A polysaccharide extract from the medicinal plant Maidong inhibits the IKK–NF-κB pathway and IL-1β–induced islet inflammation and increases insulin secretion

Abstract

The herb dwarf lilyturf tuber (Maidong, Ophiopogonis Radix) is widely used in Chinese traditional medicine to manage diabetes and its complications. However, the role of Maidong polysaccharide extract (MPE) in pancreatic β-cell function is unclear. Here, we investigated whether MPE protects β-cell function and studied the underlying mechanisms. We treated db/db and high-fat diet (HFD)-induced obese mice with 800 or 400 mg/kg MPE or water for 4 weeks, followed by an oral glucose tolerance test. Pancreas and blood were collected for molecular analyses, and clonal MIN6 β-cells and primary islets from HFD-induced obese mice and normal chow diet–fed mice were used in additional analyses. In vivo, MPE both increased insulin secretion and reduced blood glucose in the db/db mice but increased only insulin secretion in the HFD-induced obese mice. MPE substantially increased the β-cell area in both models (3-fold and 2-fold, p < 0.01, for db/db and HFD mice, respectively). We observed reduced nuclear translocation of the p65 subunit of NF-κB in islets of MPE-treated db/db mice, coinciding with enhanced glucose-stimulated insulin secretion (GSIS). In vitro, MPE potentiated GSIS and decreased interleukin 1β (IL-1β) secretion in MIN6 β-cells. Incubation of MIN6 cells with tumor necrosis factor α (TNFα), interferon-γ, and IL-1β amplified IL-1β secretion and inhibited GSIS. These effects were partially reversed with MPE or the IκB kinase β inhibitor PS1145, coinciding with reduced activation of p65 and p-IκB in the NF-κB pathway. We conclude that MPE may have potential for therapeutic development for β-cell protection. The herb dwarf lilyturf tuber (Maidong, Ophiopogonis Radix) is widely used in Chinese traditional medicine to manage diabetes and its complications. However, the role of Maidong polysaccharide extract (MPE) in pancreatic β-cell function is unclear. Here, we investigated whether MPE protects β-cell function and studied the underlying mechanisms. We treated db/db and high-fat diet (HFD)-induced obese mice with 800 or 400 mg/kg MPE or water for 4 weeks, followed by an oral glucose tolerance test. Pancreas and blood were collected for molecular analyses, and clonal MIN6 β-cells and primary islets from HFD-induced obese mice and normal chow diet–fed mice were used in additional analyses. In vivo, MPE both increased insulin secretion and reduced blood glucose in the db/db mice but increased only insulin secretion in the HFD-induced obese mice. MPE substantially increased the β-cell area in both models (3-fold and 2-fold, p < 0.01, for db/db and HFD mice, respectively). We observed reduced nuclear translocation of the p65 subunit of NF-κB in islets of MPE-treated db/db mice, coinciding with enhanced glucose-stimulated insulin secretion (GSIS). In vitro, MPE potentiated GSIS and decreased interleukin 1β (IL-1β) secretion in MIN6 β-cells. Incubation of MIN6 cells with tumor necrosis factor α (TNFα), interferon-γ, and IL-1β amplified IL-1β secretion and inhibited GSIS. These effects were partially reversed with MPE or the IκB kinase β inhibitor PS1145, coinciding with reduced activation of p65 and p-IκB in the NF-κB pathway. We conclude that MPE may have potential for therapeutic development for β-cell protection. More than 450 million people are affected worldwide by diabetes, the majority having type 2 diabetes (T2D) (1World Health OrganizationGlobal Report on Diabetes. World Health Organization, Geneva2016: 6Google Scholar, 2Cho N.H. Shaw J.E. Karuranga S. Huang Y. da Rocha Fernandes J.D. Ohlrogge A.W. Malanda B. IDF Diabetes Atlas: global estimates of diabetes prevalence for 2017 and projections for 2045.Diabetes Res. Clin. Pract. 2018; 138 (29496507): 271-28110.1016/j.diabres.2018.02.023Abstract Full Text Full Text PDF PubMed Scopus (3693) Google Scholar). Although there are many classes of antidiabetic drugs, Chinese medicines remain a popular remedy for diabetes in China (3Tahrani A.A. Bailey C.J. Del Prato S. Barnett A.H. Management of type 2 diabetes: new and future developments in treatment.Lancet. 2011; 378 (21705062): 182-19710.1016/S0140-6736(11)60207-9Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). Among these remedies, dwarf lilyturf tuber (Maidong, Ophiopogonis Radix) is widely used, with experimental data supporting its antidiabetic effects. In OLEFT T2D rat models, Maidong improved glucose homeostasis, inhibited abdominal fat accumulation, and enhanced hepatic glucose uptake (4Kim J.E. Hwang I.S. Choi S.I. Lee H.R. Lee Y.J. Goo J.S. Lee H.S. Son H.J. Jang M.J. Lee S.H. Kang B.C. Hwang D.Y. Aqueous extract of Liriope platyphylla, a traditional Chinese medicine, significantly inhibits abdominal fat accumulation and improves glucose regulation in OLETF type II diabetes model rats.Lab. Anim. Res. 2012; 28 (23091518): 181-19110.5625/lar.2012.28.3.181Crossref PubMed Google Scholar). In diabetic mice, Maidong fractions delayed carbohydrate digestion and absorption with improvement of lipid metabolism (5Ding L. Li P. Lau C.B. Chan Y.W. Xu D. Fung K.P. Su W. Mechanistic studies on the antidiabetic activity of a polysaccharide-rich extract of Radix Ophiopogonis.Phytother. Res. 2012; 26 (21560174): 101-10510.1002/ptr.3505Crossref PubMed Scopus (16) Google Scholar, 6Liu Y. Wan L. Xiao Z. Wang J. Wang Y. Chen J. Antidiabetic activity of polysaccharides from tuberous root of Liriope spicata var. prolifera in KKAy mice.Evid. Based Complement. Alternat. Med. 2013; 2013 (23762123): 34979010.1155/2013/349790PubMed Google Scholar). 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Insulin resistance, mainly mediated through obesity and inflammation, as well as insulin insufficiency contribute to the development of T2D, with β-cell dysfunction being particularly relevant in Asians (11Yabe D. Seino Y. Type 2 diabetes via beta-cell dysfunction in east Asian people.Lancet Diabetes Endocrinol. 2016; 4 (26577717): 2-310.1016/S2213-8587(15)00389-7Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Factors that contribute to β-cell dysfunction include but are not limited to glucolipotoxicity, inflammation, and endoplasmic reticulum stress (12Cerf M.E. Beta cell dysfunction and insulin resistance.Front. Endocrinol. (Lausanne). 2013; 4 (23542897): 1-1010.3389/fendo.2013.00037Crossref PubMed Scopus (464) Google Scholar, 13Yang H. Li X. The role of fatty acid metabolism and lipotoxicity in pancreatic β-cell injury: identification of potential therapeutic targets.Acta Pharm. Sin. 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IL-1β can activate the NF-κB signaling cascade followed by Fas dysregulation and DNA fragmentation, resulting in β-cell dysfunction (15Maedler K. Sergeev P. Ris F. Oberholzer J. Joller-Jemelka H.I. Spinas G.A. Kaiser N. Halban P.A. Donath M.Y. Glucose-induced beta cell production of IL-1β contributes to glucotoxicity in human pancreatic islets.J. Clin. Invest. 2017; 127 (28368291): 1589-186010.1172/JCI92172Crossref PubMed Scopus (35) Google Scholar). IFN-γ or TNFα can activate the c-Jun N-terminal kinase and protein kinase C pathways to impair β-cell function (16Cunha D.A. Hekerman P. Ladrière L. Bazarra-Castro A. Ortis F. Wakeham M.C. Moore F. Rasschaert J. Cardozo A.K. Bellomo E. Overbergh L. Mathieu C. Lupi R. Hai T. Herchuelz A. et al.Initiation and execution of lipotoxic ER stress in pancreatic beta-cells.J. Cell Sci. 2008; 121 (18559892): 2308-231810.1242/jcs.026062Crossref PubMed Scopus (471) Google Scholar). 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Polysaccharides from many Chinese herbs, such as Curcuma longa, Morinda citrifolia Linn, and Wedelia chinensis, are found to have beneficial effects in a broad spectrum of diseases, such as inflammatory bowel disease and extremity and ear infections. These beneficial effects are mediated through various mechanisms, including reduced production of inflammatory cytokines and inhibition of leukocytes migration (21Sousa S.G. Oliveira L.A. de Aguiar Magalhães D. de Brito T.V. Batista J.A. Pereira C.M.C. de Souza Costa M. Mazulo J.C.R. de Carvalho Filgueiras M. Vasconselos D.F.P. da Silva D.A. Barros F.C.N. Sombra V.G. Freitas A.L.P. de Paula R.C.M. et al.Chemical structure and anti-inflammatory effect of polysaccharide extracted from Morinda citrifolia Linn (Noni).Carbohydr. Polym. 2018; 197 (30007642): 515-52310.1016/j.carbpol.2018.06.042Crossref PubMed Scopus (56) Google Scholar, 22Illuri R. Bethapudi B. Anandakumar S. Murugan S. Joseph J.A. Mundkinajeddu D. Agarwal A. Chandrasekaran C.V. Anti-inflammatory activity of polysaccharide fraction of Curcuma longa extract (NR-INF-02).Antiinflamm. Antiallergy Agents Med. Chem. 2015; 14 (26013326): 53-6210.2174/1871523014666150407150533Crossref PubMed Scopus (15) Google Scholar, 23Gao C. Liu L. Zhou Y. Bian Z. Wang S. Wang Y. Novel drug delivery systems of Chinese medicine for the treatment of inflammatory bowel disease.Chin. Med. 2019; 14 (31236131): 23-4010.1186/s13020-019-0245-xCrossref PubMed Scopus (24) Google Scholar). Against this background, we selected IL-1β as a possible target to explore the antidiabetic effect of MPE. We hypothesized that MPE might lower blood glucose by reducing inflammation in β-cells and thus increasing insulin secretion. To test this hypothesis, we used multiple, complementary in vivo and ex vivo models. First, db/db mice and high-fat diet (HFD)-induced obese mice were used to examine the antidiabetic effect of MPE in vivo. Next, we used clonal MIN6 β-cells (24Miyazaki J. Araki K. Yamato E. Ikegami H. Asano T. Shibasaki Y. Oka Y. Yamamura K. Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms.Endocrinology. 1990; 127 (2163307): 126-13210.1210/endo-127-1-126Crossref PubMed Scopus (1053) Google Scholar) to explore changes in gene expressions in vitro by microarray analysis. MIN6 cells and primary islets from HFD-induced obese mice and C57 BL6/J mice were also used to examine the expression and release of inflammatory markers. Treatment of db/db mice for 4 weeks with MPE at 400 or 800 mg/kg or with water as a control reduced fasting blood glucose levels in a dose-dependent manner. At week 4, fasting blood glucose increased from a mean ± SEM of 10 ± 0.49 mmol/litre to 20.2 ± 1.1 mmol/liter in the control group, compared with 17.3 ± 0.5 mmol/liter in the MPE 400 mg/kg group (p < 0.05) and 15.3 ± 0.7 mmol/liter in the MPE 800 mg/kg group (p < 0.01) (Fig. 1A). There was no further reduction in blood glucose during the oral glucose tolerance test (OGTT) (Fig. S2D) or insulin tolerance test (ITT) in both treatment groups (Fig. S2C). Peak insulin secretion was 5.06 ± 0.36 in the 400 mg/kg group and ± 0.36 in the 800 mg/kg group, both of which were significantly than that in the control group ± (Fig. There was no effect of MPE on or levels the of blood glucose were increased during and 10 weeks of HFD compared with normal chow diet mice (Fig. MPE there was no in fasting blood glucose levels the groups (Fig. Treatment with MPE not blood glucose (Fig. and but enhanced glucose-stimulated insulin secretion during in HFD-induced obese mice compared with control mice (p < 0.01) (Fig. treatment for 4 weeks, were from both mouse models for of and 2 and of and in group of db/db mice and HFD-induced obese mice, In both db/db mice and HFD-induced obese mice, the β-cell as a of the pancreatic increased substantially and 2-fold, and significantly treatment with of MPE (Fig. C and compared with the control There was no in with MPE in model (Fig. and the of pancreatic area by β-cells in db/db mice and HFD-induced obese mice. and of insulin and in the of db/db mice and HFD-induced obese mice in the and the pancreatic area in of The and islets in the selected area of the pancreatic area in the of and The is in the 100 C and of in db/db mice and HFD-induced obese mice by in of β-cells in db/db mice and HFD-induced obese mice by in are as mean ± with data in p < p < We used MIN6 cells as an in vitro model with GSIS and as of β-cell function and We treated MIN6 cells with or MPE at of and 50 for and followed by of GSIS and Insulin secretion not MPE treatment for and (Fig. and but increased at ± ± of p < 0.05) (Fig. Treatment with MPE increased the in MIN6 cells ± ± p < 0.01) (Fig. In primary islets from HFD-induced obese mice, MPE treatment for a in both GSIS ± ± of p < 0.01) (Fig. and (Fig. ± 0.49 ± p < We microarray to MIN6 cells to explore the underlying with and MPE The expressions of and were reduced treatment with MPE (Fig. In vivo, MPE reduced p65 nuclear translocation in islets of db/db mice (Fig. and In MPE also reduced the expression of IL-1β in islets from both models (Fig. expression microarray and pathway inhibition of pathway by MPE analysis. expression of MPE-treated MIN6 cells are The 1 the and the and are gene analysis. and insulin pathways are pathways in the in MPE-treated MIN6 cells are gene expression levels and changes of receptor and are in control in MPE-treated MPE treatment attenuated the inflammatory pathway. the in the pathway. of expression of and by of MPE on NF-κB p65 nuclear and IL-1β of p65 in the of of db/db mice and both the and in the in the 50 respectively). in the of in the of db/db mice. of expression levels in MIN6 cells with treatment of MPE or control in or IL-1β protein levels in treatment of MIN6 cells with IL-1β or IL-1β TNFα of expression levels in primary islets from HFD-induced obese mice with treatment of MPE or IL-1β protein levels in treatment of primary islets of HFD-induced obese mice with IL-1β or IL-1β GSIS was to the primary islet function from C57 mice with normal chow treatment with IL-1β or IL-1β in control and MPE of expression levels were treatment with IL-1β or IL-1β TNFα in primary islets of C57 mice in control and MPE are as mean ± with data in p < p < of MPE on NF-κB p65 nuclear and IL-1β of p65 in the of of db/db mice and both the and in the in the 50 respectively). in the of in the of db/db mice. of expression levels in MIN6 cells with treatment of MPE or control in or IL-1β protein levels in treatment of MIN6 cells with IL-1β or IL-1β TNFα of expression levels in primary islets from HFD-induced obese mice with treatment of MPE or IL-1β protein levels in treatment of primary islets of HFD-induced obese mice with IL-1β or IL-1β GSIS was to the primary islet function from C57 mice with normal chow treatment with IL-1β or IL-1β in control and MPE of expression levels were treatment with IL-1β or IL-1β TNFα in primary islets of C57 mice in control and MPE are as mean ± with data in p < p < In vitro, expression of IL-1β in MIN6 cells and primary islets was by MPE treatment inflammatory IL-1β production by IL-1β or a TNFα was attenuated with MPE treatment compared with cells with MPE (Fig. We the of IL-1β in 10 in to the secretion. To whether production was in β-cell dysfunction and was by we GSIS in primary islets from mice treated with the inflammatory cytokines shown in MPE not effect on islets from mice IL-1β and the inhibited insulin and these effects were attenuated by the of MPE At the MPE increased insulin secretion treated with IL-1β and the in primary islets from mice in vitro, MPE reduced levels of (Fig. by IL-1β or the MPE also decreased the production of IL-1β in MIN6 cells and primary islets from HFD-induced obese mice. However, MPE not proliferation in islets of HFD-induced obese mice (Fig. with its can activate the pathway to expression of to further impair β-cell function K. Sergeev P. Ris F. Oberholzer J. Joller-Jemelka H.I. Spinas G.A. Kaiser N. Halban P.A. Donath M.Y. Glucose-induced beta cell production of IL-1beta contributes to glucotoxicity in human pancreatic islets.J. Clin. Invest. 2002; 110 (12235117): 851-86010.1172/JCI200215318Crossref PubMed Scopus (1024) Google Scholar). To test the potential effects of MPE on this we used MIN6 cells in or for and treated with or MPE. Treatment was followed by of and IL-1β shown in with glucose insulin secretion and reduced by of IL-1β at both the and protein In and treatment of MIN6 cells with inhibitor in glucose attenuated the inflammatory response and improved β-cell This was by increased GSIS and in MIN6 with the in insulin secretion and by MPE. In the control group treated with a of the of to insulin secretion and by blocking the production of IL-1β (Fig. and In MIN6 cells treated with reversed the reduction of IL-1β by MPE with increased expression and protein of IL-1β (Fig. C and Incubation of MIN6 or islet cells in increased of the NF-κB subunit p65 and NF-κB Treatment with MPE decreased p65 and IκB in MIN6 cells with reduced activation of NF-κB (Fig. MPE reduced p65 and IκB in primary islets from HFD-induced obese mice (Fig. In primary islets from mice, IL-1β or the increased and as shown in with MPE inhibited the of of both p65 and However, MPE not the effects of cytokines on islet possible for signaling pathways protein kinase protein and of A. The of protein kinase pathways in and diabetes: from the pancreatic Metab. Drug Discov. 2017; 10 PubMed Scopus Google In this we used in vivo models and in vitro models to the effects of MPE. MPE increased insulin secretion in both mice models and reduced blood glucose in db/db mice. of the effects of MPE in the systems is in 1 and is in of MPE on in various experimental obese islets from HFD-induced obese islets from (Fig. week (Fig. (Fig. in (Fig. and in (Fig. GSIS (Fig. (Fig. GSIS (Fig. (Fig. of β-cell area (Fig. and of area (Fig. of β-cell area (Fig. and of area (Fig. in microarray (Fig. 4, p65 nuclear translocation (Fig. and expression (Fig. IL-1β protein expression with inflammatory treatment (Fig. IL-1β expression (Fig. IL-1β protein expression (Fig. GSIS with inflammatory treatment (Fig. expression with inflammatory treatment (Fig. (Fig. and (Fig. (Fig. and (Fig. C and (Fig. with inhibitor GSIS (Fig. (Fig. expression (Fig. IL-1β protein expression (Fig. IL-1β expression in (Fig. and IL-1β expression in (Fig. and by (Fig. by (Fig. with inflammatory treatment by (Fig. cell proliferation in (Fig. by in (Fig. lipid α-glucosidase activity (Fig. with GSIS (Fig. in a new

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