Ginsenoside Re, Produces Multi-targeted Potent Antidiabetic Effects Via Estrogen Receptor Signaling Pathway in Rat, an Alternative Multi- Targeted Therapeutic for Type 2 Diabetes

Aims: To identify more effective ginsenoside for type 2 diabetes (T2D) and clarify whether the ginsenoside characterizing estrogenic multi-targeted antidiabetic effects. Study Design: Identifying more effective ginsenoside through preclinical evaluation of antidiabetic effects of representative ginsenosides with T2D rat model, and further test pharmacological mechanism underlying the potent antidiabetic effects of the ginsenoside in the same model. Place and Duration of Study: Key laboratory for Pharmacy, Inner Mongolia Medical University, March 2018 to November 2020. Methodology: Used a total of 240 female adult rats. Rat model of T2D induced by high-fat diet fed and streptozotocin. Five tapes of representative ginsenosides (Rb1, Rd, Rg3, Re, Rg1) administrated at low (20 mg/kg daily) and high (40 mg/ kg daily) doses to T2D rats with orally for 4 weeks. Detect testing indexes with biochemical, histological, Quantitative Real-Time PCR, and western blots analysis. Results: Ginsenoside Re (Re), very significantly lowered blood glucose (P<0.01), lipids (P<0.001), free fatty acid (P<0.001), and glucagon (P<0.01) levels, markedly improved impaired insulin sensitivity (P<0.01), ameliorated oxidative stress (P<0.01) and inflammation (P<0.01) in T2D rats, exhibited potent antidiabetic effects. Moreover, Re, phosphorylate serine/threonine kinase (Akt) (P<0.01) and endothelial nitric oxide synthase (eNOS) (P<0.01), up regulates B-cell lymphoma-2 (P<0.01) and insulin gene expression (P<0.01), down regulates glucagon gene expression(P<0.01), reverse impaired glucagon-like peptide 1 (P<0.01); exhibits multi-targeted effects; these effects of Re were inhibited by estrogen receptor (ER) inhibitor (ICI-182,780) (P<0.01). Functionally, the antidiabetic effects of Re were sequentially inhibited by inhibitor of ER, Akt, and eNOS, respectively (P<0.01). Conclusion: These findings, revealed a novel pharmacological property of Re that characterized in multi-targeted potent antidiabetic effects mediated by ER/Akt/eNOS/NO signaling pathway, provide the first evidence for the potential use of Re, as a multi-targeted therapeutic for T2D, particularly, a novel candidate for replacement of estrogen therapy and NO therapy in diabetes.


INTRODUCTION
Type 2 diabetes (T2D) is one of the leading causes of morbidity and mortality in world-wide. Pathogenesis of T2D is involved multifactor such as dysfunction of pancreatic beta cell (β-cell) as well as alpha cell (α-cell), dysregulated insulin as well as glucagon (Gcg), and insulin resistance (IR) [1,2,3], which induced by multiple pathological processes including oxidative stress, inflammation, glucose and lipids toxicity, and apoptosis [4,5]. The multifactorial pathogenesis of T2D means that treatment needs to involve multiple targeted therapies characterized in more effective and less adverse effects [6,7]. However, developing multi-targeted more effective therapeutic measures for the multifactorial T2D has been challenging [8,9,10].
Recently, numerous clinical and experimental studies have demonstrated that estrogen and 17β-estradiol (E2) characterize insulinotropic and multi-targeted antidiabetic effects that including anti-oxidative stress, anti-inflammation, antiapoptosis, enhances β-cell survival and generation , increases pancreatic insulin content, gene expression and insulin release ，reducing IR, glycemic and lipids control in various animal models and diabetes [11,12] by signaling through estrogen receptor (ER) alpha (ERα) /or ERβ [13,14]. It strongly suggests that estrogen and ER signaling pathway are potential multitargeting therapeutic candidate for diabetes therapy [15,16], but it has serious adverse events [17].
Moreover, currently, available pharmacological agents for T2D, however, have many limitations including lack of multi-targeted Original Research Article synergistic beneficial effects, and adverse effects [18,19]. Therefore, considering complementary and alternative approaches, including use of medicinal herbs that proposed synergistic multiple actions on diabetes [20,21]. In fact, ginseng is the best choice of herbal medicines that show promising result in the treatment of diabetes [22,23]. Interestingly, ginseng also characterize estrogenic and insulinotropic properties [21,23]. The active components of ginseng are considered to ginsenosides, sixteen types of ginsenosides were found to have antidiabetic effects, such as ginsenoside Re, Rg1, Rg3, Rg5, Rb1, Rb2, Rb3, and compound K [24]. The molecular mechanisms underlying the antidiabetic effects and medical applications of ginsenosides have attracted much attention [24]. Multiple molecular targets including glucose transporters (GLUTs), glucagon-like peptide 1 (GLP-1), peroxisome proliferator-activated receptor gamma (PPAR-γ), phosphatidylinositol 3-kinase (PI3K)/threonine kinase (Akt) signaling, and AMP-activated protein kinase (AMPK) are involved in the antidiabetic effects of ginsenosides [24]. However, the potential systemic pharmacological mechanisms of ginsenosides are still unclear, which was major hindrance for use of ginseng or ginsenoside in diabetes therapy.
Estrogenic activities [25] and nitric oxide (NO) actions [26] have suggested as mechanisms of ginseng's actions, whether these actions involve in the antidiabetic effects of ginseng and ginsenosides have not been tested. Several studies indicated that antidiabetic effects of ginseng extract could be attributed to ginsenoside Re (Re) which the most representative and a major bioactive component in the ginseng [27,28]. Our previous study reveals that Re, a main phytosterol of panax ginseng, activates cardiac potassium channels via a nongenomic pathway of sex hormones [29], and involve NO actions [30].
In the present study, therefore, we examined and compared the antidiabetic efficacies of Re to those of ginsenosides in ginseng, to test whether Re is more effective component of ginseng regarding its antidiabetic effects; more importantly, to test the possibility that whether molecular mechanism underlying the antidiabetic effects of Re mediated by ER signaling pathway. Re could mimics the multi-targeted antidiabetic effects of estrogen that can greatly facilitate developing multi-targeted new treatments for T2D from natural products.

Experimental Design
Rat experiments carried out using two different protocols (Protocols 1 and 2).

Protocol 1
Preparation of type 2 diabetes (T2D) model: The rats of the T2D model group were feded a HFD diet for 8 weeks and then received an intraperitoneal (IP) injection of Streptozotocin (STZ) (40 mg/kg, dissolved in citrate buffer, pH 4.5; Sigma-Aldrich, USA), twice at an interval of 24 h. After 72 h, collection of blood from tail vein of rats after 12 h of fasting, fasting blood glucose (FBG) was measured with spectrophotometrybased assays using commercially available kits (Invitrogen, USA) at once a day for three consecutive days, each measurement of FBG level in all of rats is ≥16.7 mmol/L and insulin sensitivity index decreased (compared with normal control group rats) significantly (P value <0.05), considered as model established. One hundred and twenty (120) rats in the HFD group fulfilled this T2D criterion. Ten rats of the normal control group (NC) were fed standard rat chow and received an IP injection of the same volume of citrate buffer. The T2D rats that induced by HFD and STZ were subsequently apportioned randomly and equally to a T2D model control group (MC, administered saline, 4 ml/d by gavage ) and eleven different treatment groups: administered ginsenoside Re, Rg1, Rg3, Rb1and Rd at low (20 mg/kg/day), high (40 mg/ kg/day) doses by gavage (qd) for 4 weeks, respectively, hereafter referred to as the Re, Rg1,Rg3,Rb1and Rd low (Re-l, Rg1-l,Rg3-l,Rb1-l, Rd-l) and high (Re-h, Rg1-h,Rg3-h,Rb1-h, Rd-h) groups; administered Rosiglitazone (30m/kg/day) to rats of positive control group (PC); each group has 10 rats, respectively.

Preparation
of tissues samples and biochemical analysis: At the end of the 4 weeks treatment, rats were sacrificed after a 12 h fasting. The pancreas and intestine (ileum and colon) were dissected immediately and frozen at -20℃ until analysis. Tissue was homogenized on ice and centrifuged at 12000 rpm for 20 min, then the supernatants were collected and stored at -20℃ until analysis. Pancreatic Insulin and ileocolonic glucagon-like peptide 1 (GLP-1) concentrations were determined using enzymelinked immunosorbent assay (ELISA) kits (Millipore, St. Charles, MO, USA).

Oral Glucose Tolerance Test (OGTT) and Insulin tolerance Test (ITT)
In the Protocol 1 experiment, at the end of the three weeks, the rats (in the normal control group-NC, T2D model control group-MC, positive control group-PC treated with Rosiglitazone, treated with high dose of panaxadiols and panaxatriols groups, each group has five rats) were performed OGTT and ITT, respectively. In the OGTT experiment, the rats were fasted for 12 h and then orally administrated glucose 2g/kg. Blood glucose levels were measured from tail blood samples at 0 min (before glucose administration), 30 min, 60 min, and 120 min after glucose administration. Analyze the area under blood glucose curve (AUC). AUC was calculated according to the formula: AUC= [(0 min blood glucose + 2×30 min blood glucose + 3×60 min blood glucose +2 ×120 min blood glucose)]/4. In the ITT experiment, the rats were fasted for 10 h and then received intraperitoneal (IP) administration of insulin (0.75U/kg). Blood glucose levels were measured from tail blood samples at 0 min (before glucose administration), 30 min, 60 min, and 120 min after insulin administration. Analyze the area under blood glucose curve (AUC). AUC was calculated according to the formula: AUC= [(0.25×0 min blood glucose + 0.5×30 min blood glucose + 0.5×60 min blood glucose +0.25×120 min blood glucose)]/4.

Histological Analysis and Morphometry
Pancreas tissue was fixed in 10% formalin for 1 month and then embedded with paraffin. Tissue sections (10 um) were cut with a microtome (Leica, Germany) and mounted on super frost /plus microscope slides. After being air-dried, they were stained with hematoxylin and eosin (HE), and photographed at 100 × magnification. At least two fields per slice and six slices per islet mass were analyzed for the purpose of quantifying tissue size.

Western Blots Analysis for Akt and eNOS Protein
Extraction of protein from pancreas of rats (in NC, MC, Re-l, Re-h, Re-h+ ICI 182780, Re-h+ MK-2206, and E2 treated group, respectively), and immunoblotting followed standard procedures. Briefly, those of 30 ug total protein were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to nitrocellulose membranes. Then, the membranes were blocked and subjected to immunoblot analysis by incubation with the following antibodies. Using a 1:1000-diluted anti-phospho Akt ( 473 Ser) antibody (CAS#9271) (Cell Signaling, Danvers, MA, USA) or a 1:1000-diluted anti-Akt antibody (CAS#9272) (Cell Signaling) for detection of Akt in pancreas. Using a 1:1000-diluted anti-phospho eNOS antibody (CAS#S1177) (Cell Signaling, Danvers, MA) or a 1:1000-diluted anti-eNOS antibody (eNOS/NOS Type III, CAS#610296; BD Biosciences) for detection of eNOS in pancreas. Followed incubation with a 1:20,000-diluted horseradish peroxidase-conjugated anti-rabbit IgG (Dako Japan Co. Ltd., Kyoto, Japan). Betaactin (anti-beta actin antibody, CAS# sc1616; Santa Cruz, CA, USA) was detected for verification of protein loading. Proteins were detected using an advanced enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, UK) and quantified by densitometry using IPP, version 6.0, software (Media Cybernetics, Silver Spring, MD, USA). The reagents for electrophoresis were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).

Statistical Analysis
Origin Pro 8.5 analysis software was used for Statistical analysis. All data are expressed as mean ± SD. The differences between two groups were analyzed by Student's t test. More than two conditions were analyzed by repeated-measures analysis of variance (rANOVA). Differences were considered statistically significant when P < 0.05.

Effects of Panaxadiols and Panaxatriols
Ginsenosides on Glucose Metabolism in Type 2

Diabetes Rats
High-fat diet (HFD) feeding and injection of Streptozotocin (STZ) very significantly increased fasting blood glucose (FBG), fasting blood insulin (FINS), glycosylated hemoglobin (HbA1c) and glucagon (Gcg) levels, and very significantly decreased insulin sensitivity index (ISI), in the type 2 diabetes (T2D) model control group rats (MC) that compared with normal control (NC) (Fig. 1B-E). This indicated that glucose metabolic disorder established in the T2D rats.
Compared panaxadiols and panaxatriols treated 2TD rats with those of the non-treated MC , the differences in FBG, FINS, ISI, HbA1c, and Gcg level are no significant change in Rd-l and Rd-h treated 2TD group rats (P>0.05); significant decrease of FBG and FINS observed in Rb1-l, Rb1-h, Rg3-l, Rg3-h, and Rg1-l treated (P<0.05); significant increase of ISI observed in Rb1-l, Rb1-h, Rg3-l, Rg3-h ,Rg1-l and Rg1-h treated (P<0.05); significant decrease of HbA1c observed in Rb1-l, Rg3-l, and Rg1-l treated (P<0.05); significant decrease of Gcg observed in the Re-l treated only (P<0.05); more significant decrease of FBG and FINS observed in the Rg1-h, Re-l and Re-h treated (P<0.01); more significant increase of ISI observed only in the Re-h treated (P<0.01); more significant decrease of HbA1c observed in the Rb1-h, Rg3h, Rg1-h and Re-l treated (P<0.01); more significant decrease of Gcg observed in the Re-h treated only (P<0.01); The most significant decrease of FBG and HbA1c observed in the Reh treated only (P<0.001); compared Re-l treated rats with those of rats treated by other ginsenosides at low dose, respectively; the differences in FBG, ISI, HbA1c, and Gcg level are significant (P<0.05) or very significant (P<0.01); similar significant (P<0.05) or very significant (P<0.01) differences are observed in compared Re-h treated rats with those of rats treated by other ginsenosides at high dose, respectively; compared between Re-l and Re-h treated group rats , the differences in FBG, ISI, HbA1c, and Gcg level are significant (P<0.05) ; as shown in Fig1B-F. In sum of above, the intensity of the regulative actions induced by panaxadiols and panaxatriols ginsenosides on the glucose metabolic disorder are: Re-h>Re-l>Rg1-h>Rg1-l>Rg3-h>Rg3-l>Rb1-h. This suggesting that the panaxatriols (Re and Rg1), especially Re elicit potent regulative effect on glucose metabolic disorder. Rosiglitazone showed similar regulation to that of Re ( Fig. 1B-E).

Effects of Panaxadiols and Panaxatriols Ginsenosides on Glucose Tolerance and Insulin Tolerance in 2TD Rats
In the OGTT, compared with NC rats, the rats in MC have very significant higher level of FBG (P<0.01) at the all point of test tiame and very significant increase of AUC (P<0.01); compared panaxadiols and panaxatriols treated 2TD rats with those of the non-treated MC , the difference in FBG level and AUC are no significant change in Rd-h treated 2TD group rats (P>0.05); significant decrease of FBG observed in Rb1-h, Rg3-h, and Rg1-h treated (P<0.05); more significant decrease of FBG and AUC observed in the Re-h treated (P<0.01), more significant decrease of AUC also observed in the Rg1-h treated (P<0.01); rosiglitazone showed similar effect of Re; at the all point of test time, respectively; compared Re-h treated rats with those of rats treated by other ginsenosides at high dose, respectively; the differences in FBG level and AUC are significant (P<0.05) ; as shown in Fig. 2A

Effects of Panaxadiols and Panaxatriols Ginsenosides on Lipid Metabolism in 2TD Rats
In the above experiments, compared with the NC group rats, the rats in the MC group had very significantly higher mean serum levels of total cholesterol (TC) (P<0.01), triglyceride (TG) (P<0.001), low-density lipoprotein cholesterol (LDL-C) (P<0.001), and free fatty acid (FFA) (P<0.001), as shown in Figure 3A-D; and had very significantly lower mean serum levels of high-density lipoprotein cholesterol (HDL-C) (data not shown). This indicated that lipid metabolic disorder established in the 2TD model rats. The model rats treated by the panaxadiols and panaxatriols for 4 weeks, respectively; compared panaxadiols and panaxatriols ginsenosides treated rats with those of nontreated MC group rats, respectively; the mean serum levels of TC were no significant change in the Rd-l treated group rats, significant decrease in Rd-h, Rb1-l, Rb1-h, Rg1-l, and Rg1-h treated group rats (P<0.05); more significant decrease in the Rg3-h, Re-l and Re-h treated (P<0.01); the Rosiglitazone showed similar regulation to that of Rb1-l (Fig. 3A). The mean serum levels of TG were no significant change in the Rd-l and Rd-h treated group rats, significant decrease in Rg1-l and Rg1-h treated (P<0.05), more significant decrease in the Rb1-l and Rb1-h treated (P<0.01), the most significant decrease in the Rg3-l, Rg3-h, Re-l, and Re-h treated group rats (P<0.001); as shown in Fig. 3B. The intensity of the lowering blood TG by various ginsenosides is: Rg3-l > Re-h >Rg3-h > Re-l> Rb1-h > Rb1-l > Rg1-h> Rg1-l. Rosiglitazone showed similar regulation efficacy to that of Rb1-h (Fig. 3B). The mean serum levels of LDL-C that were no significant change in the Rd-l and Rd-h treated group rats (P>0.05), significant decrease in Rb1l, Rb1-h, Rg3-l, and Rg3-h treated(P<0.05), more significant decrease in the Rg1-l, Re-l and Re-h treated(P<0.01), the most significant decrease in the Rg1-h treated group rats (P<0.001); as shown in Fig. 3C. The intensity of the lowering blood LDL-C by various ginsenosides is: Rg1-h > Re-l > Rg1-l > Re-h > Rg3-h >Rg3-l >Rb1-h >Rb1-l. Rosiglitazone showed similar regulation efficacy to that of Re-l (Fig. 3C).The mean serum levels of FFA that were no significant change in the Rd-l and Rd-h treated group rats (P>0.05), significant decrease in Rb1-l, Rb1-h, Rg3-l, and Rg3-h treated (P<0.05), more significant decrease in the Rg1-l, Rg-h and Re-l treated (P<0.01), the most significant decrease in the Re-h treated group rats (P<0.001); as shown in Fig. 3D. The intensity of the decreasing serum FFA level by various ginsenosides is: Re-h > Rel > Rg1-h > Rg1-l > Rg3-h >Rg3-l >Rb1-h >Rb1-l. Rosiglitazone showed similar regulation efficacy to that of Rg1-l (Fig. 3D). The various ginsenosides had significant increasing effects on the serum HDL-C, high dose of them showed more obvious effects except the Re-1also has obvious effects (data not shown). In sum of above, data suggesting the Rg3 and Re has potent regulative effects on lipid metabolic disorder.

Effects of Panaxadiols and Panaxatriols Ginsenosides on Oxidative Stress and Inflammation in 2TD Rats
The same the above experiments, the effects of various ginsenosides on serum superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) levels showed as in Fig. 4A-C; The intensity of antioxidative stress by various ginsenosides is: Re-h> Rg1-h >Re-l> Rb1-h > Rg3-h> Rg1-l >Rd-h> Rb1-l >Rg3-l> Rd-l. As showed in the Fig.e 4D-F, the Re more significantly decreased serum tumor necrosis factor alpha (TNF-α), nuclear factor кB (NF-кB) and interleukin 6 (IL-6) levels (P<0.01) compared with those of other ginsenosides tested (P<0.05); especially, very significant decrease in serum IL-6 observed only in the Re-l and Re-h treated rats (P<0.01). These suggesting that the Re has potent anti-oxidative stress and anti-inflammation in 2TD rats.

Effects of Ginsenoside Re on Bcl-2, Bax and Caspase3 mRNA Expression and Histological Change in pancreas of 2td Rats
As showed in the Fig. 5A-D, compared with the NC rats, the rats in MC had very significant lower level of B-cell lymphoma-2 (Bcl-2) mRNA expression (P<0.01), very significant higher level of B-cell lymphoma-2 associated X protein (Bax) (P<0.01) and caspase3 mRNA (P<0.01) expression in pancreas. Compared with the MC, Re very significantly up regulates Bcl-2 mRNA (P<0.01), down regulates Bax (P<0.01) and Caspase3 mRNA (P<0.05 for Re-l, P<0.01 for Re-h) expression in the pancreas of 2TD rats, exhibit potent anti-apoptosis effects. The histological analysis and morphometry data were shown in Fig. 5E-I, the pancreas of NC rats are mostly oval or round cell clusters, with different sizes, regular structures, and a large number. The islet boundaries are clear, the cells are abundant in the islets, the size is regular, full, and the arrangement is regular and compact. Compared with NC, the pancreas of MC rats is atrophy, the number of islets is significantly reduced, the distribution of islets is loose, sparse, and irregular, the structure of the islets is disordered, the boundary is unclear, the cells in the islets are deformed, swollen, and the cytoplasm is vacuolated or shallow, which can be seen Apoptotic or dead cells with missing or pyknotic nuclei. Compared with MC rats, the islet damage of the rats treated with Re and Rosiglitazone were significantly repaired, the islets and the cells in the islets increased significantly, the structure of the islets was more regular and the atrophy was significantly improved, the islet cells were arranged more uniformly, and the morphology was more regular. The arrangement is relatively neat, swelling, vacuolating cells, and apoptotic and dead cells are reduced; the effect of large doses of Re is better than that of small doses.
Altogether, these data suggesting that Re produces potent antidiabetic effects in T2D rats. To understand the molecular mechanisms by which Re produce potent antidiabetic effects, we employed following experiments.

Ginsenoside Re Activates Akt via ER Signaling Pathway in T2D rats
Estrogenic activities have suggested as a mechanism of ginseng's actions, and Re was one of the major component that possess estrogenic activities in ginseng [25,31]. Moreover, our previous report demonstrated that Re activates cardiac potassium channels via a nongenomic pathway of sex hormones (including estrogen) [29]. Importantly, sex hormones and sex hormone receptor, particularly, estrogen and estrogen receptor (ER), play an important role in the pathogenesis and remission of T2D [16]. These findings encouraged us to hypothesis that whether Re produce potent antidiabetic effects via ER signaling pathway. To test this hypothesis, we, first, proceeded western blot analysis to determine whether Re phosphorylate threonine kinase (Akt) that was a critical node cascade in the classical sex hormone receptors signaling pathway, and test the effects of inhibitors of ER and Akt. Results of western blot analysis were shown as in Fig. 6A

Ginsenoside Re Activates Endothelial Nitric Oxide Synthase via ER/Akt Signaling and the Antidiabetic Effects of Ginsenoside Re Involve Nitric Oxide Actions
It has been suggested that nitric oxide (NO) action as a mechanism of ginseng's actions [26]. Moreover, our previous report demonstrated that Re activates endothelial nitric oxide synthase (eNOS) through sex hormone receptor including estrogen receptor alpha (ERα) and androgen receptor (AR) signaling, Re-induced delayed rectifier K + current enhancement and L-type Ca 2+ current suppression involved NO (release from eNOS) actions in guinea-pig cardiomyocytes [29,30]. Importantly, results from present study strongly suggested that eNOS might be a main downstream signal cascade in the ER/Akt signaling pathway. We, therefore, reasoned that NO derived from eNOS should be an important effector in the ER/Akt signaling pathway and the antidiabetic effects of Re involve the NO actions.
To test the propose, first, we employed western blot analysis to determine whether Re activates eNOS via Akt signaling that a critical node in the downstream of the ERα signaling pathway and upstream of the eNOS activation. The results of western blot analysis shown as in the Fig. 7A, compared with NC rats, the rats in MC had very significant lower phosphorylated eNOS proteins (P<0.01) in the pancreas of T2D rats, Re-treated very significantly increase the impaired phosphorylated eNOS protein levels (P<0.01), MK-2206 (a Akt inhibitor) treated completely inhibited Re-induced eNOS phosphorylation (P>0.05). Consistently, these data led us to conclude that Re activate eNOS through Akt signaling. Second, we examined whether the antidiabetic effects of Re involve the NO actions. We use eNOS inhibitor (L-N5 -(l-iminoethyl) ornithine, L-NIO) and NO donor (S-nitroso-Nacetylpenicillamine, SNAP) to determine NO involved in the antidiabetic effects of Re. As shown in Fig. 7B

Regulative Effects of Ginsenoside Re on Insulin and Glucagon in T2D Rats
Interestingly, it has been demonstrated that estrogen and ER signaling pathway regulate devers targets molecules and signaling pathway including insulin ang glucagon-like peptide 1 (GLP-1) that paly important roles in the development of T2D and insulin resistence [16]. These findings make us to speculate that whether Re could mimic the regulative effects of estrogen on insulin and GLP-1. To examine the speculations, we, first, proceeded Quantitative Real-Time PCR analysis to determine regulative effects of Re on insulin and glucagon (Gcg) mRNA expression in pancreas and measured pancreatic insulin content (PINS). As shown in  (Fig..8D). These results indicate that Re reverse impaired GLP-1 through ER signaling.
Overall, these data led us to speculate that not only NO (release from eNOS) acts as a main effector, but also alternative effectors or signal molecules such as insulin, Gcg, and GLP-1 participated in ER signaling pathway that mediated potent antidiabetic effects of Re.

DISCUSSION
In the present study, our data indicate that Re produces potent antidiabetic effects that characterized in integrated multi-targeted actions, integrating of the multi-targeted actions of Re mediated by ER signaling pathway. These findings revealed a novel molecular mechanism that activation of ER/Akt/eNOS/NO signaling pathway by Re was mainly responsible for the multi-targeted potent antidiabetic effects of Re on T2D (Fig. 8E).

Ginsenoside Re Produces Potent Antidiabetic Effects
More than 40 different ginsenosides that have been identified and isolated from the root of Panax ginseng were generally divided into two major groups panaxadiols (e. g. Rb1, Rb2, Rc, Rd, Rg3, Rh2, Rh3) and panaxatriols (e. g. Re, Rf, Rg1, Rg2, Rh1) based on their chemical structure [32]. In the present study, we firstly evaluated antidiabetic efficacies of five tapes of representative panaxadiols (Rb1, Rd, Rg3) and panaxatriols (Re, Rg1) on T2D in the same experimental system, and the Re was identified more effective component. The five tapes of ginsenosides that are quantitatively the most important derivatives among the all ginsenosides in the ginseng root, have the higher absolute bioavailability in the oral administration of ginseng, as well as the commonly studied ginsenosides derivatives regarding metabolic syndromes including diabetes [24,33]. We evaluated the effects of five tapes of ginsenosides on 2TD with examine the main indexes including FBG, ISI, HbA1c, TC, TG, LDL-C, FFA, SOD, MDA, GSH-Px, TNF-α, NF-κB and IL-6 could reflect the main aspects of pathogenesis and therapeutic targets for T2D [1,4,5,6]. Results of comprehensive analysis from the main indexes indicated that the Re has the potent antidiabetic effects that characterized in the most significantly lowering blood glucose and glucagon, improving insulin sensitivity, antiinflammation, and anti-oxidative stress, compared with the others tested (Figs. 1-4); whereas regulative actions of Rb1and Rg3 on the glucose metabolic disorder and inflammation were less powerful, compared with the Re , the powerful actions of Rb1and Rg3 characterized in the antihyperlipidemic efficacies (Fig. 3), it is supported by previous reports [34,35,36]. Rd, a main component of panaxadiols, has weaker efficacies on anti-hyperglycemia and antihyperlipidemia. Rg1, a main component of panaxatriols, exhibits various biological activities [37], characteristics of the actions are similar the actions of ginsenoside Re, but the potency of actions was weaker than that of ginsenoside Re ( Fig. 1-4). Our study also found the common feature in the actions of panaxadiols and panaxatriols are significant anti-oxidative stress. Sum of above, we reasoned that Re was the most effective component for T2D among the panaxadiols and panaxatriols derivatives were tested.

The Potent Antidiabetic Effects of Ginsenoside Re Characterized by Multi-targeted Therapeutic Actions
Our data not only confirmed Re exhibiting pharmacological effects of anti-hyperglycemia, anti-hyperlipidemia and anti-oxidative stress on metabolic syndrome including diabetes [38,39], but also firstly showed more potent efficacies of Re among the representative panaxadiols and panaxatriols derivatives on T2D (Figs 1-4). These results are supported by several studies [27,28]. Moreover, our data also firstly showed Re decrease serum IL-6, TNF-α and NF-κB levels in T2D rats ( Figure 4D-F), and provide more evidences for Re possesses significant anti-inflammatory actions. These results are supported by independent reports that Re inhibits NF-κB and antagonizes TNF-α in cultured 3T3-L1 cells [40,41]. In addition, our experiments firstly explored the anti-apoptotic activities of Re that characterized in the up regulation of Bcl-2 mRNA and down regulation of Bax and caspase-3 mRNA expression in the pancreas of T2D rats (Fig. 5A-D). It has demonstrated that Re has anti-apoptotic effects via regulation of Bcl-2, Bax, inducible nitric oxide synthase (iNOS) and caspase-3 in substantia nigra neurons of Parkinson's disease mouse model [42]. More importantly, our results also first time showed antidiabetic effects of Re involve NO (release from eNOS) actions (Fig.e 7), reports from our previous study and others have demonstrated the NO actions involve beneficial effects of Re in several other tissues [30,43]. Our data also confirmed Re enhances GLP-1 secretion in ileum and colon of T2D rats (Fig.8D) [44]. It should be noted that our data also firstly showed Re normalized the dysregulated insulin and Gcg gene expression, and reverse impaired insulin content in pancreas of T2D rats (Fig. 8A-C). Collectively, our study demonstrated that Re produces multiple therapeutic effects on T2D , such as anti-oxidative stress, anti-inflammation , anti-apoptosis, restoration of pancreatic β-cell as well as α-cell functions , and normalizing dysregulated insulin and Gcg, results in improving insulin sensitivity, anti-hyperglycemia, and anti-hyperlipidemia; in which Re regulates multiple target molecules including ER, Akt, eNOS, NO, insulin , Gcg, GLP-1 , Bcl-2, SOD, TNF-α, IL-6 that were closely associated with pathogenesis and remission of 2TD.

Integrating of the Multi-targeted Potent Antidiabetic Effects of Ginsenoside Re Mediated by ER Signaling Pathway
It has demonstrated that several signaling molecules including AMPK, GLUT4, GLP-1, PPAR-γ, SOD, Bcl-2, TNF-α, and NF-кB act as potential targets for antidiabetic effects of Re [38][39][40][41][44][45][46]. However, signaling pathway by which Re regulate the multiple signaling molecules is not yet clearly elucidated. Accumulating evidence has implicated that many biological actions of ginsenosides are mediated by plasma membrane receptors /or intracellular receptors signaling, particularly steroid receptors, and some ginsenosides including Re have been shown to exhibit estrogen-like activity via acting as a functional ligand of ER [29,31]. Re acts as full agonist for sex steroid receptors including ER in several biological systems [29,47]. In the present study, our data show that Re mimic the multi-targeted antidiabetic activities and insulinotropic properties of estrogen [12,16,48,49]; functionally, the activities of antihyperglycemia and improving insulin sensitivity induced by Re were inhibited by the ERα inhibitor (ICI-182,780) (Fig.5); indicating that the actions of Re are mediated by ERα. Structurally, Re and its metabolic derivatives in the intestines possesses potent affinities to bind human ERα [25,29,31]. Consequently, we confirmed Re activates Akt that was a critical node cascade in the downstream of ER α signaling pathway; functionally, the actions of Re were inhibited by Akt inhibitor (Fig. 6). These results are strongly supported by several reports that Re activates ER/or Akt signaling pathway in several other biological systems and pathophysiological conditions including diabetic complication [29,50,51]. Thus, we reasoned that ERα/Akt signaling pathway appears to be a main signaling pathway that is responsible for the antidiabetic effects of Re. Furthermore, our data showed Re activates eNOS via ERα/Akt signaling, and Reinduced antidiabetic actions are inhibited by a relatively selective inhibitor (L-NIO) of eNOS, and NO donor (SNAP) treatment did not affect Reinduced antidiabetic actions (Fig. 7); suggesting that the antidiabetic effects of Re involve NO (release from eNOS) actions. Re induce release of NO from eNOS via activation of ERα /Akt signaling pathway in several other tissues [29,43,51]. These evidences encouraged us to speculate that the NO might be a main effector that is mainly responsible for the antidiabetic effects of Re in the ERα/Akt signaling pathway. This concept could be further supported by diverse biological actions of NO that play very important role in the pathogenesis and remission of T2D [52], ERα/ Akt /eNOS/NO signaling pathway that was a main classical mechanism for estrogen actions [53], and Akt /eNOS/NO signaling pathway is essential for IR, obesity, and diabetes [52]. In addition, our data also speculated that several candidate molecules such as insulin, Gcg, and GLP-1 regulated by Re through ERα signaling. We did not test whether NO that released by Re via ERα/ Akt /eNOS signaling pathway regulates insulin, Gcg, and GLP-1. It has been demonstrated that ERα signaling and NO (release from eNOS) regulate many signaling cascades including insulin, AMPK, PPAR-γ, GLUT4, GLP-1, ATP sensitive potassium channel (K ATP ), Bcl-2, TNFα and cGMP that are closely associated to obesity and 2TD [16,52,54]. Therefore, NO release from eNOS act as multi-targeted effector and messenger in the regulation of glucose homeostasis and insulin sensitivity, represents the potential clinical therapeutic importance of NO-based interventions and NO targeted therapies that can hopefully facilitate the development of new treatments named "NO therapy" for T2D. Altogether, we reasoned that activation of ERα/Akt/eNOS/NO signaling pathway by Re appears to be the main mechanism for the multi-targeted antidiabetic effects of Re.

Ginsenoside Re is an Alternative
Multi-targeted Therapy for T2D Developing multi-targeted effective therapeutic measures for multifactorial T2D are on the hunt, estrogen therapy and NO therapy could be imaging as potential multi-targeted therapeutic strategy for T2D [52,54,55]. However, systemic delivery of estrogen /or NO agent potentially increase its serious adverse effects of estrogen/or NO agent toxicity [17,56]. Thus, it is potentially important in the clinical setting that Re produces multi-targeted potent antidiabetic effects via activation of ERα signaling pathway in the NO dependent manner described in the present study. It is supported by estrogen induces eNOS-NO has potential therapeutic importance [57]. Moreover, Re did not stimulate proliferation of estrogen-responsive human breast cancer cell line MCF-7; rather, Re partially inhibited E2-induced MCF-7 proliferation [29], and it has anticancer effects that without mediated by ER [58]. In addition, our data imply that Re is a naturally harvested, mechanisms specific agonist of ERα. Thus, we expect that Re is a novel effective and safety multi-targeted therapeutic for T2D, particularly, a novel candidate for replacement of both estrogen therapy and NO therapy in the prevention and treatment of T2D. It was further supported by Re has many beneficial effects on the diabetic complications [38,51,59].

CONCLUSION
In summary, in the present study, identified the multi-targeted potent antidiabetic effects of Re, demonstrated a novel pharmacological mechanism that activating ERα/ Akt /eNOS/NO signaling pathway by Re was mainly responsible for the integrated multi-targeted antidiabetic effects of Re. Our data provide the first evidence for the potential use of Re, as a multi-targeted synergistic therapeutic for T2D, particularly, a novel candidate for replacement of estrogen therapy and NO therapy in diabetes. Further studies are necessary to clarified crosstalk mechanism between ERα signaling and G protein-coupled estrogen receptor GPER (formerly GPR30) signaling underlying the antidiabetic effects of Re.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
This study followed the guideline to the Declaration of Helsinki, animal care and experiments were approved by the Animal Ethics Committee of Inner Mongolia Medical University, which abides by the law of the State (the People's Republic of China) Administration of Experimental Animals(ethical clearance number, YKD2017150).