**Xiang Cui, Da-Wei Qian, Shu Jiang \*, Er-Xin Shang, Zhen-Hua Zhu and Jin-Ao Duan \***

Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing 210023, China; 15951975518@163.com (X.C.); qiandw@njucm.edu.cn (D.-W.Q.); shex@sina.com (E.-X.S.); 18913133908@163.com (Z.-H.Z.)

**\*** Correspondence: jiangshu2020@126.com (S.J.); dja@njucm.edu.cn (J.-A.D.); Tel./Fax: +86-25-8581-1516 (S.J.); +86-25-8581-1291 (J.-A.D.)

Received: 11 October 2018; Accepted: 12 November 2018; Published: 18 November 2018

**Abstract:** *Aim* Scutellariae Radix (SR) and Coptidis Rhizoma (CR) have often been combined to cure type 2 diabetes mellitus (T2DM) in the clinical practice for over thousands of years, but their compatibility mechanism is not clear. Mitogen-activated protein kinase (MAPK) signaling pathway has been suggested to play a critical role during the process of inflammation, insulin resistance, and T2DM. This study was designed to investigate their compatibility effects on T2DM rats and explore the underlying mechanisms by analyzing the metabolic profiling and MAPK/PI3K/Akt signaling pathway. *Methods* The compatibility effects of SR and CR were evaluated with T2DM rats induced by a high-fat diet (HFD) along with a low dose of streptozocin (STZ). Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed to discover potential biomarkers. The levels of pro-inflammatory cytokines; biochemical indexes in serum, and the activities of key enzymes related to glycometabolism in liver were assessed by ELISA kits. qPCR was applied to examine mRNA levels of key targets in MAPK and insulin signaling pathways. Protein expressions of p65; p-p65; phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K); phosphorylated-PI3K (p-PI3K); protein kinase B (Akt); phosphorylated Akt (p-Akt) and glucose transporter 2 (Glut2) in liver were investigated by Western blot analysis. *Results* Remarkably, hyperglycaemia, dyslipidemia, inflammation, and insulin resistance in T2DM were ameliorated after oral administration of SR and CR, particularly their combined extracts. The effects of SR, CR, low dose of combined extracts (LSC) and high dose of combined extracts (HSC) on pro-inflammatory cytokine transcription in T2DM rats showed that the MAPK pathway might account for the phenomenon with down-regulation of MAPK (P38 mitogen-activated protein kinases (P38), extracellular regulated protein kinases (ERK), and c-Jun N-terminal kinase (JNK)) mRNA, and protein reduction in p-P65. While mRNA levels of key targets such as insulin receptor substrate 1 (IRS1), PI3K, Akt2, and Glut2 in the insulin signaling pathway were notably up-modulated, phosphorylations of PI3K, Akt, and expression of Glut2 were markedly enhanced. Moreover, the increased activities of phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase (FBPase), glucose 6-phosphatase (G6Pase), and glycogen phosphorylase (GP) were highly reduced and the decreased activities of glucokinase (GK), phosphofructokinase (PFK), pyruvate kinase (PK), and glycogen synthase (GS) in liver were notably increased after treatment. Further investigation indicated that the metabolic profiles of plasma and urine were clearly improved in T2DM rats. Fourteen potential biomarkers (nine in plasma and five in urine) were identified. After intervention, these biomarkers returned to normal level to some extent. *Conclusion* The results showed that SR, CR, and combined extract groups were normalized. The effects of combined extracts were more remarkable than single herb treatment. Additionally, this study also showed that the metabonomics method is a promising tool to unravel how traditional Chinese medicines work.

**Keywords:** SR; CR; Compatibility; T2DM; metabolic profiling; MAPK/PI3K/Akt signaling pathway

#### **1. Introduction**

Type 2 diabetes mellitus (T2DM), characterized by increased blood glucose level resulting from disturbances of insulin secretion, insulin action or both, is a complex disorder influenced by both lifestyle and genetic factors. T2DM and its serious complications such as nerve damage [1], kidney disease [2], and cardiovascular disease [3,4] have significantly increased society's medical burden over the past few decades and it is estimated that their global numbers in adults will rise to 592 million by 2035 [5]. Thus, its prevalence has become a major public health issue throughout the world, especially in developing countries.

Currently, drugs clinically used to treat T2DM include insulin sensitizers, insulin secreting drugs, insulin, etc. Although current diabetes treatments have exhibited some success in lowering blood glucose levels, their effects are not always sustained and their use may be associated with undesirable side effects such as hypoglycemia [6], gastrointestinal discomfort [7], etc. So it is urgent to discover new and effective drugs with fewer side effects to cure T2DM. Luckily, successful therapies for T2DM have been available from TCM practitioners. Thus, many researchers have focused on developing potent therapies and drugs from Chinese herbs with fewer side effects on T2DM patients.

SR, the dry root of *Scutellaria baicalensis* Georgi, with a number of biological activities such as anti-inflammation [8], anti-cancer [9], and anti-oxidation [10], has been used to treat various types of diseases. The major pharmacologically-active components of SR are flavonoids such as baicalin, wogonoside, baicalein, and wogonin. Accumulating researches have revealed that baicalin can significantly improve insulin resistance [11,12] and suppress gluconeogenesis [13]. CR, the dried rhizome of *Coptis chinensis* Franch, mainly containing alkaloids such as berberine, coptisine, and palmatine, has been used in China for thousands of years to treat diarrhea. Moreover, recent researches have showed that CR has anti-bacterial [14], anti-cancer [15] activities, etc. Especially, berberine has been shown to possess remarkable effects on lowering blood glucose and promoting the secretion of insulin [16,17]. In China, SR and CR are often used together at a ratio of 1:1 to obtain a synergistic effect for treating diabetes and its complications, yet the underlying mechanism on the therapy of T2DM remains unclear.

Due to insulin resistance, the liver excessively releases glucose into the blood as a result of increased glycogenolysis and gluconeogenesis. Besides, decreased glycolysis and glycogenesis lead to reduced consumption of blood glucose, contributing to hyperglycemia eventually [18]. Inflammatory cytokines such as TNF-α and IL-6 promote the development of insulin resistance, and a recent study has reported that anti-inflammation agents showed efficacy in reducing blood glucose level [19]. Moreover, T2DM is often present for years before becoming clinically apparent. Current clinical predictors such as fasting blood glucose are helpful in gauging diabetes risk, but they only reflect extant disease and provide little additional insight regarding pathophysiologic mechanisms. Earlier identification of individuals at risk is necessary and emerging technologies have made it possible through metabolomics. Among the varieties of analytical platforms used for metabonomic analysis, the use of ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) is steadily increasing due to its better reproducibility and detection limits, and increased chromatographic resolution, which can assess all metabolites in biological samples and provide insights into the holistic efficacy of TCMs. Besides, the extent of metabolic changes and types of metabolites could be applied as good markers during insulin resistance [20].

Thus, in this study, T2DM rats induced by HFD along with a low dose of STZ were used to assess the efficacy of individual and combined extracts. The levels of biochemical indexes, pro-inflammatory cytokine, key targets in MAPK, and insulin signaling pathways as well as the activities of key enzymes were determined. To elucidate the mechanism, the metabolic changes in plasma and urine from T2DM

rats based on UPLC-Q-TOF/MS were investigated. Potential biomarkers and metabolic pathways were also identified. These data would provide sound scientific evidence for the clinical treatment of T2DM.
