Study on the Mechanism of Lupenone for Treating type 2 Diabetes by Integrating Pharmacological Evaluation and Network Pharmacology

doi:10.21203/rs.3.rs-783505/v1

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Study on the Mechanism of Lupenone for Treating type 2 Diabetes by Integrating Pharmacological Evaluation and Network Pharmacology

https://doi.org/10.21203/rs.3.rs-783505/v1

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published 28 May, 2022

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Background: Lupenone (LUP) is the active ingredient of Rhizoma Musae, which has good anti-diabetes effects, but the underlying mechanism is unclear. In this study, animal experiments combined with network pharmacology were used to explore the mechanism of LUP for treating diabetes.

Methods: Type 2 diabetic rats with insulin resistance (IR) were induced by a high-fat diet and streptozotocin. the fasting blood glucose (FBG), index of oxidative stress, blood lipids, and IR-related targets in skeletal muscle and adipose were detected. a network pharmacology-based strategy was also adopted to clarify the mechanism of LUP for treating diabetes by improving IR.

Results: LUP decreased the FBG levels and synthesis of glycogen, improved oxidative stress and lipid metabolism disorders, and increased the gene and protein expression of insulin receptor, insulin receptor substrate (IRS)-1, IRS-2, glucose transporter type 4 (GLUT-4) in skeletal muscle and peroxisome proliferator-activated receptor γ, IRS-1, IRS-2, GLUT-4 in adipose. Network pharmacology analysis revealed that LUP improves IR by multiple targets (like INS, TP53, TNF, SRC and ESR1) and signal pathways.

Conclusion: These results suggested that the mechanism of LUP for treating diabetes is closely related to improving IR. LUP has the potential to develop as a new drug for the treatment of type 2 diabetes.

Integrative & Complementary Medicine

Toxicology

Lupenone

Insulin resistance

Type 2 diabetes

Blood lipids

Oxidative stress

Diabetes mellitus is a chronic disease and it is characterized by hyperglycemia [1]. There are three main types of diabetes including type 1 diabetes, type 2 diabetes (T2DM), and gestational diabetes. Among them, T2DM is the most common. The T2DM put a huge burden on the global health-care systems [2]. Drugs currently used clinically for the treatment of T2DM have many limitations and adverse reactions, such as gastrointestinal reactions, hypoglycemia, edema, osteoporosis, and lactic acidosis [3]. Therefore, it is necessary to find an ideal drug for the safe and effective prevention and treatment of T2DM. Insulin resistance (IR) is one of the main factors leading to T2DM and its complications [4].

IR refers to a decrease in the response of biological target tissues (mainly including liver, muscle, and adipose) to normal physiological concentrations of insulin. Modern research finds that improving IR is an effective way to treat T2DM. The traditional Chinese medicine could treat T2DM through improving the IR, especially in increasing the sensitivity of muscle, adipose tissue, and liver to insulin [5–7]. Our research team has confirmed that Rhizoma Musae (the dried rhizome of Musa basjoo Sieb. et Zucc.), a traditional Chinese medicine, has a good anti-diabetic effect, and the lupenone (LUP) is isolated from the Rhizoma Musae, has good inhibitory effect on protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase in vitro [8–10]. Furthermore, LUP could reduce the fasting blood glucose (FBG) in diabetic mice and improve the oral glucose tolerance and insulin tolerance in T2DM rats [11–13]. These results indicate that LUP has definite anti-diabetic effects. However, the mechanism of LUP in the treatment of T2DM has not been elucidated. Here, the aim of this study is to elucidate the mechanism of LUP to improve IR using animal experiments and network pharmacology methods.

Reagents

LUP was isolated from Rhizoma Musae by chromatographic separation on a silica gel column, and its structure was elucidated by comparison of its spectral data (mp, MS, ¹H-NMR, ¹³C-NMR) with reference standards [9]. The purity of LUP was ≥ 98%. Streptozotocin (STZ) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used in the study were of analytical grade.

Animals

All experiments were performed on 6-week-old male Sprague-Dawley rats (200 ± 20 g) obtained from Chongqing Tengxin Biological Technology Co., Ltd. (Chongqing, PR China). The colony was maintained under controlled conditions of temperature at 23 ± 2°C, 50 ± 5% humidity, 12 h light/dark cycle. All the animals in the study were cared for and treated humanely according to the national legislations of China, as well as local guidelines. The animal experiments were approved by Guizhou University of Traditional Chinese Medicine and the Ethics Committee for Animal Experiments of West China Hospital of Sichuan University (under registration: 2013029A).

Induction Of Type 2 Diabetic Rats And Treatment

After acclimatization for one week, the rats were randomly divided into normal group and high-fat diet (HFD) group. The rats in normal group were fed with standard diet ad libitum, and the HFD group was fed with high-fat diet (15% cholesterol, 15% coconut oil, 15% granulated sugar, and 55% standard diet ad libitum). After feeding for 4 weeks, all rats were fasted for 12 h, then the HFD group animals were injected twice with low-dose streptozocin (STZ; first with 30 mg/kg, i.p.; second with 28 mg/kg, i.p. 24 h later) to induce partial insulin deficiency, and the normal group were intraperitoneal injected with sodium citrate buffer. After injection 72 h, the FBG levels of the rats were measured, and the rats with FBG of ≥ 11.1 mmol/L were regarded as T2DM rats. T2DM rats were further randomly divided into T2DM group, positive group, and three LUP treatment groups. The normal group and T2DM group were received the vehicle treatment (0.03% Tween 80; Twice daily), while the positive group were administered the rosiglitazone (1.00 mg/kg; Twice daily), and the LUP groups were administered with 2.0, 4.0, and 8.0 mg/kg LUP (Twice daily), respectively. After 2 weeks of treatment, 6 rats in each group were sacrificed by cervical dislocation, and the skeletal muscle and adipose tissues were preserved at -80°C until use for quantitative real-time PCR test; After 4 weeks of treatment, the rest rats were fasted overnight, and then blood samples were collected under anesthesia. The blood samples were centrifuged at 3500 rpm for 10 min, and then the serum was collected for biochemical analyses. The rats were sacrificed by cervical dislocation, the pancreas was fixed with 10% neutral buffered formalin for the histological analysis, and the liver tissues were fixed with Carnoy fixative solution used to perform periodic acid-Schiff staining, and the skeletal muscle and adipose tissues were preserved at -80°C until use for testing protein expression. No death was recorded and no rats were dropped out of the analysis.

Determination Of Body Weight And Fbg

After the animal starts to dose, body weight of rats in all groups was measured every three days. All rats were fasted for 12 h every weekend to measure the FBG levels using the One-touch Ultra Blood Glucose Monitoring System (Johnson & Johnson Medical (China) Ltd., Shanghai, PR China).

Biochemical Parameter Analysis

The levels of serum leptin (LEP), hemoglobin A1c (HbA1c), superoxide dismutase (SOD), Malondialdehyde (MDA), Glutathione peroxidase (GSH-PX), Adiponectin (ADPN), and free fatty acid (FFA) in serum was analyzed by commercial ELISA kit according to the manufacturer’s instructions (Abcom, Cambridge, UK). The levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) were detected by automatic biochemical analyzer (Olympus AU640, Japan).

Periodic acid-schiff (PAS) staining

PAS staining for glycogen and examined with light microscopy at 100× magnification were performed. Briefly, liver was subsequently routinely processed, embedded in paraffin. Sections of 5 µm thickness were cut. The sections of the liver tissue were deparaffinized and hydrated, and then immersed in 0.5% periodic acid solution for 10 min and rinsed in distilled water for another 5 min, twice. Then the sections were placed in Schiff reagent for 10 min and wash in water for 5 min before counterstained in hematoxylin for 2 min. Stained reddish dots or threadlike structures in the liver cells were considered to be a positive result.

Histological Analysis

The pancreas were fixed in 10% neutral buffered formalin for 48 h. Samples were subsequently routinely processed, embedded in paraffin. Sections of 5 µm thickness were cut, and stained with hematoxylin and eosin (HE) for general histopathological examination under a light microscope.

Quantitative Real-time Pcr

The total RNA of skeletal muscle and adipose tissue was isolated using trizol reagent (Invitrogen, Carlsbad, CA), and total RNA was used to synthesize cDNA using the PrimeScript RT reagent kit according to the manufacture’s protocol (TaKaRa Bio, Dalian, China), then the amplification reactions were carried out in 96 well reaction plates (Bio-Rad, Hercules, CA, USA) with 20 µL reaction volume. GAPDH was used as endogenous control, and the fold change in the level of target mRNA between control and treatment groups were normalized by the level of GAPDH. The change in gene expression was calculated by 2^−∆∆Ct [14]. Primer sequences for analysis of insulin receptor (InsR), insulin receptor substrate (IRS)-1, IRS-2, and glucose transporter type 4 (GLUT-4) mRNA in skeletal muscle and proliferator-activated receptor (PPAR)-γ, IRS-1, IRS-2, and GLUT-4 mRNA in adipose tissue were described in Table 1.

Table 1

PCR primers used in all quantitative PCR assays
Primer: gene	Forward	Reverse
Gapdh	5′-CCA AGG TCA TCC ATG ACA AC-3′	5′-TGT CAT ACC AGG AAA TGA GC-3′
Insr	5′-GCT GGT GTT TTC CTC ATA GAT TG-3′	5′-CTG TAG GTC AGG TTT GGT TTT TG-3′
Irs1	5′-CCT GAC ATT GGA GGT GGG TCT T-3′	5′-GAA TCT TCG GCA GTT GCG GTA T-3′
Irs2	5′-TGC GAA CAG CCG TCG GTG AC-3′	5′-GAC CGG TGA CGG CTG AAC GG-3′
Glut4	5′-AGC CAG CCT ACG CCA CCA TA-3′	5′-GGA CCC ATA GCA TCC GCA AC-3′
Pparγ	5′-CTG GCC TCC CTG ATG AAT AA-3′	5′-GGC GGT CTC CAC TGA GAA TA-3′

Western Blotting

Proteins were extracted from skeletal muscle and adipose tissue by using lysis buffer. The proteins were then separated by SDS-PAGE (10% or 15%) and electrophoretically transferred onto polyvinylidene fluoride membranes. The membranes were probed with anti-INSR (1:20000; Abcam, Cambridge, UK), anti-IRS-1 (1:500; Abcam, Cambridge, UK), anti-IRS-2 (1:5000; Abcam, Cambridge, UK), anti-GLUT-4 (1:2000; Abcam, Cambridge, UK), and anti-PPAR-γ (1:5000; Abcam, Cambridge, UK) overnight at 4°C, and then incubated with a HRP-coupled secondary antibody. Detection was performed using a ChemiDoc XRS⁺ (BioRad, Hercules, CA, USA) image analysis system.

Compound And Ir Related Targets

The targets of LUP were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, http://tcmspw.com/tcmsp.- php), Swiss Target Prediction database (http://www.swisstargetprediction.ch/), and BATMAN-TCM (http://bionet.ncpsb.org/batman-tcm/index.php). The genes targets associated with IR were screened by comprehensive database of human genes and gene phenotypes (OMIM, http://www.omim.org/), DurgBank database (https://www.drugbank.ca/), and GeneCards (https://www.genecards.org/).

Construction Of "component-target-disease" Network

The LUP-related targets and IR-related targets were linked into a "component-target-disease" network, and the interaction between proteins was realized by STRING database (http://string-db.org/). The above network was visualized using Cytoscape ver. 3.7.1 software (http://www.cytoscape.org/).

Biological Process Analysis And Pathway Analysis

Gene Ontology (GO) functional annotation and KEGG pathway analysis were performed to explore related biological processes and signaling pathways.

Ppi Network Analysis And Screening Of The Core Targets

Protein protein interaction (PPI) network analysis was performed by STRING database. In "component-target-disease" network, there are three key topology parameters in the network including betweenness centrality, closeness centrality, and degree, which can be used to screen the more important nodes [15]. Thus, the nodes with degree and betweenness centrality value two times higher than the median value of all nodes, and simultaneously satisfy the closeness centrality value greater than median value of nodes were chosen as the core targets.

Molecular Docking

The *mol2 format of LUP was downloaded at the TCMSP database, and the 3D structure (*PDB format) format of the targets were obtained from the PDB protein database (https://www.rcsb.org/). PyMol software was used to perform dehydration and hydrogenation of the proteins, and the docking was performed using AutoDock Vina software, and the docking results were analyzed and visualized.

LUP improves body weight loss and decreases the FBG in T2DM rats

LUP inhibited the body weight loss induced by STZ. The results were shown in Fig. 1a. After 2 weeks of treatment, the LUP and rosiglitazone significantly decreased the FBG level (P < 0.05). After 3 weeks of treatment, LUP (2.0 and 8.0 mg/kg) decreased the FBG level (P < 0.05). After 4 weeks of treatment, LUP (4.0 and 8.0 mg/kg) could significantly decrease the FBG level of rats (P < 0.05). The results showed that LUP has a stable hypoglycemic effects in T2DM rats (Fig. 1b).

LUP decreases the levels of LEP, HbA1c, MDA and increases the ADPN levels and GSH-PX activity in T2DM rats

After treated with LUP for 4 weeks, LUP (8.0 mg/kg) and rosiglitazone significantly reduced the levels of LEP in serum (Fig. 2A). In addition, rosiglitazone and LUP (4.0 and 8.0 mg/kg) significantly reduced the serum HbA1c levels in T2DM rats (Fig. 2C). Rosiglitazone and LUP (8.0 mg/kg) also significantly reduce serum MDA levels and increase the serum GSH-PX activity (Fig. 2D and Fig. 2F). Furthermore, rosiglitazone and LUP (2.0 and 8.0 mg/kg) increased the serum ADPN levels (Fig. 2B). However, LUP had only a slight effect on serum SOD activity (Fig. 2E).

Lup Improves The Lipid Metabolism Disorder In T2dm Rats

Compared to the normal group, the levels of FFA, TG, and TC of the DM group were significantly increased, suggesting that the lipid metabolism disorder occurred in the T2DM rats. LUP (8.0 mg/kg) significantly reduced the levels of FFA, TG, and TC in serum (P < 0.05). In addition, LUP (2.0 and 4.0 mg/kg) significantly decreased the TC levels and LUP (4.0 mg/kg) reduced the levels of TG in serum (Fig. 3a-3c). Furthermore, rosiglitazone and LUP (2.0 mg/kg) significantly reduced the ratio of TC/HDL-C (Fig. 3f). However, LUP had no significant effect on LDL-C and HDL-C levels in serum (Fig. 3d and Fig. 3e).

LUP increases the synthesis of hepatic glycogen in T2DM rats

The liver cell morphology of T2DM group was complete, but the interstitial was hyperplasia, the cell gaps of hepatocytes were large, cells were irregularly arranged, and the intercellular and intracellular PAS staining was low and relative homogeneity was poor. While, in the normal, rosiglitazone, and the LUP (8.0 mg/kg) group, the structure of hepatocytes was tight and neat, with no cell gaps. Furthermore, the intercellular and the intracellular PAS staining were diffusely colored and had good homogeneity. Compared to the T2DM group, in the LUP (2.0 and 4.0 mg/kg) group, the interstitial hyperplasia was less, cells gap was smaller, the cells were arranged regularly, and PAS-positive cells were significantly increased. The rosiglitazone and LUP could increase the synthesis of hepatic glycogen in T2DM rats (Fig. 4A).

LUP improves the pathological changes of the pancreas in T2DM rats

The structure of the exocrine part of pancreatic tissue in rosiglitazone and LUP (2.0, 4.0, and 8.0 mg/kg) group was normal, the endocrine part of pancreatic tissue had a small amount of fatty degeneration and no obvious inflammatory cell infiltration was observed (Fig. 4B).

LUP increases the mRNA and protein expression of InsR, IRS-1, IRS-2, GLUT-4 in skeletal muscle and PPAR-γ, IRS-1, IRS-2, GLUT-4 in adipose of T2DM rats

The mRNA expression of InsR, IRS-1, IRS-2, and GLUT-4 in skeletal muscle and PPAR-γ, IRS-1, IRS-2, and GLUT-4 in adipose of T2DM rats was decreased (Fig. 5a-5h). LUP (4.0 and 8.0 mg/kg) and rosiglitazone significantly increased the expression of InsR, IRS-1, IRS-2, and GLUT-4 mRNA in skeletal muscle (P < 0.05). In addition, LUP(2.0 mg/kg) significantly increased the expression of IRS-1 mRNA in skeletal muscle (P < 0.05). Furthermore, LUP (8.0 mg/kg) and rosiglitazone significantly increased the expression of PPAR-γ, IRS-1, IRS-2, and GLUT-4 mRNA in adipose tissue (P < 0.05). In addition, LUP (2.0 and 4.0 mg/kg) significantly increased the expression of IRS-1 and GLUT-4 mRNA in adipose, and LUP (4.0 mg/kg) could significantly increase the expression of PPAR-γ mRNA in adipose tissue (P < 0.05).

The protein expression of InsR, IRS-1, IRS-2, and GLUT-4 in skeletal muscle and PPAR-γ, IRS-1, IRS-2, and GLUT-4 in adipose of diabetic rats was significantly decreased (Fig. 6A-6H). Rosiglitazone significantly increased the protein expression of IRS-1, IRS-2, and GLUT-4 in skeletal muscle and PPAR-γ, IRS-2, and GLUT-4 in adipose tissue (P < 0.05). LUP (8.0 mg/kg) significantly increased the protein expression of InsR, IRS-1, IRS-2, and GLUT-4 in skeletal muscle and PPAR-γ, IRS-1, IRS-2, and GLUT-4 in adipose tissue (P < 0.05). In addition, LUP (4.0 mg/kg) increased the protein expression of PPAR-γ in adipose tissue (P < 0.05).

Network Analysis

An interactive network of LUP to improve IR was constructed, visualized using different color and shape patterns (Fig. 7). Among them, the red triangle is the chemical component, the pink diamond is disease, and the green circle is the common target genes of the component and the disease.

Go Functional Annotation And Kegg Pathway Analysis

GO functional annotation was performed. 139 potential targets were used to perform the biological functional enrichment using the R packages ggplot2 and cluster Profiler. A total of 2724 biological processes with a total P value of ≤ 0.01 were enriched. Biological process was most strongly correlated with response to steroid hormone, protein kinase B signaling, gland development, etc. (Fig. 8A); In cellular component, there was 110 biological processes with P value ≤ 0.01, including neuronal cell body, dendritic spine, neuron spine, Golgi lumen, etc. (Fig. 8B); In molecular function, there were 150 biological processes with P value ≤ 0.01, among which G protein-coupled receptor binding, histone deacetylase binding, 1-phosphatidylinositol-3-kinase activity were the stronger (Fig. 8C).

A total of 164 pathways were screened using the KEGG enrichment analysis (P ≤ 0.01), and some of the signal pathways are shown in Fig. 9. The LUP might improve IR by acting on Prostate cancer, Endocrine resistance, AGE-RAGE signaling pathway in diabetic complications, Proteoglycans in cancer, Kaposi sarcoma-associated herpesvirus infection, etc. The targets of LUP's direct effect on IR is shown in Fig. 10.

Ppi Network Analysis And The Core Targets Results

The targets were introduced into the STRING database, and the specie was “homo sapiens”, then the interaction network of the protein targets was obtained, as shown in Fig. 11. There was a total of 139 nodes and 1357 edges in the network. In addition, the results of topological analysis in the network showed that the median value of Betweenness Centrality, Closeness Centrality, and Degree were 0.000577, 0.534351, and 18, respectively. According to the screening criteria, 24 hub targets of LUP were obtained. The results are shown in Table 2.

Table 2

Topological analysis of core targets in " lupenone -target-disease" network
Gene name	BetweennessCentrality	ClosenessCentrality	Degree
INS	0.068121	0.756757	95
TP53	0.02875	0.679612	74
TNF	0.023754	0.669856	71
SRC	0.022054	0.660377	68
ESR1	0.013141	0.633484	59
JUN	0.009126	0.625	56
PTGS2	0.008946	0.611354	51
HSP90AA1	0.01071	0.608696	50
CTNNB1	0.009412	0.606061	49
MTOR	0.008296	0.606061	49
AR	0.007686	0.598291	46
MAPK14	0.005711	0.595745	45
IL10	0.006232	0.59322	44
SIRT1	0.005683	0.59322	44
PIK3CA	0.005147	0.588235	42
IL4	0.005161	0.585774	41
KIT	0.004878	0.585774	41
RELA	0.003582	0.585774	41
HIF1A	0.004726	0.583333	40
EDN1	0.004736	0.578512	38
PIK3R1	0.004544	0.578512	38
MDM2	0.004322	0.578512	38
AGT	0.006154	0.576132	37
CRP	0.005963	0.576132	37

Molecular Docking Results

The conformation with the highest score (lowest affinity value) was selected, and the docking conformation was visualized using PyMol software. The results showed that the LUP had good affinity with the proteins corresponding to the genes including INS, TP53, TNF, SRC, ESR1, JUN (Fig. 12). These six genes were the most relevant genes for LUP to improve IR according to the network analysis.

In this study, we found that LUP could inhibit the body weight loss and decreased the FBG levels. Furthermore, LUP improved the lipid metabolic disorders and reduced the excessive oxidative stress in T2DM rats. Moreover, LUP promoted the synthesis of hepatic glycogen and improved the pathological changes of pancreas. LUP could improve the IR via regulation of gene and protein expression in T2DM rats. In addition, network pharmacology analysis revealed that LUP improve IR by multiple targets and signal pathways. These results indicate that LUP has the potential to be developed as a new drug for the treatment of T2DM.

T2DM rats induced by HFD/STZ are often accompanied by weight loss [16, 17], but there are conflicting reports on the changes in body weight of T2DM rats after drug treatment [18]. In this study, LUP could inhibit the body weight loss of T2DM rats. In addition, LUP decreased the LEP level, which is closely related to the appetite, body weight, neuroendocrine functions and glycaemia [19]. The above results suggested that LUP has a good effect on weight maintenance of T2DM rats. As we know, HbA1c was formally included in the diagnostic index of T2DM by the American Diabetes Association (ADA) in 2010. LUP could reduce the FBG and HbA1c levels in T2DM rats, and the result is consistent with our previous work [11]. Furthermore, the IR is closely related to the hepatic glycogen metabolism disorders, LUP could increase the synthesis of hepatic glycogen, so LUP might improve the IR by regulating gluconeogenesis in T2DM rats.

Lipid metabolism disorders are closely related to the cardiovascular diseases and T2DM [20], and TC/HDL-C ratio is a reasonable blood lipid index in predicting the cardiovascular risk of T2DM patients. Our results showed that LUP significantly decrease the levels of TC, TG, and TC/HDL-C ratio, which indicates that LUP can improve the lipid metabolism disorders and may reduce cardiovascular risk in T2DM.

The oxidative stress caused by obesity, hyperglycemia, FFA, and inflammation could lead to the islet β cell damage [21]. As report, long-term exposure to high concentrations of FFA will damage islet β cell function and reduce the secretion of insulin, and then the gluconeogenesis is increased, eventually IR is observed [22]. Moreover, ADPN can increase the muscle and liver sensitivity to insulin by increasing FFA oxidation [23], and ADPN level is an important marker for improvement of IR [24]. LUP could decrease FFA levels and increase ADPN secretion in T2DM rats, indicating that LUP could improve the IR of T2DM rats in this work. Furthermore, levels of SOD, MDA, and GSH-PX are closely related to the islet β cells damage [25, 26]. LUP increased the GSH-PX activity and reduced MDA levels in T2DM rats. Interestingly, LUP also inhibited the mild inflammatory response in pancreas tissue. Taken together, LUP might protect the T2DM via inhibiting the inflammation, and the result is consistent with our previous work [27].

InsR/PI3K/GLUT-4 signaling pathway plays an important role in improving IR in skeletal muscle and adipose tissue [28]. This study found that LUP could increase the gene and protein expression of InsR in skeletal muscle and GLUT-4 in skeletal muscle and adipose tissue. Network pharmacology analysis results showed that LUP can improve IR by acting on PI3K (Fig. 10). These results indicate that LUP may increase the sensitivity of skeletal muscle and adipose to insulin, at least partly, by activating the InsR/PI3K/GLUT-4 signaling pathway. According to the report, the expression of IRS-1 in skeletal muscle and adipose tissue of T2DM patients is significantly decreased [29, 30]. Both IRS-1 and IRS-2 function in peripheral carbohydrate metabolism, and IRS-2 has the major role in β-cell development and compensation for peripheral IR [31]. In present work, LUP up-regulated the gene and protein expression of IRS-1 and IRS-2 in skeletal muscle and adipose tissue, suggesting that LUP may improve the IR via acting on the family of insulin substrates in T2DM. In addition, PPAR-γ was known to play a major role in the improvement of insulin sensitivity through stimulating differentiation of small adipocytes from preadipocyte [32]. Here, LUP might improve the IR by up-regulating the expression of PPAR-γ. All in all, the above results showed that LUP improves the skeletal muscle and adipose tissue sensitivity to insulin through increasing the gene and protein transcription related to IR in T2DM.

Combining the drug-target network with the biological system network can provide new approaches and strategies for new drug development [33]. Network pharmacological studies have found that LUP improve IR through multiple targets and multiple pathways. The biological process is mainly related to the regulation of cell proliferation, signal transduction, metabolism, cell differentiation, etc. the result suggested that LUP could improve IR by regulating multiple biological processes. KEGG pathway analysis also showed that LUP improve IR involving multiple signaling pathways, like AGE-RAGE signaling pathway in diabetic complications, IR, Type II diabetes mellitus. Advanced glycation end products (AGEs) play a significant role in the pathogenesis of multiple diabetic complications [34]. These results indicated that LUP might be used for the prevention and treatment of T2DM and diabetic complications. Of course, many signaling pathways related to cancer, tumor, atherosclerosis, and infection were predicted, indicating that LUP may have various pharmacological activities, which deserves further research.

LUP could improve the lipid metabolism disorder and alleviate the excessive oxidative stress in T2DM. Furthermore, as expected, LUP improved the IR via increasing the sensitivity of skeletal muscle and adipose tissue to insulin by regulating related gene and protein expression in T2DM. Our preliminary results indicate that LUP has the potential to be developed as a new drug for the treatment of T2DM.

ADPN: Adiponectin; FBG: Fasting blood glucose; FFA: Free fatty acid; GLUT-4: Glucose transporter type 4; GO: Gene Ontology; GSH-PX: Glutathione peroxidase; HbA1c: Hemoglobin A1c; HDL-C: High-density lipoprotein-cholesterol; HE: Hematoxylin and eosin; HFD: High-fat diet; InsR: Insulin receptor; IRS: Insulin receptor substrate; LDL-C: Low-density lipoprotein-cholesterol; LEP: Leptin; LUP: Lupenone; MDA: Malondialdehyde; OMIM: Comprehensive database of human genes and gene phenotypes; PPAR: Proliferator-activated receptor; PPI: Protein protein interaction; PTP1B: Protein tyrosine phosphatase 1B; SOD: Superoxide dismutase; STZ: Streptozotocin; TC: Total cholesterol; TCMSP: Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform; TG: Triglyceride; T2DM: Type 2 diabetes

Acknowledgements

The authors would like to thank Zhendong Zhong from the Chengdu University of TCM, Chengdu, China, for technical support.

Authors' contributions

Xiangpei Wang, Feng Xu, and Hongmei Wu contributed to the study conception and design, acquisition, analysis and interpretation of data, and writing of the article. Yuanmin Wang and Ye Yang contributed to the acquisition and interpretation of data and also to the final critical revision of the article. All authors read and approved the manuscript.

Funding

This work was supported by NSFC (81260686), the Special Funds of Outstanding Young Scientists in Guizhou, China (2013, No.46), Guizhou Univerisity of Traditional Chinese Medicine Doctor Startup Fund (2017), Research project of Chinese medicine and ethnic medicine science and technology of Guizhou Provincial Administration of Traditional Chinese Medicine (No. QZYY-2019-004), Academy of Guizhou University of Traditional Chinese Medicine ([2019] No. 15), the Scholarship Fund from the China Scholarship Council (No. 201908525058), and Guizhou Province General Colleges and Universities Youth Science and Technology Talent Growth Project (Qianjiao He KY Zi [2021] 209).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request

Ethics approval and consent to participate

All experiments in the present study were approved by the were approved by Guizhou University of Traditional Chinese Medicine and the Ethics Committee for Animal Experiments of West China Hospital of Sichuan University (under registration: 2013029A).

Consent for publication

The manuscript is approved by all authors for publication.

Competing interests

The authors declare that they have no competing interests.

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published 28 May, 2022

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Study on the Mechanism of Lupenone for Treating type 2 Diabetes by Integrating Pharmacological Evaluation and Network Pharmacology

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Journal Publication

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Abstract

Figures

Background

Materials And Methods

Reagents

Animals

Induction Of Type 2 Diabetic Rats And Treatment

Determination Of Body Weight And Fbg

Biochemical Parameter Analysis

Periodic acid-schiff (PAS) staining

Histological Analysis

Quantitative Real-time Pcr

Western Blotting

Compound And Ir Related Targets

Construction Of "component-target-disease" Network

Biological Process Analysis And Pathway Analysis

Ppi Network Analysis And Screening Of The Core Targets

Molecular Docking

Results

LUP improves body weight loss and decreases the FBG in T2DM rats

Lup Improves The Lipid Metabolism Disorder In T2dm Rats

Network Analysis

Go Functional Annotation And Kegg Pathway Analysis

Ppi Network Analysis And The Core Targets Results

Molecular Docking Results

Discussion

Conclusion

Abbreviations

Declarations

References

Status:

Journal Publication

Version 1