Antidiabetic and antioxidant effects of catalpol extracted from Rehmannia glutinosa (Di Huang) on rat diabetes induced by streptozotocin and high-fat, high-sugar feed

Diabetes mellitus is a heterogeneous metabolic disorder characterized by chronic hyperglycemia. It is associated with long-term complications and results in various bodily dysfunctions [1]. Diabetes often leads to the vascular complications of coronary artery disease and cerebrovascular disease and can cause renal failure, blindness, limb amputation, neurological complications, and premature death. With rapid therapeutic advancements, novel treatments with fewer side effects have become more feasible for long-term management of this disorder [2].

Rehmannia glutinosa (Di Huang) is a widely used herb in Chinese medicine (CM) that belongs to the Scrophulariaceae family. It has a high medicinal value and is taken to nourish yin and invigorate the kidney in CM [3]. R. glutinosa is often used to treat xiao ke [4], both xiao ke and diabetes patients experience the “3Ps”: polyuria (frequent urination), polydipsia (increased thirst), and polyphagia (increased hunger). CM theory attributes yin deficiency to diabetes [4] and R. glutinosa is often clinically used to ameliorate patients’ symptoms.

Catalpol, an iridoid glucoside of R. glutinosa, has many biological effects, including anticytotoxic and anti-inflammatory properties. Catalpol protected mesencephalic neurons from 1-methyl-4-phenylpyridinium [MPP(+)]-induced oxidative stress [5]. It also protected against hydrogen peroxide-induced apoptosis by preventing cytochrome c release and inactivating the caspase cascade, [6] against hydrogen peroxide-induced oxidative damage [7], and against ischemia-induced damage in astrocytes. Moreover, in an animal model, catalpol treatment ameliorated cognitive deficits and attenuated oxidative damage in brains of aging mice induced by d-galactose, increased the activities of SOD and GSH-PX, and reduced MDA levels in the liver and spleen [8].

Recent works have demonstrated catalpol’s anti-hyperglycemic effect in streptozotocin (STZ)-induced [2] and alloxan-induced rat diabetes [9, 10]. Catalpol could increase glucose use to lower plasma glucose in diabetic rats lacking insulin [2] through increasing beta-endorphin secretion from the adrenal gland in STZ-diabetic rats [11]. Moreover, oral supplementation of catalpol ameliorates diabetic encephalopathy by attenuating oxidative stress and increasing nerve growth factor concentration [1]. It ameliorated diabetic nephropathy [12] in rats by reducing the expression of pro-inflammatory mediators, such as monocyte chemotactic protein-1, tumor necrosis factor-alpha, inducible nitric oxide synthase, and receptors for advanced glycation endproducts (AGE). Catalpol suppressed AGE-induced phosphorylation of mitogen activated protein kinases, degradation of IkappaBalpha, and the nuclear localization of nuclear factor-kappaB [13]. Most studies of the effects of catalpol on diabetes mellitus have used normal diets in experimental models of chemical diabetes. However, type 2 diabetes mellitus is usually accompanied by glycolipid metabolism disorders and the effect of catalpol on type 2 diabetes mellitus with hyperlipid syndrome after a high-fat diet is still unknown.

This study aims to investigate the antidiabetic and antioxidant effects of catalpol from R. glutinosa on rat diabetes induced by STZ and high-fat, high-sugar feed. STZ and alloxan have been used to induce a diabetes mellitus model and damage the pancreas. Oxidative stress produced by STZ and alloxan is involved in diabetes mellitus and catalpol seems to have good anti-oxidative stress properties. In the present study, STZ-induced diabetes mellitus with a high-sugar and high-fat diet was used to imitate human type 2 diabetes mellitus. We investigated the effects of catalpol on glucose and fat metabolism in STZ-induced diabetic rats and measured oxidative enzyme and antioxidative enzyme plasma levels to explore the possible mechanisms of catalpol on type 2 diabetes mellitus.

In this study, STZ-induced type 2 diabetes with a high-fat and high sugar diet was used to study the antidiabetic and antioxidant effects of catalpol. STZ is synthesized by Streptomycetes achromogenes and is used to induce both insulin-dependent and non-insulin-dependent diabetes mellitus based on the STZ dose [22, 23]. The most frequently used single intravenous dose to induce insulin-dependent diabetes mellitus in adult rats is between 40 and 60 mg/kg by body weight [24]. STZ is also efficacious after intraperitoneal administration of a similar or higher dose, but a single dose below 40 mg/kg body weight may be ineffective [25]. STZ can also be given in multiple low doses. In this paper, rat diabetes was induced by a small dose of STZ (30 mg/kg) for 3 days after the rats were given high-fat and high-sugar feed for 3 weeks; this modeled more closely human type 2 diabetes because it accompanied the lipid abnormality. Before STZ administration, rats were given a high-sugar and high-fat diet for 3 weeks to induce fat and lipid abnormities, and their fasting blood sugar was near normal (less than 7 mmol/L). The plasma sugar and lipids were high after STZ administration for 2 weeks. STZ-diabetic body weight obviously decreased and was accompanied by increased thirst, frequent urination, and increased hunger. These changes indicated diabetes was successfully induced.

We found that catalpol had positive effects on type 2 diabetes. A dose-dependent anti-hyperglycemic action was observed for intravenous injection of catalpol at the dose range 10–50 mg/kg for 2 weeks. An increase in dosage beyond 50 mg/kg produced no additional effects. We chose a catalpol dose of 50 mg/kg for further study. The data showed that diabetic rats consumed more food and water and lost body weight. Interestingly, 50 mg/kg catalpol treatment for 2 weeks significantly ameliorated body weight loss and attenuated water intake and food consumption. Although we did collect urine or measure urine volume in each group, STZ-fat-diabetes rats became increasingly thinner, and the padding was often wet, which meant diuresis was markedly obvious in STZ-fat-diabetic rats; however, catalpol obviously ameliorated these changes.

As a glucose load test, the OGCT was used to test glucose clearance, which reflects pancreatic β cell function. Catalpol treatment 3 h after oral glucose administration obviously reduced plasma glucose and levels returned to normal in 6 h. The OGCT results showed that catalpol treatment improved diabetic rats’ ability to adjust blood sugar, perhaps through improved islet β cell function. Histological evaluation of the pancreas showed clearly (Fig. 9c) that the pancreatic tissue was atrophic with the presence of fibrosis. There was infiltration of inflammatory cells and these were markedly distributed in the interlobular and intralobular areas, but there was minimal distribution of inflammatory cells and fibrosis in the catalpol group. These results indicate that catalpol might facilitate glucose uptake, which is in accordance with previous findings [2].

Diabetes can cause glucose-lipid metabolism disturbance. Many diabetes patients show long-term abnormal lipid metabolism. In this study, the animal model induced by STZ with high-sugar and high-fat feed was close to human diabetes type 2. The rat model indicated that high-sugar and high-fat food with STZ produced type 2 diabetes accompanied by lipid metabolism disturbances with increases in TC and TG and decreases in HDL-C. Catalpol lowered plasma TC and TG and increased HDL-C (Fig. 5) when the blood sugar was lowered (Fig. 6). Catalpol not only ameliorated abnormal glucose tolerance, but also relieved the glucose-lipid metabolism disturbance.

Oxidative stress plays an important role in diabetes mellitus induced by STZ. STZ enters β cells via a glucose transporter and causes alkylation of DNA. DNA damage induces the activation of poly adenosine diphosphate-ribosylation, a process that is more important for the diabetogenicity of STZ than DNA damage itself [26]. Poly adenosine diphosphate-ribosylation leads to the depletion of cellular nicotinamide adenine dinucleotide and adenosine triphosphate. Enhanced adenosine triphosphate dephosphorylation after STZ treatment supplies a substrate for xanthenes oxidase resulting in the formation of superoxide radicals, hydrogen peroxide and hydroxyl radicals. Furthermore, STZ liberates toxic amounts of nitric oxide that inhibit aconitase activity and contribute to DNA damage. STZ can cause β cell necrosis [22, 23, 26]. In addition to a plasma lipid and sugar increase in STZ-fat-diabetic rats, the present findings show that endogenous antioxidants, such as SOD, GSH-PX, and CAT, were decreased and MDA levels were increased, which indicates that oxidative stress was strengthened in STZ-fat-diabetic rats. However, catalpol at 50 mg/kg for 2 weeks significantly reversed the lowered antioxidant levels and the increase of oxidative stress induced by STZ with sugar-lipid abnormities. SOD, GSH-PX, and CAT in plasma increased and MDA levels reduced to recover the balance of endogenous oxidants and antioxidants. The main active ingredients of catalpol, which is an iridoid glucoside, are isolated from the roots of R. glutinosa and used for treatment of diabetes and its complications. Furthermore, catalpol is a strong antioxidant [5, 27, 28] used to treat aging, stroke, and demyelination disease. The main effects of catalpol are on endogenous oxidant damage [1, 6, 7, 28] and inflammatory reactions [29, 30]. The antioxidant effects of catalpol found here are in accord with previous findings [1, 6, 7, 28]. However, catalpol possessed a stronger anti-inflammatory action and reduced inflammatory cytokines in senescent mice [29, 30], inhibited lipopolysaccharide plus interferon-gamma-induced inflammatory responses in astrocyte primary cultures [29, 30], and suppressed advanced glycation end-products-induced inflammatory responses through the inhibition of reactive oxygen species [13]. Based on the anti-hyperglycemic effect of catalpol and anti-hyperlipid abnormal coupling with markedly reduced oxidative stress in plasma, morphological pancreatic changes were examined by a light microscope (Fig. 9) after treatment with catalpol. Pancreatic damage induced by STZ was manifested as inflammation: infiltration of inflammatory cells was noted in the hypertension and markedly dilated intralobular and periductal areas. The atrophic, fibrotic, and inflammatory cells were markedly distributed in the interlobular and intralobular areas (Fig. 9c). After treatment with catalpol for 14 days, injury was ameliorated and blood glucose was decreased, and lipid levels also decreased as catalpol attenuated oxidative stress in STZ-fat-diabetic rats. In addition, inflammatory cells and fibrosis were minimal (Fig. 9d) and the pancreatic morphological changes were similar to those of the high-fat, high-sugar group (Fig. 9b). This suggests that catalpol might improve insulin levels in plasma through mitigation of pancreas inflammation induced by STZ and preserve pancreas islet structure and function.

We observed protective effects of catalpol on STZ-fat-diabetic rats. To date, only two studies have investigated the effect of catalpol on STZ-induced diabetes; these have focused on catalpol’s actions on preventing diabetic encephalopathy [1] and lowering plasma glucose through increasing muscle glucose use [2]. To our knowledge, this is the first study to investigate the effect of catalpol on diabetes induced by STZ with a high-sugar and high-fat diet. Catalpol reduced the damage to the pancreas in STZ-fat-diabetic rats. Catalpol’s protective effects are mainly related to decreased oxidative stress and the attenuation of the “3P” symptoms in a severely diabetic animal model. Further mechanisms of action, such as those involving insulin or glycated hemoglobin levels, should be investigated further.

Catalpol protected against STZ-induced diabetes with high-fat and high-sugar feed with ameliorated structural impairment of the pancreas and restored balance between oxidative enzymes and antioxidative enzymes.