μg/ml of total flavonoids of P. elongate were chosen for the maximum test concentration.
Liyan LI et al. Anti-inflammatory Activity and Mechanism of Total Flavonoids from the Phloem of Paulownia elongate S.Y. Hu in LPS-stimulated RAW264.7 Macrophages
Effect of total flavonoids of P. elongate on NO production in LPS-stimulated RAW264.7 cells
After the treatment with total flavonoids of P. elongate followed stimulation of LPS, NO concentration in the cultured medium was determined here. For control group, the basal concentration of NO in RAW264.7 macrophages was 0.095 μM, but this value was significantly increased to 4.96 μM after the treatment with 0.5 μg/ml of LPS, indicating inflammatory response in RAW264.7 macrophages. However, NO concentration by LPS treatment was significantly decreased by pretreatment with total flavonoids of P. elongate in a dose-dependent manner and it was1.5, 2.08 and 3.27 μM, respectively, and which was 0.35 μM in aspirin group (Fig. 2).
Effect of total flavonoids of P. elongate on inflammatory factors
We also examined whether total flavonoids of P. elongate could regulate production of pro-inflammatory cytokines by ELISA assay. As shown in Fig. 3a-c, LPS treatment significantly increased the expression level of IL-6 and IL-1β compared with those of the nontreatment group, which was consistent with the results of mRNA expression (Fig. 3b and Fig. 3c) and protein level (Fig. 3a). While, the pretreatment of total flavonoids of P. elongate significantly inhibited the expression of proinflammatory cytokines in a dose-dependent manner. The present results indicate that total flavonoids of P. elongate may be beneficial for preventing inflammatory disorder.
Western blot analysis
To determine whether the blockade of JAK/STAT signaling activation plays a role in mediating the anti-inflammatory effect of total flavonoids of P. elongate, we investigated the effects of total flavonoids of P. elongate on LPS-stimulated activation of p-JAK2 (Tyr1007/1008) and p-STAT3 (Tyr705) in macrophages by western blot. As illustrated in Fig. 4a, total flavonoids of P. elongate attenuated the phosphorylation of p-JAK2 induced by LPS (1 μg/ml) stimulation for 4 h in a dose-dependent manner. Subsequently, total flavonoids of P. elongate were also shown to attenuate LPS-induced STAT3 phosphorylation levels in a concentration-dependent manner (Fig. 4b). It suggested that total flavonoids of P. elongate prevented the JAK/STAT signaling pathway in LPS-treated inflammatory condition. Discussion
The juice leaked from the broken phloem of Paulownia elongate has been used to cure acute inflammation resulted from stung injury by poisonous insects in folk of China for longtime. However, the mechanism through which the effective constituent exerts anti-inflammatory activity is not elucidated. Flavonoids in plant always show anti-inflammatory activity. In this study, therefore, we investigated the anti-inflammatory activity and mechanism of total flavonoids from phloem of P. elongate in LPS-stimulated murine macrophages, RAW264.7 cells.
The process of inflammation response include extensive leukocytes infiltration, secretion of inflammatory mediators, and abnormal activation of signaling pathways regulating inflammation, which may result in the development and ongoing of various diseases［7］. Macrophages are crucial cells that play a variety of roles in the inflammatory process, through produce a variety of proinflammatory mediators such as NO, IL-1β and IL-6 in response to LPS stimuli. Thus, we evaluated whether flavonoids of P. elongate exerts its anti-inflammatory properties by inhibiting other inflammatory mediators and cytokines, as well as NO, and investigate the signaling pathways involved in its mechanism in RAW 264.7 cells stimulated by LPS, which is widely used for searching proinflammatory agents.
In this study, we observed that total flavonoids of P. elongate significantly lowered LPS-induced NO levels, a key biomarker of oxidative stress in inflammatory reactions, in RAW264.7 cells［8］. Our study also indicate that total flavonoids of P. elongate displays great potency in deceasing LPS-stimulated proinflammatory cytokines IL-1β and IL-6 in RAW264.7 cells without exhibiting cytotoxicity. This suggested that it may be a promising candidate as a novel anti-inflammatory drug.
Furtherly, we investigated the possible mechanism of the anti-inflammatory effect of total flavonoids of P. elongate in RAW264.7 cells.
The transcriptional activation of JAK2/STAT3 signal pathway
has been reported to be essential in mediating inflammatory diseases［9-11］. The transcriptional activity of STATs could be activated by upstream inflammatory cytokines, and activated STATs protein mediates a positive feedback loop and results in the overproduction of inflammatory mediators and proinflammatory cytokines［12-13］. We observed a decreasing of phosphorylated JAK2 and phosphorylated STAT3 proteins expression level in LPS-activated macrophages following pretreatment with flavonoids of P. elongate, which in turn might inhibit the proinflammatory factors, such as IL-1β and IL-6. Conclusions
Our study demonstrated that total flavonoids of P. elongate significantly inhibited the production of the pro-inflammatory mediators such as NO, IL-1β and IL-6 in LPS-stimulated RAW264.7 cells. Total flavonoids of P. elongate exerted potential anti-inflammatory activity through the regulation of several signaling pathways. Total flavonoids of P. elongate inhibited JAK/STAT by blocking JAK2 and STAT3 phosphorylation levels. These findings suggest that flavonoids of P. elongate may have great potential for the development of anti-inflammatory drug to treat inflammatory disorders. In further, the experiments in vivo need to be done to further identify to supply a probability of flavonoids of P. elongate to develop as an anti-inflammatory drug.
References
［1］ CHEN L, DENG H, CUI H, et al. Inflammatory responses and inflammation-associated diseases in organs［J］. Oncotarget, 2017, 9(6): 7204-7218.
［2］ FANG H, PENGAL RA, CAO X, et al. Lipopolysaccharide-induced macrophage inflammatory response is regulated by SHIP［J］. J. Immunol. (Baltimore, Md.: 1950), 2004, 173(1): 360-366.
［3］ KHANSARI N, SHAKIBA Y, MAHMOUDI M. Chronic inflammation and oxidative stress as a major cause of agerelated diseases and cancer［J］. Recent Patents on Inflammation & Allergy Drug Discovery, 2009, 3(1): 73-80.
［4］ LIU BX, DONG GP, LIU HY, et al. Traditional application and modern research and development of skin phytomedicine in dermatosis treatment［J］. China Journal of Traditional Chinese Medicine and Pharmacy, 2018, 33(12): 5654-5659.
［5］ SITTISART P, CHITSOMBOON P, KAMINSKI NE. Pseuderanthemum palatiferum leaf extract inhibits the proinflammatory cytokines, TNF-α and IL-6 expression in LPS-activated macrophages［J］. Food Chem. Toxicol., 2016(97): 11-22.
［6］ OH Y, AHN CB, YOON NY, et al. Protective effect of enzymatic hydrolysates from seahorse (Hippocampus abdominalis) against H2O2-mediated human umbilical vein endothelial cell injury［J］. Biomed. Pharmacother, 2018(108): 103-110.
［7］ LEE DH, SHIN JS, KANG SY, et al. Iridoids from the roots of Patrinia scabra and their inhibitory potential on LPS-induced nitric oxide production［J］. J. Nat. Prod., 2018, 81(6): 1468-1473.
［8］ XUAN YT, GUO Y, ZHU Y, et al. Mechanism of cyclooxygenase-2 upregulation in late preconditioning［J］. J. Mol. Cell. Cardiol., 2003, 35(5): 525-537.
［9］ YU X, KENNEDY RH, LIU SJ. JAK2/STAT3, not ERK1/2, mediates interleukin-6-induced activation of inducible nitric-oxide synthase and decrease in contractility of adult ventricular myocytes［J］. J. Biol. Chem., 2003, 278(18): 16304-16309.
［10］ GORINA R, FONT-NIEVES M, MRQUEZ-KISINOUSKY L, et al. Astrocyte TLR4 activation induces a proinflammatory environment through the interplay between MyD88-dependent NFκB signaling, MAPK, and Jak1/Stat1 pathways［J］. Glia, 2011, 59(2): 242-255.
［11］ BOLLI R, DAWN B, XUAN YT. Role of the JAK–STAT pathway in protection against myocardial ischemia/reperfusion injury［J］. Trends Cardiovas. Med., 2003, 13(2): 72-79.
［12］ MORI T, MIYAMOTO T, YOSHIDA H, et al. IL-1β and TNFα-initiated IL-6-STAT3 pathway is critical in mediating inflammatory cytokines and RANKL expression in inflammatory arthritis［J］. Int. Immunol., 2011, 23(11): 701-712.