Antidiabetic Properties and Possible Mechanism of the Traditional Chinese Medicine Yi Qi Yang Yin Recipe in Db/db Mice

a clinical that ameliorates type 2 diabetes. The aim of the present study was to comprehensively evaluate the ecacy of YQYY granules and explore their mechanism of action. db/db mice were studied as an animal model of type 2 diabetes. After administered with YQYY for eight weeks, food and water consumption, levels of fasting blood glucose, glycosylated serum protein, and liver and pancreas tissue morphology were investigated. In addition, RT-PCR and Westernblot analysis were used to determine the expression of genes and proteins related to glycogen synthesis and gluconeogenesis pathways in the liver. leptin. By being sated, anabolism becomes greater than catabolism, causing animals to accumulate fat and become obese, gradually developing severe diabetes with signicant hyperglycemia. db/db mice exhibit characteristics similar to the clinical symptoms of T2DM in humans, including polydipsia, polyphagia, polyuria, obesity, hyperglycemia, hyperinsulinemia, insulin resistance, and abnormal lipid metabolism 9, 19 . These are consistent with the symptoms of T2DM as described in TCM. In the present study, UHPLC-Orbitrap MS technology was used to identify the principal chemical components within the YQYY prescription. The effects on improving diabetic phenotype of YQYY and the possible mechanisms of these effects were explored in a db/db animal model.


Introduction
Type 2 diabetes mellitus (T2DM) is a complex and severe metabolic disease caused by peripheral insulin resistance and islet β-cell dysfunction. It is a prevalent global disease with serious consequences to health.
Epidemiological studies suggest that 9.3% of the global population suffers from diabetes, the proportion likely to rise to 10.2% by the end of 2030, of which 90% will be T2DM 1, 2 . The effectiveness of the majority of hypoglycemic synthetic drugs declines during long-term treatment. For example, glycosylated hemoglobin can be reduced to 7% when metformin is rst administrated. However, it is effective in only 42% of patients after 2-5 years of use 3 . In recent years, combination drug therapy has been the focus of attention among medical researchers, providing a more e cacious response, for example, thiazolidinedione with metformin, glimepiride with metformin, GLP-1RA with SGLT2i, or GLP-1RA with DPP-4 [4][5][6][7] . Although the oral administration of additional Western medicines generally increases the incidence and severity of side effects, TCM has been widely used in clinical treatments (in China, Japan, Korea, and other East Asian countries) for thousands of years 8 . TCM provides a unique approach to therapy through synergistically treating metabolic diseases via a multi-targeting approach using multiple compounds. By virtue of their inherent compatibility, multiple compounds synergistically target the pathophysiological mechanism and not only avoid side effects such as hypoglycemia, the problem of drug resistance is also fundamentally prevented.
In conventional concepts of TCM, T2DM is regarded as "xiaoke". A de ciency of Yin and Qi, and excessive dryness and heat are the main contributors to the pathogenesis of "xiaoke" 9 . YQYY is a clinical prescription for the treatment of T2DM in patients that have a syndrome in which there is a de ciency of Qi and Yin. It is formed from a decoction of Angelica Liuhuang, as recorded in "The Hoard of Lan Shi" by Li Dong-Yuan. YQYY has been used clinically for many years and consists of seven herbs, including Astragalus mongholicus Bunge, Rehmannia glutinosa (Gaertn.) DC, Coptis chinensis Franch, Crataegus pinnati da Bunge, Mori fructus of Morus alba L. etc. It not only reduces blood glucose in T2DM, but also improves clinical manifestations caused by excessive drinking, over eating, fatigue, irritability, etc. Previous studies have shown that Astragalus mongholicus, an important herb with in YQYY, inhibits the apoptosis of β-cells in islets, the principal pathological mechanism resulting in their functional degeneration in T2DM, through immune regulation 10,11 . Rehmannia glutinosa functions by nourishing Yin and reducing re, manifested mainly as the regulated release of glucocorticoids (GC), inhibition of the excessive secretion of gastric acid, gluconeogenesis, and lipolysis 12,13 . The main active ingredient in Coptis chinensis, the adjuvant herb in the prescription, is berberine, which decreases in ammatory factors, improves insulin resistance, and inhibites glycogenolysis 10,14,15 . The active ingredients in Crataegus pinnati da and Mori fructus are organic acids, avonoids, and polyphenols which signi cantly reduce serum cholesterol and triglycerides, thereby effectively preventing and treating atherosclerosis 16-18 . db/db mice are widely used in animal models of T2DM. They have a mutation in the leptin receptor (Lepr) gene that prevents a response to the satiety hormone, leptin. By never being sated, anabolism becomes greater than catabolism, causing animals to accumulate fat and become obese, gradually developing severe diabetes with signi cant hyperglycemia. db/db mice exhibit characteristics similar to the clinical symptoms of T2DM in humans, including polydipsia, polyphagia, polyuria, obesity, hyperglycemia, hyperinsulinemia, insulin resistance, and abnormal lipid metabolism 9,19 . These are consistent with the symptoms of T2DM as described in TCM.
In the present study, UHPLC-Orbitrap MS technology was used to identify the principal chemical components within the YQYY prescription. The effects on improving diabetic phenotype of YQYY and the possible mechanisms of these effects were explored in a db/db animal model. preparing the prescription was as follows: the herbs in the recipe were decocted in a 10-fold volume of water 3 times, each for 1 h. The boiled liquid was centrifuged while hot (6000 g, 10min) and then concentrated to a relative density of 1.25-1.30 at reduced pressure (60°C). The concentrate was vacuum dried (80°C), then crushed, and ltered through a no. 5 screen (80 mesh).

Materials And Methods
Analysis of ingredients using UHPLC-Orbitrap MS Chromatographic and mass spectrometric conditions Analysis was performed using a Thermo Scienti c Vanquish Ultra Performance LC system coupled to a Thermo Scienti c Q Exactive Plus mass spectrometer equipped with a heated electrospray ionization (HESI) source (Thermo Scienti c, Santa Clara, CA, USA). Separation of the samples was performed through an Aquity UPLC®BEH C18 column (2.1 mm*100 mm, 1.7µm). Gradient elution was employed using a 0.1% aqueous solution of formic acid as solvent A and acetonitrile as solvent B. The following gradient was used: 0-5min (5% Fasting blood glucose level At the same time every week, animals in each group were fasted but with ad libitum access to drinking water for 12 h. They were weighed and fasting blood glucose (FBG) measured using a glucose meter in blood sampled from their tails (Accu-Chek® Performa, Roche Diagnostics GmbH, Mannheim, Germany).

Serum biochemistry
Blood samples were collected from the ocular vein of the mice. Serum was obtained by centrifugation of whole blood at 3500g for 10 min. The following parameters in the samples were measured using a CX4 Pro automatic biochemical analyzer (Beckman, Brea, CA): glycosylated serum proteins (GSP), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C).

Histopathology of pancreases and liver tissue
At the end of the experiment, necropsies were performed on all animals. The pancreas and liver were xed in 10% neutral buffered formalin and embedded in para n. Subsequently, the tissue samples were sliced into 3 μm thick sections using a rotary microtome, stained with hematoxylin-eosin (H&E), then observed and examined using an optical microscope at 200× magni cation (Carl Zeiss Meditec AG, Germany).

Liver oil red O staining and determination of glycogen content
The liver tissue samples were analyzed by Oil red O staining according to the manufacturer's recommendations. (Solarbio Life Sciences, Beijing, China). The sections were sealed then observed using an Olympus BX41 optical microscope. The glycogen content of the liver tissue was determined in accordance with the instructions of the glycogen kit (Solarbio Life Sciences, Beijing, China).

Immunohistochemistry of pancreas
Pancreas tissue was xed in 4% formalin, embedded in para n wax then sectioned for immunohistochemistry.
The sections were depara nized in xylene then rehydrated through a gradient of alcohol concentrations.
Secondly, the slides were treated with 3% H 2 O 2 to block endogenous peroxidase activity, then heated for 2.5 min in 95°C citrate buffer (10 mmol/L, pH 6.0) to retrieve the antigens. Thirdly, the slides were incubated with 10% BSA at 4°C overnight to reduce nonspeci c binding. Finally, Power-Vision TM two-step histostaining reagent and a 3,3-diaminobenzidine tetrahydrochloride substrate kit (ZSGB-Bio, China) were used to visualize antigen localization, in accordance with the manufacturer's instructions.

RNA Isolation and Real-Time RT-PCR
The liver tissue was disrupted in liquid nitrogen, and total RNA was isolated using Trizol reagent (Invitrogen, USA), following the manufacturer's instructions. Equivalent quantities of RNA were reverse transcribed using HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) (Vazyme, China). Real-time quantitative PCR was performed with ChamQ TM SYBR®qPCR Master Mix (Vazyme, China) using a Bio-Rad CFX96 Touch system.
Comparative gene expression analysis was performed using the ∆∆CT method, with GAPDH used as the endogenous control, for normalization. The primers used are as follows: Total protein was prepared from tissues using ice-cold RIPA buffer and quanti ed using a bicinchoninic acid (BCA) protein quanti cation kit (Dingguo, China). Equal quantities of protein were separated using SDS-PAGE then transferred to polyvinylidene uoride membranes. Each membrane was incubated with a primary antibody overnight at 4°C, then with an anti-rabbit or anti-mouse IgG-HRP secondary antibody at room temperature for 2h.

Statistical analyses
The results are presented as means ± SD. Data in multiple groups were analyzed using a one-way analysis of variance (ANOVA) using SPSS (version 16.0) software. Differences among means were analyzed using a Dunnett T3 multiple comparisons test or by post hoc analysis. P < 0.05 was considered a signi cant difference.

UHPLC-Orbitrap MS analysis of the components in YQYY
Peaks relating to the principal ingredients of YQYY are displayed in Figure 1. A total of 41 compounds were identi ed after comparison with standards (Table 3), MS fragmentation behavior, or through exploration of the literature [20][21][22][23][24] . The components identi ed were mostly triterpene glycosides, alkaloids, avonoids, oligosaccharides, which have been reported to have a therapeutic effect on T2DM.
YQYY improves the diabetic phenotype in db/db mice As shown in Figure 2A, the majority of mice in each administration group displayed a trend of decreased food intake compared with the model group. After 4 and 5 weeks of administration, consumption in the YQYY-L group was signi cantly different from that of the model group (p< 0.05). As displayed in Figure 2B, the daily water consumption of the db/db mice in the model group displayed an increasing trend as the experiment proceeded.

Liver histopathological observation and hepatic glycogen measurement
The results indicated that the liver tissue in the control and all db/db mice displayed varying degrees of hepatocyte steatosis and/or hepatocyte cytoplasmic vacuity ( Figure 3A). Liver glycogen content was measured in each group since glycogen synthesis in the livers of db/db mice affects FBG levels, and cytoplasmic vacuolation of hepatocytes is induced by glycogen synthesis. As displayed in Figure   3D, compared with control mice, glycogen in the model group was signi cantly higher under the in uence of high glucose (p< 0.01). Compared with the model group, metformin and all YQYY dose groups displayed increased liver glycogen content, the YQYY-H and YQYY-M groups exhibited a 1.51 and 1.54-fold greater quantity of glycogen than the model group, differences that were statistically signi cant (p< 0.01).
YQYY prevents morphological changes in pancreatic islets in db/db mice As shown in Figure 4A, histopathological examination of pancreas tissue demonstrated that islets in the control group displayed clear structural morphology and boundaries, mostly round or oval in shape. The islets in the model group were irregular, with scattered structures and broad distribution of new islet cells. In addition, apparent parenchymal cell proliferation was observed in the islets. Compared with the model group, lesions were less apparently resolved in the MET group and each YQYY dose group.
To further investigate the proliferation of islet parenchymal cells, islet α and β cells were stained in sectioned pancreatic tissue. In the control group, β cells were widely distributed in the inner islet and arranged densely in clusters, while α cells were distributed within the outer islet, mostly in monolayers ( Figures 4B, C). In the model group, proliferating β cells were also observed, mainly scattered within pancreatic exocrine tissues. The pancreatic islet structures in the model group had signi cantly higher numbers of α cells, which were scattered in the interior of the islets. Additionally, the relative insulin and glucagon concentrations in the pancreatic islets increased signi cantly in the db/db mice compared with the control group. However, the administration of YQYY and metformin signi cantly ameliorated the relative intensity levels of insulin and glucagon in the pancreatic islets of the db/db mice (Figures 4D, E).
YQYY ameliorates hyperglycemia in db/db mice via regulation of the expression of genes or proteins associated with glucose homeostasis Following observations that glycogen content in the livers of db/db mice were raised by YQYY, the molecular mechanism of the antidiabetic effect due to YQYY was investigated by measuring glycogen synthesis and the expression of proteins and genes related to the gluconeogenesis pathway. As presented in Figure 5, compared with the model group, the expression of G6Pase and PEPCK was signi cantly lower in the YQYY-L group (p< 0.01) and the MET group (p<0.05). The expression of IRS-1 and Akt2 in db/db mice were signi cantly lower than in the control group (p< 0.01), while the lower expression of IRS-1 and Akt2 were elevated by the treatment of YQYY and MET. As displayed in Figure 6A, the ratio of p-AMPK/AMPK in the model group was signi cantly lower than in the control group (p< 0.01). Compared with the model group, p-AMPK/AMPK ratio was higher in all the treatment groups, differences that were signi cant in the YQYY-M and YQYY-L groups (p< 0.01). In addition, p-GSK-3β and GS levels in the livers of YQYY groups and MET group were signi cantly higher than model group( Figures 6B, C).

Discussion
The etiology and pathogenesis of T2DM are complex and interrelated, resulting in the effectiveness of Western drugs declining after long-term use as they achieve a hypoglycemic effect by targeting a single pathology. In the early stages of the study, the pharmacological function of each herb in YQYY was analyzed. Using the compatibility rules of TCM, we speculated that YQYY may target multiple pathways for the synergistic treatment of T2DM, including the degradation of islet β cells 11 , decreased glucose uptake in muscle tissue 25,26 , and increased liver glucose output 15,27 .
The results of the study demonstrated that each dose of YQYY resulted in reduced food and water consumption in db/db mice. YQYY-L displayed the most signi cant reduction in food and water consumption, and signi cantly reduced FBG and GSP. Due to the lack of dietary control caused by gene de ciency in the db/db mice, we hypothesized that increased satiety, and thus reduced food and water consumption, could be a mechanism by which blood glucose was reduced in the YQYY-L group.
Obesity caused by over-consumption in db/db mice is accompanied by disrupted lipid metabolism, elevated levels of free fatty acids (FFAs), TG, and LDL-c. Excess FFAs and TG in plasma result in ectopic lipid accumulation, such as in the liver, pancreas, or kidneys 28 . In the present study, although YQYY had no apparent effect on resolving serum lipoprotein levels, histopathological observation of H&E and Oil red O-stained sections indicates that each dose of YQYY ameliorated steatosis and the degree of fat accumulation in liver tissue. The speci c reasons require further exploration of the expression of lipid metabolism-related genes and proteins.
Insulin resistance induces a compensatory secretion of insulin by islet β cells to maintain normoglycaemia, resulting in their proliferation and hypertrophy of islets 29 . This pathological process was also con rmed in the present study. Insulin storage in the pancreatic islets of db/db mice increased signi cantly, in addition to the islet hypertrophy and the proliferation of islet β cells, compared with the control group. However, morphological changes to the pancreatic islets and islet β cell proliferation were prevented by the administration of YQYY, suggesting that YQYY could ameliorate islet β cell function and improve insulin sensitivity. Additionally, the α cells of proliferating islets tend to secrete an excess of glucagon, leading to increased blood glucose. In the present study, YQYY also signi cantly inhibited the proliferation of islet α cells in db/db mice.
The results of UHPLC-Orbitrap MS analysis indicated that the principal components of YQYY were triterpenoid glycosides, alkaloids, avonoids, iridoids, phenylpropanol glucoside, the representative active components of each category being astragaloside 30 , berberine 26 , calycosin-7-glucoside 31 , catalpol 13,32 , and acteoside 33 , respectively. Based on previously published studies and the theory of TCM, we speculated that these components were the main active components that improve the function of islet β cells, ameliorate the steatosis of liver tissue, prevent gluconeogenesis and increase glycogen synthesis,increase satiety, and inhibit the proliferation of islet α cells in db/db mice.
The liver plays a key role in the maintenance of blood glucose homeostasis and the development of diabetes. As blood glucose rises, the liver converts glucose into glycogen 34 . As blood glucose levels decline, glucose is produced by glycogenolysis and gluconeogenesis to maintain blood glucose homeostasis. Promotion of glycogen synthesis and inhibition of gluconeogenesis is an effective method of ameliorating T2DM. The IRS/PI3K/AKT/GS pathway is key to glycogen synthesis 35 . GS is a rate-limiting enzyme for glycogen, whose activity is inhibited by GSK-3β 36 . Activation of PI3K activates Akt, a key kinase in the insulin signaling pathway.
Akt inactivates GSK-3β by phosphorylation of the Ser-9 locus in GSK-3β. The inactivation of GSK-3β promotes the dephosphorylation of GS, thereby enhancing GS activity and increasing glycogen synthesis 37  PEPCK and G6Pase are rate-limiting enzymes in gluconeogenesis, and the conversion of oxaloacetic acid into phosphoenolpyruvate by PEPCK, the rst key step in gluconeogenesis. G6Pase converts glucose 6-phosphate into glucose and is the nal stage of gluconeogenesis 38 .In addition, AMPK activation reduces the expression of gluconeogenic enzymes in the liver, including PEPCK and G6Pase. In the present study, YQYY-L signi cantly reduced gene expression levels of PEPCK and G6Pase in the liver tissue of db/db mice, resulting in decreased glucose production in the liver. Moreover, it has been shown that AMPK activation inhibits the dephosphorylation of p-GSK-3β, which in turn promotes glycogen synthesis 37,39 . These observations are consistent with the results here, with the levels of p-AMPK, the activated form of AMPK, higher following the administration of YQYY in db/db mice, resulting in the activation of GS. The results demonstrate that YQYY can reduce FBG levels in db/db mice by promoting the synthesis of liver glycogen and inhibiting gluconeogenesis.
In addition, there is a phenomenon worthy of discussion that is TCM formulas usually have the characteristics of the non-linear dose-effect relationship. In our study, low-dose of YQYY had the best ameliorative effect on food intake, water consumption, FBG, GSP, and the mRNA levels of G6Pase and PEPCK of db/db mice. Medium and high doses of YQYY had the better effect on improving the liver steatosis, increasing glycogen content of livers and the expression of proteins and genes related to the glycogen synthesis. We speculated that it was caused by the synergistic therapeutic effects of TCM via a multi-targeting approach using multiple compounds. The content of catalpol and berberine in low-dose of YQYY was the effective dose in inhibiting gluconeogenesis, while the content of the ingredients such as astragaloside in medium and high doses of YQYY had the effect on improving glycogen synthesis.

Conclusions
In summary, the results suggest that YQYY granules greatly attenuate the diabetic phenotype in db/db mice including excessive drinking and over eating. In addition, we established the effects of YQYY on antidiabetic and the possible mechanisms involved. The administration of YQYY reduced the levels of FBG and GSP by activating transduction of the IRS/PI3K/AKT signaling pathway and AMPK, and via inhibition of PEPCK and G6Pase gene expression. Therefore, we anticipate that YQYY could be utilized for the prevention of diabetes and its treatment.
However, the research on the mechanism of YQYY was not comprehensive and in-depth. the test of genome-wide DNA methylation will be conducted in the next for further reveal the mechanism of YQYY on T2DM by analyzing the level of methylation about diabetes-related genes to con rm the multi-targeting action of YQYY in achieving the antidiabetic effect.