ZHENG Xiu-wen, WEN Huan, ZHANG Jun-qing, HUANG Yu-fang, ZHANG Yu-xin, LIU Ai-xia, LI Xiang-yi, GAO Ya-nan, ZHANG Xu-guang

1. Key Laboratory of Tropical Translational Medicine of Ministry of Education; Hainan Provincial Key Laboratory for Research and Development of Tropical Herbs; Haikou Key Laboratory of Li Nationality Medicine; School of Pharmacy, Hainan Medical University, Haikou 571199, China

Keywords:Alpinia officinarum Hance Insulin resistance HepG2 cells GLUT4

ABSTRACT Objective: To study the effects of characteristic Li-medicine Alpinia officinarum Hance on improving insulin resistance (IR), and provide scientific evidence for the adjuvant treatment T2DM. Methods: CCK8 kit was used to detect the viability of HepG2 cells with 0-200 μg/mL Alpinia officinarum Hance extract (AOE) and 0-100 μmol/L galangin, and determine the

1. Introduction

In recent years, with the development of society, people's diets and lifestyles have undergone tremendous changes. The long-term intake of high-sugar and high-fat diets and the reduction of physical activity have made diabetes a major global disease affecting the world's public health. In China, the prevalence of diabetes from 2015 to 2017 was as high as 11.2%, and it is estimated that by 2040,the number of diabetic patients will reach 159.7 million. Type 2 diabetes (T2DM) is the main type of diabetes, accounting for more than 90% of the disease [1]. A large number of studies have shown that insulin resistance (IR) is the central link in the occurrence and development of T2DM, accompanied by the entire course of the onset of T2DM [2-4]. The occurrence of IR reduces the sensitivity and response ability of peripheral tissues (including skeletal muscle,liver and adipose tissue, etc.) to insulin, resulting in a reduction in the efficiency of insulin to promote glucose uptake and utilization by cells at normal physiological doses, and cannot regulate blood sugar balance. The physiological function of [5-7]. The low content of glucose transporter 4 (GLUT4) in cells is considered to be a key factor leading to IR [8]. Studies have shown that GLUT4 promotes glucose uptake and insulin utilization through transmembrane transport, maintains the body's blood sugar homeostasis and improves IR [9-11]. At present, drug therapy is an effective means to improve IR, but the only drugs that can directly improve IR are metformin and thiazolidinediones, and long-term use will produce obvious side effects, such as drug resistance or heart failure [12-13] .Therefore, the discovery of new hypoglycemic drugs that are safe,effective and non-toxic and side-effects is very necessary.

Galangain is the dried rhizome of Alpinia officinarum Hance,a plant of the ginger family of the ginger family. Its functions include warming the stomach and relieving vomiting, dispelling cold and relieving pain. It is used for abdominal cold pain, stomach cold, vomiting, belching and so on[14]. Galangain is a commonly used medicinal material in the Li nationality area of Hainan,and it is included in "Li Nationality Medicine Annals" and "Li Pharmaceuticals" [15,16] as a Li nationality medicine; The main chemical components in ginger have been studied. Galangin is the main chemical component of galangal. It has been reported that galangin can improve IR through the Akt/mTOR signaling pathway.The mechanism of action needs to be further studied [17,18]. In summary, this study explored the effect of galangal and galangin,the main chemical component, on improving IR, and provided a scientific basis for the further application of galangal, a characteristic of the same medicine and food, to the adjuvant treatment of diabetes.

2. Materials and Methods

2.1 laboratory apparatus

Spectra Max19 full-wavelength microplate reader (U.S.);NovoCyte3130 flow cytometer (U.S.); CRYSTAL191L air-jacketed CO2 cell incubator (U.S.); Thermo Micro 21R refrigerated highspeed centrifuge (U.S.); electrophoresis system (U.S. Bio-Rad) );Chemidoc xrs+ high-sensitivity chemiluminescence imaging system(Bio-Rad, USA).

2.2 Materials and reagents

Galangal medicinal material (produced in Haikou City, Hainan Province); column chromatography silica gel (200 mesh to 300 mesh, produced by Qingdao Ocean Chemical Plant); 80%ethanol, petroleum ether (boiling range 60 to 90), ethyl acetate(Tianjin University Mao Chemical Reagent Factory); HepG2 cells(Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.); Fetal Bovine Serum (Yikesai, FSP500); Cell Counting Kit-8 (CCK8,APExBIO, K1018); Penicillin-Streptomycin (Xinsai United States,C125C5); 2-NBDG (fluorescent glucose analogue, APExBIO,B6035); Glucose determination kit (Nanjing Jiancheng Institute of Bioengineering); Rosiglitazone (Shanghai Yuanye Biotechnology Co., Ltd., Y05F9C54480); DMEM high Sugar medium (Gibco,C11995500BT); PBS Ph7.4 (Gibco, C10010500BT); 0.25% Trypsin Digestion Solution (Xinsaimei, C125C1); RIPA Lysis Solution(Biyuntian, P0013C); GLUT4 antibody (Proteintech, 668461- 1-lg).

2.3 Experimental method

2.3.1 Preparation of Galangal Extract and GalanginTake the dried and crushed galangal rhizome (1.5 kg) and extract 3 times with 80% ethanol under reflux for 1 h each time, filter the combined filtrate, concentrate and cool to room temperature to obtain the extract (200 g), which is the galangal extract (AOE).

The AOE was eluted with a gradient of petroleum ether-ethyl acetate system (1:0→0:1) to obtain 3 components, namely Fr.1(14 g), Fr.2 (14 g), Fr.3 (56 g). Fr.3 was separated and enriched repeatedly and purified by recrystallization to obtain galangin (15 mg). The chemical structure of galangin was determined using the physical and chemical properties of the compound and thin layer chromatography. Needle-shaped yellow crystals, soluble in methanol, slightly soluble in chloroform, developed by a thin layer of ferric chloride, yellow fluorescence observed under ultraviolet light. Three different developing reagents with different ratios were prepared and compared with the standard galangin for thin-layer chromatographic analysis. The results showed that the compound and the standard galangin had the same ratio shift value and spot color. Therefore, the compound was determined to be galangin. The structural formula is shown in Figure 1.

2.3.2 Cell culture

HepG2 cells were cultured in DMEM complete medium(containing 10% fetal bovine serum and 1% penicillin-streptomycin)in a cell culture incubator at 37°C and 5% CO2. When the number of cells reaches about 90%, discard the culture medium, add 1 ml of PBS solution to wash twice, and then add 0.25% trypsin digestion solution for digestion, stop the digestion with complete medium after 1 to 2 minutes, and proceed with cell passage , Take the cells in the log phase for follow-up experiments.

2.3.3 CCK8 detects cell viability

The HepG2 cells were pipetted and mixed to make a cell suspension and added to a 96-well plate with about 1×104cells per well, and placed in a cell incubator for 24 h. When the cells are over 70%, each well is replaced with a medium containing 100 μL of galangal extract (50, 100, 200 μg/mL) and different concentrations of galangin (1, 2.5, 5, 10, 25, 50, 100 μM) were incubated for 48 h,and a control group (zero well) and a blank group were set up at the same time, with 6 replicate wells in each group. After the incubation,add 10 μL of CCK8 reagent to each well, and incubate for 1.5 h in the incubator in the dark. Detect the OD value with a microplate reader at 450 nm wavelength, and calculate the survival rate of each group of cells.Cell survival rate (%)=[(OD value of experimental group-OD value of blank group)/(OD value of control group-OD value of blank group)] × 100%

2.3.4 Glucose uptake experiment

Divide HepG2 cells into 6 groups, namely blank group (CON),model group (MOD), positive control group (rosiglitazone, ROSI; 25μM), and galangal extract group (AOE; 50 μg/mL) , Different doses of galangin group (10, 20 μM), each group has 3 repetitions. Except that the blank group used 5.5 mM low-sugar medium, the rest of the groups were all incubated with 50 mM high-sugar medium to incubate HepG2 cells to establish an IR cell model. According to the cell grouping, the corresponding drugs were given and incubated for 48 h. Then discard the medicated medium, wash twice with PBS, add 25 μM 2-NBDG solution, incubate at 37℃ for 20 min in the dark, and use flow cytometry to detect the flow cytometric fluorescence intensity in HepG2 cells to obtain the glucose uptake .

2.3.5 Glucose consumption experimentThe experiment grouping was the same as experiment 1.3.4, divided into 6 groups, each group had 6 multiple holes, and the cells of each administration group were incubated in a cell incubator for 48 h.After the incubation, the absorbance (OD) value of the cell culture medium of each group was measured in a microplate reader using a glucose determination kit (glucose oxidase method) at a wavelength of 505 nm. At the same time, the OD value in each well was measured through the CCK8 experiment, and normalized to correct the error caused by the number of cells. The result is expressed as glucose consumption/CCK8 (GC/CCK8).

2.3.6 Western blot detection of cell GLUT4 protein expression

According to experiment 1.3.4, the cells were grouped and incubated with the drug for 48 h. The drug-containing culture medium was discarded, washed twice with PBS, and the cells were digested and transferred to a centrifuge tube, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded; The RIPA lysate was placed on ice for 30 min for protein lysis, and then centrifuged at 4℃, 14000 rpm for 15 min, and the supernatant was taken out,and it was denatured for later use. Separate the protein samples with 8% SDS-PAGE gel electrophoresis, transfer the protein to PVDF membrane, and block with skimmed milk powder solution, incubate the primary antibody GLUT4 (diluted 1:2000) overnight; the next day, incubate the secondary antibody, and develop imaging , Image Lab software analyzes GLUT4 protein expression.

2.4 Statistical methods

Graphpad Prism 8.0 software was used for statistical analysis of data. All statistical data are expressed as mean±standard deviation,the comparison between the two groups adopts t test, and the comparison among multiple groups adopts single-factor analysis of variance. P<0.05 indicates that the difference is statistically significant.

3 Results

3.1 The effect of AOE and galangin on the viability of HepG2 cells

As shown in Table 1, the cell viability of HepG2 cells incubated with 0～200 μg/ml AOE and 0～50 μM galangin did not change significantly, while galangin at a concentration of 100 μM significantly reduced the cell viability (P<0.01). Therefore, this study determined that the administration concentrations of AOE and galangin were 50 μg/mL and 10 and 20 μM, respectively.

3.2 Effects of AOE and galangin on glucose uptake of IRHepG2 cells

It can be seen from Tab 2 that compared with the CON group, the glucose uptake of the MOD group cells was significantly reduced(P<0.01), and insulin sensitivity was reduced, indicating that insulin resistance occurred, indicating that the model was successful. The AOE group, different doses of galangin administration group, and ROSI group all significantly increased the glucose uptake of IRHepG2 cells (P<0.05), and the 20 μM galangin dose had the best efficacy, which was better than positive Drug ROSI (P<0.01).

3.3 The effect of AOE and galangin on glucose consumption of IR-HepG2 cells

The results in Tab 3 showed that the glucose consumption of the MOD group was significantly reduced compared with the CON group (P<0.01), and the glucose consumption of IR-HepG2 cells was significantly improved after AOE and different doses of galangin were treated (P<0.05).

Tab 1 AOE(0～200 μg/mL) and galangin (0～100 μmol/L) to determine the viability of HepG2 cells

Tab 2 Effects of AOE and different doses of galangin on glucose uptake of IR-HepG2 cells

Tab3 Effects of AOE and different doses of galangin on glucose consumption of IR-HepG2 cells

3.4 The effect of AOE and galangin on the expression of GLUT4 protein in IR-HepG2 cells

The results in Figure 2 showed that the expression of GLUT4 protein in the MOD group was significantly lower than that in the CON group (P<0.01), indicating that the insulin signal transduction pathway of IR-HepG2 cells was impaired. However, compared with the MOD group, the expression of GLUT4 protein in the AOE group and the galangin administration group of different doses was significantly increased (P<0.01), indicating that AOE and galangin promoted the expression of GLUT4 protein in IR-HepG2 cells , It has a sensitizing effect on insulin.

Tab 4 Effects of AOE and different doses of galangin on the expression of GLUT4 protein

4. Discussion

Diabetes is a chronic metabolic disease characterized by elevated blood sugar. The 9th edition of the Global Diabetes Map released by the International Diabetes Federation shows that the number of adults with diabetes in the world is about 463 million in 2019, and the number of patients is increasing year by year[19] . IR is a key pathogenic factor of diabetes. Diabetes caused by IR dominates.At the same time, many metabolic diseases are related to IR [20-22].The understanding of IR in Chinese medicine mentions that IR is the disease of the spleen, which causes the spleen to have abnormal transport and chemical functions and damage the heart and kidneys over time [23]. As China's resources of traditional Chinese medicine and natural medicines are extremely abundant, it can be developed into a medicine and food homologous product by taking advantage of its unique advantages such as low price, easy availability, mild action, small side effects, and long-term use. Galangal, a plant of the ginger family, is mainly distributed in the southern region of China. It is a characteristic Li medicine of Hainan and a treasure in China's TCM treasure house. Current studies have found that the 80% ethanol extract of Galangal, a characteristic Li medicine, has a significant effect on reducing blood sugar levels in T2DM mice.After analyzing the effective part, the results show that this part is mainly composed of galangin and other 5 species. The composition of ingredients, of which galangin has the highest content [24], but the material basis of the medicinal effect of galangal for lowering blood sugar is still not very clear.

In summary, this study prepared AOE from the dried roots of galangal, and obtained galangin after further separation and purification. The IR-HepG2 cell model was constructed to explore the effects of AOE and galangin on improving IR and glucose metabolism. The results showed that after IR-HepG2 cells were incubated with AOE and different concentrations of galangin, their glucose uptake and consumption were significantly increased,maintaining the balance of glucose inside and outside the cell,confirming that the characteristic Li medicine galangal may improve the effect of IR Play the effect of lowering blood sugar. In addition,GLUT4 is a downstream molecule of the insulin signaling cascade and the rate-limiting molecule of liver glucose utilization, which is closely related to IR [9]. Studies have shown that significant glycogenolysis occurs in mice lacking GLUT4, which can cause IR [25,26]. In this study, Western blot technology was used to detect the expression of GLUT4 protein, and it was found that AOE and galangin can significantly increase the expression of GLUT4 protein,suggesting that galangal may improve IR by regulating GLUT4 related signal pathways.

In order to better prevent and treat T2DM, it is still necessary to further explore the mechanism of galangal for lowering blood sugar.The pathogenesis of diabetes is closely related to IR, oxidative stress and inflammatory factors, and the pharmacological effects of galangal include anti-inflammatory and antioxidant effects.Therefore, it is necessary to conduct a more in-depth study on the mechanism of galangal in treating diabetes. To clarify the exact mechanism of galangal in the treatment of T2DM.

Author contributions: Zheng Xiuzhen and Wen Huan conduct experimental operations, analyze data, and draft papers; Zhang Junqing designs experimental schemes and revises the papers;Huang Yufang participates in cell culture and CCK8 determination of cytotoxicity experiments; Zhang Yuxin participates in glucose uptake experiments; Liu Aixia participates in glucose Consumption experiment; Li Xiangyi participated in the Western blot experiment;Gao Yanan and Zhang Xuguang provided technical support and guidance, and corrected the paper.

This article has no conflict of interest.