Dietary polyherbal supplementation decreases CD3+ cell infiltration into pancreatic islets and prevents hyperglycemia in nonobese diabetic mice

N U TR IT ION RE S E ARCH XX ( 2 0 15 ) X XX–X XX

Available online at www.sciencedirect.com

ScienceDirect www.nrjournal.com

Dietary polyherbal supplementation decreases CD3 + cell infiltration into pancreatic islets and prevents hyperglycemia in nonobese diabetic mice Susan J. Burke a , Michael D. Karlstad a, b , Caroline P. Conley b , Danielle Reel c , Jay Whelan a , J. Jason Collier a, b,⁎ a b c

Department of Nutrition, University of Tennessee, Knoxville, TN, USA Department of Surgery, Graduate School of Medicine, University of Tennessee Medical Center, Knoxville, TN, USA Department of Biomedical and Diagnostic Sciences, University of Tennessee, College of Veterinary Medicine, Knoxville, TN, USA

ARTI CLE I NFO

A BS TRACT

Article history:

Type 1 diabetes mellitus results from autoimmune-mediated destruction of pancreatic islet

Received 20 October 2014

β-cells, a process associated with inflammatory signals. We hypothesized that dietary

Revised 17 December 2014

supplementation with botanicals known to contain anti-inflammatory properties would

Accepted 19 December 2014

prevent losses in functional β-cell mass in nonobese diabetic (NOD) mice, a rodent model of autoimmune-mediated islet inflammation that spontaneously develops diabetes. Female

Keywords:

NOD mice, a model of spontaneous autoimmune diabetes, were fed a diet supplemented

Autoimmunity

with herbal extracts (1.916 g total botanical extracts per 1 kg of diet) over a 12-week period.

Chemokine

The mice consumed isocaloric matched diets without (controls) and with polyherbal

Diabetes

supplementation (PHS) ad libitum starting at a prediabetic stage (age 6 weeks) for 12 weeks.

Inflammation

Control mice developed hyperglycemia (>180 mg/dL) within 16 weeks (n = 9). By contrast,

Nonobese diabetic mouse

mice receiving the PHS diet did not develop hyperglycemia by 18 weeks (n = 8). Insulinpositive cell mass within pancreatic islets was 31.9% greater in PHS mice relative to controls. We also detected a 26% decrease in CD3+ lymphocytic infiltration in PHS mice relative to mice consuming a control diet. In vitro assays revealed reduced β-cell expression of the chemokines CCL2 and CXCL10 after overnight PHS addition to the culture media. We conclude that dietary PHS delays initiation of autoimmune-mediated β-cell destruction and subsequent onset of diabetes mellitus by diminishing islet inflammatory responses. © 2014 Elsevier Inc. All rights reserved.

1.

Introduction

Type 1 diabetes mellitus (T1DM) results from autoimmunemediated destruction of pancreatic islet β-cells [1,2]. The initiating trigger for this disease is unknown. However, many different types of immune cells cooperate to eliminate the insulin-producing β-cells while sparing the other endocrine

cell types within the pancreatic islets [3,4]. Currently, insulin injection is the only therapeutic option to treat T1DM. Although insulin injections are adequate to prevent death, they are insufficient to prevent metabolic and other complications of the disease. Consequently, T1DM is associated with a host of sequelae, especially in individuals with poorly controlled glycemia [5–11]. Therefore, an intervention that

Abbreviations: NF-κB, nuclear factor κB; NOD, nonobese diabetic; PHS, polyherbal supplementation; T1DM, type 1 diabetes mellitus. ⁎ Corresponding author. Laboratory of Islet Biology and Inflammation, Pennington Biomedical Research Center, 6400 Perkins Rd, Baton Rouge, LA 70808, USA. Tel.: +1 225 763 2884; fax: +1 225 763 0274. E-mail address: [email protected] (J. Jason Collier). http://dx.doi.org/10.1016/j.nutres.2014.12.003 0271-5317/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Burke SJ, et al, Dietary polyherbal supplementation decreases CD3+ cell infiltration into pancreatic islets and prevents hyperglycemia in nonobese diabetic mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2014.12.003

2

N UTR IT ION RE S EA RCH XX ( 2 01 5 ) X XX–X XX

helps to decrease blood glucose levels and potentially increase or prevent losses in functional β-cell mass would be of clinical use to prevent, delay, or even assist in treating diabetes. A number of herbal remedies have been used throughout history to treat a variety of human diseases, including diabetes mellitus [12–14]. Although there are herbal drugs approved for use in China that are effective in lowering blood glucose levels [15], few clinical trials using herbal based approaches have been carried out in Western countries. However, in human cancer patients, herbal based approaches are well tolerated and effective [16,17]. The mechanisms underlying the effectiveness of individual herbal interventions (eg, berberine) are beginning to be elucidated and include effects on AMP-activated protein kinase [18–20], direct impacts on hepatic gluconeogenesis [21], and modulation of the MAP kinases [22]. Although many effects have been observed by using the biological compound of interest in the micromolar range, it is nanomolar efficacy that is sought after by the pharmaceutical and nutraceutical industries. Detection of relevant biologically active molecules within the blood of humans fed individual botanical supplements reveals concentrations in the nanomole range [23–25]. Because numerous compounds are often consumed in combination as part of a diverse diet, it is possible that biological effects at the nanomolar level can be achievable through combinatorial interactions in vivo. Specific blends of botanical extracts may be additive, synergistic, or antagonistic, depending on the compounds interacting and the parameter being measured. Although there are several methods of reporting additive and synergistic activities and the concept itself has been reviewed recently [26], specific dietary components have already been shown to have beneficial interactions that reduce both inflammation and tumor proliferation [27–30]. We hypothesized that autoimmune-mediated development of diabetes mellitus, which is driven by inflammatory responses within pancreatic islets, would also respond to dietary herbal supplementation as a therapeutic intervention. Therefore, in this study, we used a polyherbal formulation fed to nonobese diabetic (NOD) mice, a common preclinical murine model of spontaneously autoimmune-initiated diabetes mellitus, to determine whether onset of hyperglycemia was delayed by this intervention. This precise polyherbal blend was chosen due to its known anti-inflammatory effects [28,31] and because it is well tolerated in humans [17,32]. The objective of this study was to determine whether specific readouts of inflammation, relevant to onset of autoimmune forms of diabetes, could be decreased in vitro and in vivo in the presence of a specific polyherbal supplement (PHS). Because T-lymphocytes contribute to diabetes onset in NOD mice [33], we monitored their infiltration into pancreatic islets. The observed reduction in T-lymphocyte infiltration into pancreatic islets in NOD mice consuming the PHS diet was sufficient to prevent onset of hyperglycemia. These results were consistent with preservation of pancreatic β-cell mass in vivo and also with in vitro studies indicating suppression of specific nuclear factor (NF) κB target genes controlling immune cell infiltration and β-cell inflammatory responses.

2.

Methods and materials

2.1.

Diets and animals

The animals were fed human equivalent diets based on the micronutrient and macronutrient intakes of a typical Westerntype diet. All animals were maintained on a control diet for at least 1 week after arrival and then randomized into group that received either the experimental diet or the control diet. The control diet was based on the US17 Monsanto diet with slight modifications in macronutrient distributions (see Ref. [34] and Table). The diet was designed to mimic the Western (US) diet with the following distribution (% of energy): protein 16%, carbohydrates 50%, and lipids 34% (Research Diets No. D07100504, New Brunswick, NJ, USA). The experimental diet containing polyherbal components (Research Diets No. D07100505) remained isocaloric and was formulated using the control diet as the background diet but included the addition of the PHS. Water and food were provided to the mice ad libitum during the study. The dominant chemical constituents present in the botanical extracts have been

Table – Ingredient composition of the diets (g/kg) fed to mice Ingredients a

Control b

Treatment c

Casein, lactic L-Cysteine Corn starch Maltodextrin 10 Sucrose Cellulose Linseed oil Cocoa butter Palm oil Safflower oil Sunflower oil, Trisun Olive oil PHS d t-BHQ Mineral mix Calcium phosphate Calcium carbonate Potassium citrate Vitamin mix Choline bitartrate α-Tocopheryl acetate Total (g)

169.8 3.4 343.0 84.9 113.2 56.6 4.25 40.4 56.5 30.7 29.1 2.9 0 0.03 11.3 14.7 6.2 18.7 11.3 2.3 0.15 1000

169.5 3.4 342.4 84.7 113.0 56.5 4.25 40.3 56.4 30.6 29.0 2.9 1.92 0.03 11.3 14.7 6.2 18.6 11.3 2.3 0.15 1000

a

The macronutrient distribution in the diet was as follows: protein, 17.3% by weight, 16% kcal; carbohydrates, 54.1% by weight, 50% kcal; fat, 16.4% by weight, 34% kcal. b Control diet (product number D07100504; Research Diets, New Brunswick, NJ, USA). c PHS diet (product number D07100505; Research Diets). d The PHS comprises the following extracts: barberry (Berberis vulgaris; 0.098 g/kg diet), basil skullcap (Scutellaria baicalensis; 0.048 g/kg diet), Chinese goldthread (Coptis chinesis; 0.098 g/kg diet), hu zhang (P cuspidatum; 0.196 g/kg diet), ginger (Zingiber officinalis; 0.246 g/kg diet), green tea (Camellia sinesis; 0.246 g/kg diet), holy basil (Ocimum sanctum; 0.246 g/kg diet), oregano (O vulgare; 0.098 g/kg diet), rosemary (R officinalis; 0.369 g/kg diet), and turmeric (C longa; 0.271 g/kg diet). The PHS was purchased from Earth Fare (Knoxville, TN, USA) as a commercially available combination (Zyflamend).

Please cite this article as: Burke SJ, et al, Dietary polyherbal supplementation decreases CD3+ cell infiltration into pancreatic islets and prevents hyperglycemia in nonobese diabetic mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2014.12.003

N U TR IT ION RE S E ARCH XX ( 2 0 15 ) X XX–X XX

3

reported elsewhere [27]. The PHS comprises the extracts of 10 herbs whose description, formulation, and quality control have been described previously [27,28]. This particular polyherbal formulation was provided at a dose of 0.1916 weight% of the diet (ie, 1.916 g/kg diet; details given in Table). This amount is equivalent to a human dose of 3 capsules per day allometrically scaled down for inclusion into a diet formulated for mice, the details of which are provided in Ref.[35]. NOD/ShiLtJ female mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) at 5 weeks of age, allowed to acclimate for 1 week to the same photoperiod (12-hour light/12-hour dark) and temperature conditions (22 ± 1 °C) at the University of Tennessee Graduate School of Medicine animal facility. All mice were given free access to the control diet and water prior to the start of the experiment al period. At 6 weeks of age, mice were randomly sorted into 2 groups to be fed either the control diet or the diet containing herbal extracts. Mice were maintained on these isocaloric diets for 12 weeks. A baseline tail vein blood sample was taken on the first day prior to the start of each diet and subsequently once a week to measure blood glucose. Blood glucose was measured using the ACCU-CHEK Aviva Glucometer (Roche, Mannheim, Germany). Mice were euthanized by CO2 asphyxiation followed by cervical dislocation. The Animal Care and Use Committee of the University of Tennessee approved the experimental protocols in accordance with the Institute for Laboratory Animal Research Guide for Care and Use of Laboratory Animals.

2.4. Transfection of plasmids and measurements of luciferase enzyme activity

2.2.

Nonobese diabetic mice at 6 weeks of age were fed a control diet or a diet containing a blend of herbal extracts (Table). The mice were maintained on these isocaloric diets for 12 weeks, with blood glucose levels and body weights monitored each week. We found that body weights increased similarly during the 12-week feeding period, indicating that weight gain was not influenced by consumption of a diet supplemented with herbal extracts (Fig. 1A). Although body weights were not different, we observed a rise in blood glucose that was most apparent by week 16 in NOD mice consuming the control diet (Fig. 1B). However, mice eating the diet supplemented with herbal extracts were protected from the increase in blood glucose levels to 18 weeks (Fig. 1B). For example, at week 16, the control mice had an average blood glucose level of 185.3 mg/dL, whereas the PHS group averaged 117.9 (a 36% decrease in blood glucose levels relative to control; Fig. 1B). We conclude that a diet supplemented with herbal extracts prevents the development of hyperglycemia in NOD mice.

Cell culture

Culture of 832/13 rat insulinoma cells has been described [36]. The adenoviral vector encoding β-galactosidase has been described [37], and the adenovirus encoding IκKβ S177E/S181E was a generous gift from Dr Haiyan Xu (Brown University). The IκKβ S177E/S181E plasmid was described previously [38]. The coding region of this plasmid was subcloned into the pACCMV adenoviral shuttle vector using standard molecular biology techniques. Recombinant adenovirus was generated per previously documented procedures [39]. Interleukin (IL) 1β was obtained from Peprotech.

2.3.

Histology

Pancreatic tissue used for immunohistochemistry was fixed in 10% neutral-buffered formalin. Fixed tissues were embedded in paraffin wax. All slides were cut at 5 μm on charged slides, air dried, and then heated at 60 °C for 15 minutes. Slides were then deparaffinized with xylene, rehydrated through graded ethanol solutions to deionized water. For insulin and CD3+ staining, formalin-fixed paraffin-embedded mouse pancreatic sections were labeled by routine immunohistochemistry, as outlined previously [40] using primary antibodies obtained from Cell Signaling (Danvers, MA, USA) and Dako (Carpinteria, CA, USA). The stained slides were scored for percent islet endocrine cells expressing insulin and for the relative numbers of CD3 + T-lymphocytes by a board-certified veterinary pathologist blinded to the dietary groups.

832/13 cells were split into 24-well plates and grown to 50% confluence. At 50% confluence, the cells were transfected with 100 ng of a control or promoter luciferase reporter plasmid per well, as indicated in the figure legends. Transfected cells were incubated for 8 hours prior to the experimental conditions described in the figure legends. At the conclusion of the experiment, cell lysates were collected in passive lysis buffer (Promega; Madison, WI, USA) and centrifuged at 12 000 ×g for 10 minutes. The supernatants were used for luciferase assays performed in 96-well plates using the Luciferase Assay System (Promega) protocol provided by the manufacturer. All measurements were made in the GloMax plate reading luminometer (Promega) as described previously [41].

2.5.

Statistical analyses

All statistical analyses were performed using GraphPad (La Jolla, CA, USA) Prism 5.0. Data were subjected to Student t test to determine statistical differences between groups. All data are horizontal bar indicating the means ± SEM of the data set as a whole.

3.

Results

3.1. NOD mice fed a diet supplemented with herbal extracts for 12 weeks did not become hyperglycemic

3.2. NOD mice fed a diet supplemented with herbal extracts retained more insulin-positive cell mass Because the NOD mice fed the diet supplemented with herbal extracts were protected against the rise in blood glucose levels seen in control animals (Fig. 1B), we examined pancreata at the conclusion of the study (week 18; 12 weeks total for the dietary intervention). We discovered that although control animals exhibited a marked loss in insulinpositive cell mass (Fig. 2A), the animals that had consumed the diet supplemented with the herbal extract combination

Please cite this article as: Burke SJ, et al, Dietary polyherbal supplementation decreases CD3+ cell infiltration into pancreatic islets and prevents hyperglycemia in nonobese diabetic mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2014.12.003

4

N UTR IT ION RE S EA RCH XX ( 2 01 5 ) X XX–X XX

Fig. 1 – Weekly body weight and blood glucose levels in control and PHS dietary groups during 12 weeks of feeding in female NOD mice. A, The gain in body weight during the course of the study was similar between the control and the PHS groups. B, The increase in blood glucose levels in mice consuming the control diet was prevented in mice fed the PHS diet. #P < .1, *P < .05, **P < .01 vs the polyherbal diet group. n = 8-9 mice in each dietary group. Data are shown as means ± SEM.

retained a significantly higher percentage of cells staining positive for insulin (Fig. 2B). Quantitation of sections from multiple islets taken from each individual animal revealed a 31.9% average increase in insulin-positive cell mass in PHS fed animals relative to controls (Fig. 2C). Thus, the consumption of polyherbal extracts preserved functional β-cell mass in NOD mice. Development of T1DM in NOD mice is known to be a Tlymphocyte–driven event [33,42]. Thus, we examined CD3+ (a T-lymphocyte marker) cell infiltration into pancreatic islets in both control and PHS fed animals. We observed a 26% decrease in CD3+ positive cells present within pancreatic islets in PHS-fed animals relative to controls (compare Fig. 3A and B; quantification is shown in Fig. 3C). Taken together, the data in Figs. 2 and 3 are consistent with dietary herbal extract supplementation providing protection against autoimmunemediated losses in islet β-cell mass and function.

Fig. 2 – Immunohistochemical identification of insulin-positive cells within pancreatic islets. Representative pancreatic section from NOD mice consuming a control diet (A) or PHS diet (B). C, Quantification of insulin-positive mass. *P < .05 vs control. Data in panel C are presented as individual points with the horizontal bar indicating means ± SEM. Each point represents data from 1 mouse (n = 8-9).

3.3. Polyherbal extracts reduce NF-κB activity and decrease transcription driven by chemokine gene promoters Interleukin 1β is one of the main cytokines associated with alterations in pancreatic β-cell function and mass [43–45]. CCL2 and CXCL10 are secreted proteins that promote infiltration of macrophages and T-cells into pancreatic islets, and their expression is increased by IL-1β [46,47]. Because both macrophage and T-cell populations contribute to autoimmune-mediated destruction of pancreatic β-cells [48,49], we examined transcriptional readouts from CCL2 and CXCL10

Please cite this article as: Burke SJ, et al, Dietary polyherbal supplementation decreases CD3+ cell infiltration into pancreatic islets and prevents hyperglycemia in nonobese diabetic mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2014.12.003

N U TR IT ION RE S E ARCH XX ( 2 0 15 ) X XX–X XX

Fig. 3 – Immunohistochemical identification of CD3-positive cells within pancreatic islets. Pancreatic section from NOD mice consuming a control diet (A) or PHS diet (B). C, Quantification of CD3+ cell infiltrates. #P < .1 vs control. Data in panel C are presented as individual points with the horizontal bar indicating means ± SEM (n = 8-9). gene promoters. Using 832/13 rat insulinoma cells, a clonal β-cell population [36], we found that promoter activity driven by CCL2 or CXCL10 genomic regions is increased by IL-1β [50,51]. However, overnight culture of 832/13 rat insulinoma cells with 100 μg/mL herbal extract prior to IL-1β exposure decreased the activity of the CCL2 promoter by 29% (Fig. 4A). In addition, there was a 30.2% decrease in IL-1-mediated CXCL10 reporter activity using 832/13 cells cultured in the botanical extracts relative to control cells (Fig. 4B). We next used a synthetic promoter containing 5 copies of a consensus κB genomic response element controlling expression of the luciferase reporter gene, which is an efficient readout of global activation of the NF-κB signaling pathway [52,53]. Using this promoter-luciferase reporter gene, we

5

Fig. 4 – Polyherbal extracts reduce NF-κB activity and decrease transcription driven by chemokine gene promoters. A and B, 832/13 cells were transfected with CCL2 (A) or CXCL10 (B) promoter-luciferase constructs for 8 hours. Cells were then preincubated with 100 μg/mL PHS for 18 hours prior to 4-hour stimulation with 1 ng/mL IL-1β. (C), 832/13 cells were transduced concomitantly with adenoviruses expressing either AdCMV-βGal or AdCMV-CA IκKβ and a 5× NF-κB promoter driving the luciferase gene. Eight hours posttransduction, the cells were preincubated with 100 μg/mL PHS for 18 hours prior to harvesting total protein for luciferase assays. Data in panel C are normalized as the activity of NF-κB promoter in the presence of AdCMV-CA IκKβ divided by the activity of AdCMV-βGal. That value was set as 100%, and the luciferase activity in the presence of the herbal extracts was calculated in the same way and shown relative to the maximal activity. A-C, Luciferase assay activity was normalized to total cellular protein content. **P < .01 vs. control group. Data in panels A to C are shown as means ± SEM from 3 separate experiments.

Please cite this article as: Burke SJ, et al, Dietary polyherbal supplementation decreases CD3+ cell infiltration into pancreatic islets and prevents hyperglycemia in nonobese diabetic mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2014.12.003

6

N UTR IT ION RE S EA RCH XX ( 2 01 5 ) X XX–X XX

examined the ability of a constitutively active IκKβ (CA IκKβ) protein to enhance NF-κB activity after prior incubation or not with 100 μg/mL of the polyherbal extract combination. We found a 28.1% decrease in the ability of CA IκKβ protein to activate the NF-κB reporter in the presence of herbal extracts (Fig. 4C). We conclude that supplementation of clonal β-cells with a combination of herbal extracts dampens NF-κB activity, which reduces the transcription of specific chemokine gene activity. Taken together, these data are consistent with the overall decrease in CD3+ cell infiltration into the islets of NOD mice fed the PHS diet and with the preservation of insulinpositive cell mass within the pancreas.

4.

Discussion

Plant extracts have been used since Medieval times for therapeutic purposes, including treatment of diabetes mellitus [54]. Attempts to understand how these therapeutic approaches elicit their effects have been underway for centuries [55–57]. Interestingly, many therapies, including insulin [58], were initially administered to diabetic patients without a complete understanding of their biochemical mechanism(s) of action. Similarly, the investigation into the mechanisms of herbal based antiinflammatory approaches has led to discoveries of specific small molecules, including the biguanide compounds (now used as the diabetes drug metformin), that contain antidiabetic properties and have developed into current pharmaceutical use [59]. Herein, we described the dietary delivery of an herbal extract combination to NOD mice. We chose the NOD mouse because it is the most popular model of spontaneous autoimmune diabetes and thus allowed us to examine the efficacy of herbal-based approaches in a well-characterized in vivo autoimmune setting. The amount of herbal extract added to the food source was ~1.92 g herbal extract per 1 kg solid food. Because a mouse typically consumes 3 to 5 g of solid food per day, the total amount of herbal extract consumed was in the range of 6 to 10 mg/d. Therefore, the hypothesis that a small total amount of herbal extracts, delivered through the food supply, would be sufficient to decrease inflammation and prevent a rise in blood glucose levels in a rodent model of autoimmunity was confirmed by our results. Moreover, the dose used herein would be consistent with a human taking 1-g capsules (containing 400 mg herbal extracts each) approximately 3 times a day, which is a readily achievable and costeffective dosing strategy [35]. Using this approach, we report the following novel observations: (1) polyherbal dietary supplementation decreased infiltration of CD3+ cells into pancreatic islets which correlated with preservation of insulin-positive cell mass; (2) hyperglycemia did not manifest in NOD mice fed herbal extracts, whereas there was a clear increase in blood glucose by week 16 in mice consuming the control diet; and (3) there was a decrease in the ability of IL-1β and IκKβ to drive transcription of NF-κB–controlled gene promoters, including CCL2 and CXCL10, in vitro, providing molecular evidence of the anti-inflammatory activities present within herbal extracts. CCL2 can recruit monocytes, macrophages, and Tlymphocytes into sites of inflammation, including the pancreatic islets [46,60]. Synthesis and secretion of CCL2 are associated with poor function and survival of transplanted

islets [61,62]. In addition, CXCL10 promotes recruitment of T-cells into islets [47] and also contributes to rejection of transplanted tissues [63]. CCL2 and CXCL10 are both regulated at the level of transcription and are synthesized and secreted from pancreatic β-cells in response to proinflammatory cytokines [50,64–66]. We report here for the first time that a well-tolerated combination of herbal extracts decreases the IL-1β–mediated transcriptional response of the CCL2 and CXCL10 gene promoters. In addition, prior cellular exposure to these polyherbal extracts reduced IκKβ–mediated NF-κB transcriptional activity. Because this latter experiment does not require cell surface receptor activity to induce NF-κB signaling events, we speculate that the polyherbal mixture used in this study contains naturally occurring inhibitors of intracellular NF-κB activity, including potential IκKβ inhibitors. Identifying and characterizing such compounds will be the subject of future studies. Because chemokines are not typically stored in appreciable amounts and thus have to be transcribed to be synthesized and secreted, our in vitro data fit with the in vivo general decrease in CD3+ cell infiltration into pancreatic islets of NOD mice consuming the diet supplemented with the polyherbal extract. An additional positive aspect of the polyherbal dietary intervention used in this study is its safety for human consumption. This precise herbal combination has been used in human clinical trials for prostate cancer, where it was well tolerated [17,32] as well as in various mouse models [27,31]. Thus, the safety of specific herbal therapies, combined with their anti-inflammatory properties, will likely make their use as an adjuvant therapy realistic in human subjects. The advantage of using herbal blends over an individual herb is the synergistic effectiveness achieved in the combinatorial approach [67–70]. The interactions between biological molecules present within individual herbal extracts lowers the effective dosage needed for any individual herbal component, consequently improving therapeutic potential while also decreasing the quantity of extract (eg, micromolar to nanomolar efficacy) needed for a salutary outcome. The individual components of the polyherbal blend used in this study are beneficial in rodent models of obesity and diabetes. For example, resveratrol (present in Polygonum cuspidatum) protects against autoimmune-mediated β-cell destruction [71], promotes insulin secretion [72], and dampens overall inflammation [73,74]. In addition, curcumin (derived from Curcuma longa), which has anti-inflammatory effects in the tumor environment [28,75], also helps to prevent autoimmune diabetes [76], an effect that is similarly observed in our study. Constituents within oregano (eg, Origanum vulgare) and rosemary (Rosmarinus officinalis) display glucose lowering effects in various rodent models of diabetes [77,78]. We postulate that polyherbal based therapies would also be effective strategies for lowering the amount of injected insulin required to control glycemia, in addition to their ability to preserve functional insulin-positive cell mass. For example, reducing NF-κB activity decreases the synthesis and secretion of chemokines. Because chemokines promote immune cell recruitment and therefore potentially regulate destructive insulitis, reducing their overall production is likely beneficial to slowing the process of autoimmune-mediated β-cell destruction. By contrast, enhancing NF-κB pathway activity via

Please cite this article as: Burke SJ, et al, Dietary polyherbal supplementation decreases CD3+ cell infiltration into pancreatic islets and prevents hyperglycemia in nonobese diabetic mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2014.12.003

N U TR IT ION RE S E ARCH XX ( 2 0 15 ) X XX–X XX

islet-specific IKKβ overexpression reduces functional β-cell mass in vivo [79]. Consequently, inhibiting the ability of IKKβ to drive expression of genes controlled by NF-κB proteins is one strategy to prevent the loss of insulin-producing cells. In line with these observations, our findings support our hypothesis that a polyherbal combination decreases both CD3+ cell infiltration in vivo and IL-1β– and IκKβ-mediated transcriptional activity in vitro, indicating that herbal extracts may modulate both pancreatic and immune system function. A limitation of the present study is the paucity of data on the long-term efficacy of the PHS in diabetic rodent models. Whether the NOD mice consuming a PHS diet will eventually develop diabetes later in their life span is not currently known. In addition, we have no information on whether or not this particular polyherbal blend protects against ageinduced insulin resistance or obesity-associated inflammatory responses. Additional studies will be pursued to address these existing questions. In conclusion, although herbal-based approaches are often effective, the biology is not well understood. Additional and varied studies will be required to elucidate the precise combination of chemical components conferring the protective responses. Our results provide compelling evidence that dietary intervention with a combination of herbal extracts preserves functional islet β-cell mass in an autoimmune model of diabetes by suppressing inflammatory responses.

Conflict of interest There are no conflicts of interest that would bias the results of these studies or any interpretation of results presented herein.

Acknowledgments This work was supported by start-up funds provided by the University of Tennessee, Knoxville (J.J.C.) and by the Tennessee Agricultural Experiment Station (J.W.), University of Tennessee, Knoxville, TN. We thank Dr Michael F. McEntee for useful discussions and critical reading of the manuscript.

REFERENCES

[1] Burke SJ, Collier JJ. Insulitis and diabetes: a perspective on islet inflammation. Immunome Research 2014:10. http://dx. doi.org/10.4172/1745-1780.S2.e002, [In Press]. [2] Padgett LE, Broniowska KA, Hansen PA, Corbett JA, Tse HM. The role of reactive oxygen species and proinflammatory cytokines in type 1 diabetes pathogenesis. Ann N Y Acad Sci 2013;1281:16–35. [3] Lehuen A, Diana J, Zaccone P, Cooke A. Immune cell crosstalk in type 1 diabetes. Nat Rev Immunol 2010;10:501–13. [4] Bluestone JA, Herold K, Eisenbarth G. Genetics, pathogenesis and clinical interventions in type 1 diabetes. Nature 2010;464: 1293–300. [5] Christlieb AR. Diabetes and hypertensive vascular disease. Mechanisms and treatment. Am J Cardiol 1973;32:592–606.

7

[6] Kannel WB, McGee DL. Diabetes and cardiovascular disease. The Framingham study. JAMA 1979;241:2035–8. [7] Castano L, Eisenbarth GS. Type-I diabetes: a chronic autoimmune disease of human, mouse, and rat. Annu Rev Immunol 1990;8:647–79. [8] Chukwuma Sr C, Tuomilehto J. Diabetes and the risk of stroke. J Diabetes Complications 1993;7:250–62. [9] Mankovsky BN, Metzger BE, Molitch ME, Biller J. Cerebrovascular disorders in patients with diabetes mellitus. J Diabetes Complications 1996;10:228–42. [10] Chantrel F, Moulin B, Hannedouche T. Blood pressure, diabetes and diabetic nephropathy. Diabetes Metab 2000; 26(Suppl. 4):37–44. [11] Daneman D. Type 1 diabetes. Lancet 2006;367:847–58. [12] Kolasinski SL. Herbal medicine for rheumatic diseases: promises kept? Curr Rheumatol Rep 2012;14:617–23. [13] Li T, Peng T. Traditional Chinese herbal medicine as a source of molecules with antiviral activity. Antiviral Res 2013;97:1–9. [14] Li GQ, Kam A, Wong KH, Zhou X, Omar EA, Alqahtani A, et al. Herbal medicines for the management of diabetes. Adv Exp Med Biol 2012;771:396–413. [15] Jia W, Gao W, Tang L. Antidiabetic herbal drugs officially approved in China. Phytother Res 2003;17:1127–34. [16] Ryan JL, Heckler CE, Ling M, Katz A, Williams JP, Pentland AP, et al. Curcumin for radiation dermatitis: a randomized, double-blind, placebo-controlled clinical trial of thirty breast cancer patients. Radiat Res 2013;180:34–43. [17] Capodice JL, Gorroochurn P, Cammack AS, Eric G, McKiernan JM, Benson MC, et al. Zyflamend in men with high-grade prostatic intraepithelial neoplasia: results of a phase I clinical trial. J Soc Integr Oncol 2009;7:43–51. [18] Lu DY, Tang CH, Chen YH, Wei IH. Berberine suppresses neuroinflammatory responses through AMP-activated protein kinase activation in BV-2 microglia. J Cell Biochem 2010;110:697–705. [19] Lee YS, Kim WS, Kim KH, Yoon MJ, Cho HJ, Shen Y, et al. Berberine, a natural plant product, activates AMP-activated protein kinase with beneficial metabolic effects in diabetic and insulin-resistant states. Diabetes 2006;55:2256–64. [20] Jeong HW, Hsu KC, Lee JW, Ham M, Huh JY, Shin HJ, et al. Berberine suppresses proinflammatory responses through AMPK activation in macrophages. Am J Physiol Endocrinol Metab 2009;296:E955–64. [21] Xia X, Yan J, Shen Y, Tang K, Yin J, Zhang Y, et al. Berberine improves glucose metabolism in diabetic rats by inhibition of hepatic gluconeogenesis. PLoS One 2011;6:e16556. [22] Cui G, Qin X, Zhang Y, Gong Z, Ge B, Zang YQ. Berberine differentially modulates the activities of ERK, p38 MAPK, and JNK to suppress Th17 and Th1 T cell differentiation in type 1 diabetic mice. J Biol Chem 2009;284:28420–9. [23] Mullen W, Larcombe S, Arnold K, Welchman H, Crozier A. Use of accurate mass full scan mass spectrometry for the analysis of anthocyanins in berries and berry-fed tissues. J Agric Food Chem 2010;58:3910–5. [24] Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB. Bioavailability of curcumin: problems and promises. Mol Pharm 2007;4:807–18. [25] Del Rio D, Borges G, Crozier A. Berry flavonoids and phenolics: bioavailability and evidence of protective effects. Br J Nutr 2010;104(Suppl. 3):S67–90. [26] Geary N. Understanding synergy. Am J Physiol Endocrinol Metab 2013;304:E237–53. [27] Huang EC, McEntee MF, Whelan J. Zyflamend, a combination of herbal extracts, attenuates tumor growth in murine xenograft models of prostate cancer. Nutr Cancer 2012;64: 749–60. [28] Zhao Y, Collier JJ, Huang EC, Whelan J. Turmeric and Chinese goldthread synergistically inhibit prostate cancer cell

Please cite this article as: Burke SJ, et al, Dietary polyherbal supplementation decreases CD3+ cell infiltration into pancreatic islets and prevents hyperglycemia in nonobese diabetic mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2014.12.003

8

N UTR IT ION RE S EA RCH XX ( 2 01 5 ) X XX–X XX

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43] [44]

[45]

[46]

proliferation and NF-kB signaling. Funct Foods Health Dis 2014;4:312–39. Huang EC, Chen G, Baek SJ, McEntee MF, Collier JJ, Minkin S, et al. Zyflamend reduces the expression of androgen receptor in a model of castrate-resistant prostate cancer. Nutr Cancer 2011;63:1287–96. McEntee MF, Ziegler C, Reel D, Tomer K, Shoieb A, Ray M, et al. Dietary n-3 polyunsaturated fatty acids enhance hormone ablation therapy in androgen-dependent prostate cancer. Am J Pathol 2008;173:229–41. Subbaramaiah K, Sue E, Bhardwaj P, Du B, Hudis CA, Giri D, et al. Dietary polyphenols suppress elevated levels of proinflammatory mediators and aromatase in the mammary gland of obese mice. Cancer Prev Res (Phila) 2013;6:886–97. Rafailov S, Cammack S, Stone BA, Katz AE. The role of Zyflamend, an herbal anti-inflammatory, as a potential chemopreventive agent against prostate cancer: a case report. Integr Cancer Ther 2007;6:74–6. Leiter EH. The NOD mouse: a model for insulinc-dependent diabetes mellitus. Curr Protoc Immunol 2001:9 [Chapter 15:Unit 15]. Petrik MB, McEntee MF, Johnson BT, Obukowicz MG, Whelan J. Highly unsaturated (n-3) fatty acids, but not alpha-linolenic, conjugated linoleic or gamma-linolenic acids, reduce tumorigenesis in Apc(Min/+) mice. J Nutr 2000; 130:2434–43. Weldon KA, Whelan J. Allometric scaling of dietary linoleic acid on changes in tissue arachidonic acid using human equivalent diets in mice. Nutr Metab 2011;8:43. Hohmeier HE, Mulder H, Chen G, Henkel-Rieger R, Prentki M, Newgard CB. Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel–dependent and –independent glucose-stimulated insulin secretion. Diabetes 2000;49: 424–30. Herz J, Gerard RD. Adenovirus-mediated transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice. Proc Natl Acad Sci U S A 1993;90:2812–6. Mercurio F, Zhu H, Murray BW, Shevchenko A, Bennett BL, Li J, et al. IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation. Science 1997;278:860–6. Becker TC, Noel RJ, Coats WS, Gomez-Foix AM, Alam T, Gerard RD, et al. Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol 1994;43(Pt A):161–89. McEntee MF, Chiu CH, Whelan J. Relationship of beta-catenin and Bcl-2 expression to sulindac-induced regression of intestinal tumors in Min mice. Carcinogenesis 1999;20: 635–40. Burke SJ, Updegraff BL, Bellich RM, Goff MR, Lu D, Minkin Jr SC, et al. Regulation of iNOS gene transcription by IL-1beta and IFN-gamma requires a coactivator exchange mechanism. Mol Endocrinol 2013;27:1724–42. Serreze DV, Leiter EH. Genetic and pathogenic basis of autoimmune diabetes in NOD mice. Curr Opin Immunol 1994;6:900–6. Mandrup-Poulsen T. The role of interleukin-1 in the pathogenesis of IDDM. Diabetologia 1996;39:1005–29. Maedler K, Sergeev P, Ris F, Oberholzer J, Joller-Jemelka HI, Spinas GA, et al. Glucose-induced beta cell production of IL-1beta contributes to glucotoxicity in human pancreatic islets. J Clin Invest 2002;110:851–60. Larsen CM, Faulenbach M, Vaag A, Volund A, Ehses JA, Seifert B, et al. Interleukin-1-receptor antagonist in type 2 diabetes mellitus. N Engl J Med 2007;356:1517–26. Martin AP, Rankin S, Pitchford S, Charo IF, Furtado GC, Lira SA. Increased expression of CCL2 in insulin-producing cells of transgenic mice promotes mobilization of myeloid cells

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54] [55]

[56]

[57]

[58] [59] [60]

[61]

[62]

[63] [64]

[65]

from the bone marrow, marked insulitis, and diabetes. Diabetes 2008;57:3025–33. Rhode A, Pauza ME, Barral AM, Rodrigo E, Oldstone MB, von Herrath MG, et al. Islet-specific expression of CXCL10 causes spontaneous islet infiltration and accelerates diabetes development. J Immunol 2005;175:3516–24. Calderon B, Carrero JA, Miller MJ, Unanue ER. Entry of diabetogenic T cells into islets induces changes that lead to amplification of the cellular response. Proc Natl Acad Sci U S A 2011;108:1567–72. Calderon B, Suri A, Unanue ER. In CD4+ T-cell-induced diabetes, macrophages are the final effector cells that mediate islet beta-cell killing: studies from an acute model. Am J Pathol 2006;169:2137–47. Burke SJ, Goff MR, Updegraff BL, Lu D, Brown PL, Minkin Jr SC, et al. Regulation of the CCL2 gene in pancreatic beta-cells by IL-1beta and glucocorticoids: role of MKP-1. PLoS One 2012;7: e46986. Burke SJ, Goff MR, Lu D, Proud D, Karlstad MD, Collier JJ. Synergistic expression of the CXCL10 gene in response to IL-1beta and IFN-gamma involves NF-kappaB, phosphorylation of STAT1 at Tyr701, and acetylation of histones H3 and H4. J Immunol 2013;191:323–36. Collier JJ, Burke SJ, Eisenhauer ME, Lu D, Sapp RC, Frydman CJ, et al. Pancreatic beta-cell death in response to pro-inflammatory cytokines is distinct from genuine apoptosis. PLoS One 2011;6: e22485. Burke SJ, Collier JJ. The gene encoding cyclooxygenase-2 is regulated by IL-1beta and prostaglandins in 832/13 rat insulinoma cells. Cell Immunol 2011;271:379–84. Witters LA. The blooming of the French lilac. J Clin Invest 2001;108:1105–7. Chang CL, Lin Y, Bartolome AP, Chen YC, Chiu SC, Yang WC. Herbal therapies for type 2 diabetes mellitus: chemistry, biology, and potential application of selected plants and compounds. Evid Based Complement Alternat Med 2013; 2013:378657. Watanabe CK. Studies in the metabolic changes induces by administration of guanidine bases. I. Influence of injected guanidine hydrochloride upon blood sugar content. J Biol Chem 1918;33:253–65. Xie W, Du L. Diabetes is an inflammatory disease: evidence from traditional Chinese medicines. Diabetes Obes Metab 2011;13:289–301. Banting FG, Best CH. The internal secretion of the pancreas. J Lab Clin Med 1922;7:465–80. Bailey CJ, Turner RC. Metformin. N Engl J Med 1996;334:574–9. Carr MW, Roth SJ, Luther E, Rose SS, Springer TA. Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant. Proc Natl Acad Sci U S A 1994;91:3652–6. Ogliari AC, Caldara R, Socci C, Sordi V, Cagni N, Moretti MP, et al. High levels of donor CCL2/MCP-1 predict graft-related complications and poor graft survival after kidney-pancreas transplantation. Am J Transplant 2008;8:1303–11. Piemonti L, Leone BE, Nano R, Saccani A, Monti P, Maffi P, et al. Human pancreatic islets produce and secrete MCP-1/ CCL2: relevance in human islet transplantation. Diabetes 2002;51:55–65. Romagnani P, Crescioli C. CXCL10: a candidate biomarker in transplantation. Clin Chim Acta 2012;413:1364–73. Burke SJ, Goff MR, Lu D, Proud D, Karlstad MD, Collier JJ. Synergistic expression of the CXCL10 gene in response to IL-1beta and IFN-gamma involves NF-kappaB, phosphorylation of STAT1 at Tyr701, and acetylation of histones H3 and H4. J Immunol 2013;191:323–36. Chen MC, Proost P, Gysemans C, Mathieu C, Eizirik DL. Monocyte chemoattractant protein-1 is expressed in pancreatic islets from prediabetic NOD mice and in

Please cite this article as: Burke SJ, et al, Dietary polyherbal supplementation decreases CD3+ cell infiltration into pancreatic islets and prevents hyperglycemia in nonobese diabetic mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2014.12.003

N U TR IT ION RE S E ARCH XX ( 2 0 15 ) X XX–X XX

[66]

[67]

[68]

[69]

[70]

[71]

[72]

interleukin-1 beta–exposed human and rat islet cells. Diabetologia 2001;44:325–32. Schulthess FT, Paroni F, Sauter NS, Shu L, Ribaux P, Haataja L, et al. CXCL10 impairs beta cell function and viability in diabetes through TLR4 signaling. Cell Metab 2009;9:125–39. Wang X, Xu X, Tao W, Li Y, Wang Y, Yang L. A systems biology approach to uncovering pharmacological synergy in herbal medicines with applications to cardiovascular disease. Evid Based Complement Alternat Med 2012;2012: 519031. Li B, Xu X, Wang X, Yu H, Li X, Tao W, et al. A systems biology approach to understanding the mechanisms of action of chinese herbs for treatment of cardiovascular disease. Int J Mol Sci 2012;13:13501–20. Li S, Zhang B, Zhang N. Network target for screening synergistic drug combinations with application to traditional Chinese medicine. BMC Syst Biol 2011;5(Suppl. 1):S10. Gertsch J. Botanical drugs, synergy, and network pharmacology: forth and back to intelligent mixtures. Planta Med 2011;77:1086–98. Lee SM, Yang H, Tartar DM, Gao B, Luo X, Ye SQ, et al. Prevention and treatment of diabetes with resveratrol in a non-obese mouse model of type 1 diabetes. Diabetologia 2011;54:1136–46. Vetterli L, Brun T, Giovannoni L, Bosco D, Maechler P. Resveratrol potentiates glucose-stimulated insulin secretion in INS-1E beta-cells and human islets through a SIRT1-dependent mechanism. J Biol Chem 2011;286:6049–60.

9

[73] Rahman I, Biswas SK, Kirkham PA. Regulation of inflammation and redox signaling by dietary polyphenols. Biochem Pharmacol 2006;72:1439–52. [74] Cavallaro A, Ainis T, Bottari C, Fimiani V. Effect of resveratrol on some activities of isolated and in whole blood human neutrophils. Physiol Res 2003;52:555–62. [75] Kunnumakkara AB, Guha S, Krishnan S, Diagaradjane P, Gelovani J, Aggarwal BB. Curcumin potentiates antitumor activity of gemcitabine in an orthotopic model of pancreatic cancer through suppression of proliferation, angiogenesis, and inhibition of nuclear factor-kappaB–regulated gene products. Cancer Res 2007;67:3853–61. [76] Castro CN, Barcala Tabarrozzi AE, Winnewisser J, Gimeno ML, Antunica Noguerol M, Liberman AC, et al. Curcumin ameliorates autoimmune diabetes. Evidence in accelerated murine models of type 1 diabetes. Clin Exp Immunol 2014;177:149–60. [77] Ezhumalai M, Radhiga T, Pugalendi KV. Antihyperglycemic effect of carvacrol in combination with rosiglitazone in high-fat diet-induced type 2 diabetic C57BL/6J mice. Mol Cell Biochem 2014;385:23–31. [78] Yun YS, Noda S, Shigemori G, Kuriyama R, Takahashi S, Umemura M, et al. Phenolic diterpenes from rosemary suppress cAMP responsiveness of gluconeogenic gene promoters. Phytother Res 2013;27:906–10. [79] Salem HH, Trojanowski B, Fiedler K, Maier HJ, Schirmbeck R, Wagner M, et al. Long-term IKK2/NF-kappaB signaling in pancreatic beta-cells induces immune-mediated diabetes. Diabetes 2014;63:960–75.

Please cite this article as: Burke SJ, et al, Dietary polyherbal supplementation decreases CD3+ cell infiltration into pancreatic islets and prevents hyperglycemia in nonobese diabetic mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2014.12.003