Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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Effects of Scutellaria barbata extracts on serum glycolipid hormones and pancreatic-tissue structure in type 2 diabetic rats

Ruile Song1, Chunyang Tian1, Miao Xian1, Tang LI1,*, Zhengli Chen1, Fang Jing1, Huang Chao1, Qihui Luo1, Wentao Liu1, Hengmin Cui1, Kaiyu Wang1, He Min1, Geng Yi1, Ping Ouyang1, Weimin Lai1
1College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China.
The study describes the effect of Chinese herbal medicine extracts from Scutellaria barbata on serum glycolipid hormones and pancreatic-tissue structure in type 2 diabetes mellitus (T2DM) rats. Healthy Wistar rats (n=150) were divided randomly into two groups: healthy (30) and model (120). The healthy group was fed normally for 30d and injected citrate buffer on day 31. The model group was fed high-fat and high-sugar feed for 30d and injected STZ on day 31. On day 38 the model group was divided randomly into four groups: model group, low-, medium- and high-dose S. barbata (n=30 in each). 5, 10, 20 and 30d after treatment, serum levels of FGB, INS, CORT, TG, TC, HDL-C and LDL-C in all groups were measured and pancreatic-tissue sections were stained with hematoxylin-eosin and aldehyde fuchsine to observe the tissue structure. The results showed significantly decreased levels of FGB, INS, CORT and LDL-C (P<0.01) and increased level of HDL-C (P>0.05). In addition, the damaged structure of diabetic pancreatic tissue has been partially restored in the low-dose S. barbata group. These results show that low-dose S. barbata extract treatment is effective in treating T2DM.
In the past decades, type 2 diabetes mellitus (T2DM) incidence, morbidity and mortality rates have greatly increased worldwide (Khajuria et al., 2018). T2DM, the non-insulin-dependent form of diabetes, is a chronic, comprehensive disease characterized by hyperglycemia (Zhang and Li, 2012). Maintenance of homeostasis of body glycolipid hormones such as insulin (INS) and corticosterone (CORT), as well as of triglycerides (TG) and total cholesterol (TC), constitutes an important element in T2DM treatment. At present, the common treatment for T2DM mainly consists of orally administered blood-pressure medication and INS injection. These exert a beneficial short-term effect, but long-term usage leads to side effects such as reduced sensitivity to INS and low blood pressure and requires gradually increasing dosage due to decreasing effect of hormone and drugs (Skamagas et al., 2008). The treatment of T2DM using Chinese herbal medicine has been gaining attention. However, most s focus on the direct regulation of INS (Zhang and Li, 2012), with as of yet no specific medicine that can be clinically used S. barbata, a flowering plant of the mint family, produces copious amounts of flavonoids, polysaccharides and other effective bioactive constituents can regulate glycolipid metabolism and improve immunity (Wang et al., 2013; Wang et al., 2007), exert anti-oxidative protection (Yang et al., 2015) and enhance immunity (Peng, et al., 2014). However, S. barbata’s main usage is in cancer treatment. The usage and underlying mechanisms of S. barbata in regulating glycolipid hormones remain obscure. We aimed to address this in this study by investigating the correlation between T2DM rats, their secreted glycolipid hormone levels and changes in their pancreatic-tissue structure, this under the application of S. barbata extract. Our study aimed to explore the action mechanisms of S. barbata extract in the body and to promote the usage of S. barbata as a therapeutic agent.
150 Wister rats (4 weeks old, male, weighing 200-220 g) were purchased from Chengdu Dashuo Experimental Animal, Chengdu, Sichuan Province, China. All rats were treated in accordance with the Guidelines for Care and Use of Laboratory Animals and the study was approved by the Animal Ethics Committee of the Sichuan Agriculture University.
Preparation of Scutellaria barbata extracts
100 g S. barbata powder was mixed with 95% ethanol in a volume ratio of 1:10 and extracted ultrasonically at room temperature for 60 min. Following filtration, extraction was repeated, where after filtrates were combined twice, then rotated and evaporated to 10ml (Kuang et al., 2015). Extracts of 0.1 g/ml, 0.25 g/ml and 0.5 g/ml were prepared and stored at 4°C (Li et al., 2010).
Establishment of rat T2DM model
Wistar rats (n=150) were divided randomly into healthy group (30) and model group (120). The model group was fed with high-sugar and high-fat feed (Table 1) and the healthy group was fed with normal feed. All rats were fed continuously for 30 d. On day 31, all rats fasted for 24 h. On day 32, rats were weighed and 5‰ Streptozotocin (STZ) was injected intraperitoneally (45 mg/kg body weight) in the model group and citrate buffer was injected intraperitoneally (4.5 ml/kg body weight) in the healthy group (Kannan, et al., 2016). Injection was completed within 30 minutes (Chen et al., 2016). Levels of fasting blood glucose (FGB) and glycated hemoglobin (GHb) were measured in the model group on day 37. With FGB > 7.8 mmol/l and GHb > 7%, our rat T2DM model was confirmed (Xiang, 2010).

Table 1: Composition of high-sugar and high-fat diets.

Rat treatment with S. barbata extracts
Model group rats were divided into a model group (n=30) and a model treatment group (n=90), the latter further divided into a low-, medium- and high-dose extract group, 30 rats each. S. barbata extracts at 0.1 g/ml, 0.25 g/ml and 0.5 g/ml were intragastrically given to the low-, medium- and high-dose groups on day 38 (1 ml extract per 100 g body weight), respectively. Mean while, all rats of the healthy and model groups were also administered normal saline intragastri- cally (1 ml per 100 g body weight) and once a day for 30 days. During the entire process, the healthy group was fed normal feed, while the model group was fed high-sugar and high-fat feed, with all rats allowed to drink water freely. Feeding houses were regularly cleaned.
Measurements and data analysis
Six rats were selected randomly from each group to collect tail venous blood on days 43, 48, 58 and 68 (5, 10, 20 and 30 after treatment). Levels of FBG, GHb, INS, CORT, TC, TG, high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) in the blood were measured. Pancreases of rat were fixed in 4% paraformaldehyde for 24 h, followed by hematoxylin-eosin and aldehyde fuchsine staining to observe changes under an optical microscope. Data were analyzed by IBM Statistics 20 software and expressed as mean±standard deviation. Different lowercase letters indicate significant differences (p<0.05) and different uppercase letters indicate extremely significant differences (p<0.01).
Hyperlipidemia, hyperglycemia and obesity are three independent risk factors for the occurrence of T2DM and constitute clinically recognized predictors of various complications in T2DM patients (Liang et al., 2018). Therefore, reducing blood lipid and sugar levels can effectively delay the development of T2DM (Manikandan et al., 2017). Although conventional T2DM treatment can effectively reduce hyperglycemia in the short term, but long-term injection of INS can lead to reduced β-cell sensitivity with concomitant compensatory secretion of INS, leading to shortening of β-cell lifespan (Chen and Ren, 2016). Finding treatments that promote β-cell proliferation and delay their aging is therefore one of the goals of T2DM research.

S. barbata produces numerous diterpenoids, alkaloids, polysaccharides and flavonoids, with the latter representing the main active component (Li et al., 2008). It was shown that various flavonoids control FBG by reducing TG, TC and LDL-C in T2DM rats (Nicolle et al., 2011). Total flavonoids from buckwheat leaves showed a significant effect on diabetic C57BL/6 mice, promoting reduction in levels of TC, TG and LDL-C and increase in HDL-C (Bai et al., 2012). Total flavonoids from Ginkgo biloba were shown to decrease glucose and lipid levels in a rat model of INS resistance (Tang et al., 2009). Yet another study showed that flavonoids from artichoke can induce the expression of adiponectin, repair enzyme activity related to glucose metabolism, reduce blood glucose levels and help maintain lipid homeostasis (Liao et al., 2010). In the present study, CORT, INS, TC, TG and LDL-C levels in T2DM rat serum all decreased to nearly healthy levels after treatment with S. barbata extracts (Tables 4, 5, 6). FBG and GHb levels also decreased (Table 2, 3), but were still significantly (P<0.01) higher than those of the healthy group. It was previously speculated that a short experimental period and the absence of dual regulation of diet and exercise may lead to this phenomenon of FBG and GHb levels still higher than healthy (Yates et al., 2009; Zhou and Zhou, 2017). We previously showed that S. barbata can reduce blood glucose and lipid levels via the hypothalamic-pituitary-adrenalaxis (Tian, 2018).This experiment showed that S. barbata can reduce INS levels and improve the condition of the pancreas to regulate balance of glucose and lipid.

Table 2: FBG level changes in rats at different treatment time points.

Table 3: GHb level changes in rats at different treatment time points.

Table 4: INS level changes in rats at different treatment time points.

Table 5: CORT level changes in rats at different treatment time points.

Table 6: Lipid metabolism index changes in rats at different treatment time points.

Our staining of healthy rats pancreas with hematoxylin-eosin and aldehyde fuchsine staining showed clearly demarcated pancreas islets, oval or round and a large number of cells (Fig 1, 2). In contrast, model-rat pancreas islets were significantly atrophic, with many β-cells degenerated and necrotic. In the low-dose treatment group, pancreas-islet tissue structure was restored, with a clear boundary between pancreas islet and acinus. Islet cell numbers increased and cells were neatly arranged. These results suggest that S. Barbata extracts can repair the pancreas islets and induce proliferation of pancreatic islet β-cells. This study joins previous reports that demonstrate beneficial effect of S. barbata extract on the pancreas (Shen et al., 2014).

Fig 1: Pathological-histological changes in the pancreas of rats on day 30 of treatment (HE staining, bar=50 µm). “a1, b1, c1, c2, c3,” indicate, respectively, the healthy groups, model group, low-, middle- and high-dose S. barbata group. Arrows represent pancreatic islets.

Fig 2: Changes in pancreatic islet â-cells in rat on day 30 of treatment (aldehyde fuchsine staining, bar= 50 µm). “a1, b1, c3, c2, c1” indicate, respectively, the healthy group, model group, low-, middle- and high-dose S. barbata group. ”1" indicates â-cell, “2”indicates á-cell and “3” indicates collagen fibers.

It showed a favorable trend that the low-dose S. barbata group in the treatment of T2DM throughout the treatment phase. Not only were levels of INS, CORT and LDL-C in low-dose treatment serum close to those of the healthy groups, but HDL-C levels increased. The body uses the latter to rid of excess blood lipids and waste (Members, 2010). Pancreas-islet tissue recovered well in the low-dose treatment group compared with the other groups. Our study therefore show a beneficial therapeutic effect of low-dose S. Barbata extract.
To summarize, low-dose S. barbata extract treatment can significantly improve glucose and lipid hormone metabolism, as well as pancreatic-tissue structure damage caused by T2DM. The underlying mechanism is not yet clear and requires further research.

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