The Antidiabetic Activities of Neocryptotanshinone: Screened by Molecular Docking and Related to the Modulation of PTP1B

The aim of this study was to provide a practical experimental basis for the development of Neocryptotanshinone (NCTS) as an effective hypoglycemic drug and a theoretical method for the rapid screening of natural compounds with hypoglycemic effects. Molecular docking was used to screen the most suitable ligand. Hematoxylin and eosin, immunohistochemical staining, enzyme-linked immunosorbent assay and Western Blotting approved the hypoglycemic effect of NCTS. According to the free energy of binding, among 180 active compounds from the Traditional Chinese Medicine Integrated Database, NCTS was finally chose for investigation its hypoglycemic effects. In db/db mice, NCTS significantly reduced body weight and plasma glucose, improved glucose tolerance and levels of fasting plasma glucose and glycated hemoglobin A1c, and decreased insulin resistance after six-week administration. NCTS restored the pathological state in the liver of db/db mice and significantly decreased protein tyrosine phosphatase 1B (PTP1B) expression in the liver and muscle of db/db mice, which is related to the regulatory effect of NCTS on insulin receptor substrate 1. In conclusion, we successfully explored the hypoglycemic effect of NCTS in db/db mice via regulating the expression of PTP1B.


Introduction
Diabetes mellitus (DM), which is characterized by metabolic disturbances, affected more than 220 million people worldwide in 2011, and this number will double by 2030 [1]. Type II diabetes mellitus (T2DM) is the most common type of DM, accounting for approximately 95% of all diabetes cases [2]. T2DM is mainly caused by genetic and environmental factors, and/or their interaction, which leads to the dysfunction of skeletal muscle, liver, and adipose tissues to insulin and pancreatic β cells; for example, tissues fail to respond normally to insulin, and pancreatic β cells fail to produce enough insulin to compensate [3][4][5]. In T2DM, insulin resistance (IR) leads to the development of hyperglycemia, which causes oxidative stress and the activation of inflammatory pathways, ultimately increasing the morbidity and mortality of T2DM patients [3]. Protein tyrosine phosphatase 1B (PTP1B), encoded by the protein tyrosine phosphatase non-receptor type 1 (PTPN1) gene, terminates insulin signaling by dephosphorylating the insulin receptor and insulin receptor substrate (IRS) [6][7][8]. PTP1B is closely related to IR. PTPN1-deficient mice are more responsive to insulin and leptin [9,10], have lower blood glucose levels, and have more normal insulin levels [6]. Aging leads to increased levels of PTP1B in various tissues [11] and dephosphorylation of IRS1 in the liver, which inhibits insulin signaling [12]. PTP1B-deficient mice are more sensitive to insulin. In the livers of diabetic mice that were intraperitoneally treated with PTP1B antisense oligonucleotides, PTP1B and blood glucose levels were decreased [13]. In the liver of obese mice, short-term strength exercise improved insulin sensitivity by reducing PTP1B levels [14]. Biotechnology Co., Ltd., Beijing, China) and 0.6% Tween 80 (CAS NO.: 9005-65-6, T104866, Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai, China).
Animal experiments were approved by the Animal Ethics Committee of Jilin University (SY202105006). The db/db mice (male, 10-11 weeks old) and their littermates with genotype BKS.Cg-Dock7 m +/Dock7 m +/JNju (m/m) used as normal controls (male, 10-11 weeks old) were purchased from Changzhou Cavens Laboratory Animal Co., Ltd.
[Changzhou, Jiangsu, China, SCXK (SU) 2018-0002] and were housed in a controlled room with a stable and continuous temperature (23 ± 1 • C) and humidity (40-70%) under a 12-h light/dark cycle without convective wind. Mice were fed a normal chow diet (maintenance feed of SPF grade; Liaoning Changsheng Biotechnology Co., Ltd., Liaoning, China) for six weeks.
After one week of adaptive feeding, 18 db/db mice were randomly divided into three groups (n = 6 per group) and orally treated with 10 mg/kg of NCTS (NCTS-treated db/db mice), 100 mg/kg of Met (Met-treated db/db mice), and 3.33 mL/kg of normal saline (containing 0.1% DMSO and 0.6% Tween-80) (vehicle-treated db/db mice) once a day for another six weeks. Another six m/m mice were orally treated with 3.33 mL/kg of normal saline (containing 0.1% DMSO and 0.6% Tween-80) (serving as vehicle-treated m/m mice) once a day for six weeks. Body weights and plasma glucose levels were monitored weekly. At the end of the experiment, all mice were euthanized by anesthesia with carbon dioxide. Serum samples were collected from the tail vein, and organs including the heart, liver, spleen, lung, kidney, skeletal muscle, and pancreas were collected for visceral index calculations and biochemical and pathological analyses. The percentage of the organ weight to the body weight was defined as the organ indices.

Oral Glucose Tolerance Test (OGTT)
After the latter agent's administration, all mice were fasted for 12 h and then an OGTT was carried out. A glucose solution (2.0 g/kg) (B21882, Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China) was rapidly orally administered to all mice after measuring the fasting plasma glucose values. Blood samples were collected from the tail vein of mice at 0, 30, 60, 120, and 240 min after glucose administration. The plasma glucose content was measured using a detection kit (ADS-W-TDX001, Jiangsu Feiya Biotechnology Co., Ltd., Jiangsu, China) using the glucose oxidase peroxidase (GOPOD) oxidase method.

Food and Water Intake Measurement
Food and water intake can reflect the metabolic status of mice. The food and water intake of all mice at 24 h was measured using metabolic cages after the last drug administration.

Hematoxylin and Eosin (H&E) Staining
The liver was fixed in 4% paraformaldehyde (BL539A, Biosharp, Guangzhou, China), and the muscle was fixed in muscle tissue fixative (G1111, Servicebio, Wuhan, China). The fixed tissues were dehydrated via continuous immersion in 30%, 50%, 70%, 80%, 95%, and 100% ethanol. The dehydrated tissues were then immersed in xylene, embedded in wax, and cut into 5 µm sections. Paraffin sections were deparaffinized in xylene, anhydrous ethanol, and 75% alcohol following the initial preparation and stained with H&E. The histopathological state of the liver and muscle was observed after another dehydration process using an upright optical microscope (Nikon Eclipse E100; Nikon, Japan).

Immunohistochemical Examination
Consistent with the H&E staining protocol, we recovered the antigen and blocked the endogenous peroxidase activity in the paraffin sections of the liver and muscle. Then, 3% bovine serum albumin (BSA) was added to evenly cover the tissues, and they were sealed for 30 min at room temperature. The tissues were incubated with PTP1B (A1590; ABclonal Technology Co., Ltd., Wuhan, China) at 4 • C overnight. Subsequently, the tissues were incubated with peroxidase/HRP-conjugated goat anti-rabbit IgG (H+L) (E-AB-1003; Elabscience Biotechnology Co., Ltd., Wuhan, China) at room temperature for 50 min. Paraffin sections were reacted with diaminobenzidine (DAB) chromogenic reagent (DA1016; Solarbio, Beijing, China). After terminating the reaction, hematoxylin was used to counterstain the nuclei. The sections were dehydrated and sealed for observation under a microscope and a quantitative analysis was performed to determine the positive state of PTP1B in the tissues.

Statistical Analysis
All values are expressed as the mean ± Standard Deviation (S.D.). One-way analysis of variance (ANOVA) followed by the post-hoc multiple comparisons (Tukey's) test was used to determine differences using SPSS Statistics 26 (BONC, Beijing, China). Statistical significance was defined as a p value less than 0.05. AutoDock Vina software was used for docking. Protein-ligand interactions were shown by PyMOL.

High-Throughput Virtual Screening and Molecular Docking
The affinity scores of 180 ligands for PTP1B were ranked, and the top 50 ligands were selected. The lower of the free energy of binding obtained by docking is, the more suitable the binding of the inhibitor to PTP1B is. PTP1B contains both active and allosteric sites. To obtain suitable binding sites for the complexes, we tried to dock the top six ligands in terms of docking score to each site of the receptor protein ( Figure 1A-D). The binding free energy values were shown in Table 1, and the docking positions were shown in Figure 1. The results showed that the lowest binding free energy values were obtained when the six ligands were docked to the site-1 site, and among the six compounds, the NCTS had the best effect. Therefore, NCTS can be regarded as a competitive inhibitor located at the active site, and its detailed interaction with the active site is shown in Figure 1E.
of variance (ANOVA) followed by the post-hoc multiple comparisons (Tukey's) test was used to determine differences using SPSS Statistics 26 (BONC, Beijing, China). Statistical significance was defined as a p value less than 0.05. AutoDock Vina software was used for docking. Protein-ligand interactions were shown by PyMOL.

High-Throughput Virtual Screening and Molecular Docking
The affinity scores of 180 ligands for PTP1B were ranked, and the top 50 ligands were selected. The lower of the free energy of binding obtained by docking is, the more suitable the binding of the inhibitor to PTP1B is. PTP1B contains both active and allosteric sites. To obtain suitable binding sites for the complexes, we tried to dock the top six ligands in terms of docking score to each site of the receptor protein ( Figure 1A-D). The binding free energy values were shown in Table 1, and the docking positions were shown in Figure 1. The results showed that the lowest binding free energy values were obtained when the six ligands were docked to the site-1 site, and among the six compounds, the NCTS had the best effect. Therefore, NCTS can be regarded as a competitive inhibitor located at the active site, and its detailed interaction with the active site is shown in Figure 1E.

Effect of NCTS on Hypoglycemia in db/db Mice
Compared with vehicle-treated db/db mice, both NCTS and Met strongly suppressed body weight (p < 0.01, Figure 2A) without influencing food intake ( Figure 2B). Both NCTS and Met reduced the water intake of db/db mice (p < 0.001, Figure 2C), suggesting an improvement in diabetic symptoms. NCTS enhanced the organ indices, including those for the pancreas, heart, kidney, and lung (p < 0.001, Table 2), decreased the organ indices of liver (p < 0.01, Table 2), but not for the spleen; meanwhile, Met enhanced the organ indices, including those of the pancreas (p < 0.001), heart (p < 0.001), and lung (p < 0.01) in db/db mice, and decreased the organ indices of liver (p < 0.01) ( Table 2).

Effect of NCTS on Hypoglycemia in db/db Mice
Compared with vehicle-treated db/db mice, both NCTS and Met strongly suppressed body weight (p < 0.01, Figure 2A) without influencing food intake ( Figure 2B). Both NCTS and Met reduced the water intake of db/db mice (p < 0.001, Figure 2C), suggesting an improvement in diabetic symptoms. NCTS enhanced the organ indices, including those for the pancreas, heart, kidney, and lung (p < 0.001, Table 2), decreased the organ indices of liver (p < 0.01, Table 2), but not for the spleen; meanwhile, Met enhanced the organ indices, including those of the pancreas (p < 0.001), heart (p < 0.001), and lung (p < 0.01) in db/db mice, and decreased the organ indices of liver (p < 0.01) ( Table 2).  The data were analyzed using a one-way ANOVA and expressed as means ± S.D. (n = 6). ### p < 0.001 vs. vehicle-treated m/m mice; ** p < 0.01 and *** p < 0.001 vs. vehicle-treated db/db mice.  The data were analyzed using a one-way ANOVA and expressed as means ± S.D. (n = 6). ### p < 0.001 vs. vehicle-treated m/m mice; ** p < 0.01 and *** p < 0.001 vs. vehicle-treated db/db mice.
The protective effect of NCTS in the liver was confirmed by H&E staining. In the livers of db/db mice, a large number of hepatocytes with balloon-like degeneration, centered nuclei, vacuolated cytoplasm, a small number of hepatocytes with steatosis, round vacuoles of different sizes in the cytoplasm, and a small number of hepatocytes with intranuclear inclusion bodies were noted. All of these pathological changes improved after NCTS and Met treatment (Figure 2D), and the relative size of lipid droplets were significantly reduced with the action of NCTS or Met (p < 0.05, Figure S1A). Compared with vehicle-treated db/db mice, the low muscle fiber cross-sectional areas of skeletal muscle were enhanced in both NCTS and Met-treated mice (p < 0.001, Figure 2D and Figure S1B).
Compared to vehicle-treated db/db mice, both NCTS and Met suppressed the enhancement of plasma glucose after the three-week treatment and continued until the end of the experiment (p < 0.01, Figure 3A). Following a 4-h administration of 2 g/kg of glucose, the plasma glucose levels of db/db mice were stable and significantly higher than those of m/m mice (p < 0.001, Figure 3B). Both NCTS and Met decreased plasma glucose levels from 30 min after glucose administration to 4 h (p < 0.05, Figure 3B). The AUC was significantly reduced in both Met-and NCTS-treated mice compared with that in db/db mice (p < 0.01, Figure 3C). Compared with vehicle-treated db/db mice, NCTS and Met significantly suppressed the serum levels of fasting plasma glucose (FPG) (p < 0.05, Figure 3D) and GHbA1c (p < 0.05, Figure 3E), confirming the effect of NCTS on hyperglycemia.
tered nuclei, vacuolated cytoplasm, a small number of hepatocytes with steatosis, rou vacuoles of different sizes in the cytoplasm, and a small number of hepatocytes with i tranuclear inclusion bodies were noted. All of these pathological changes improved aft NCTS and Met treatment ( Figure 2D), and the relative size of lipid droplets were signi cantly reduced with the action of NCTS or Met (p < 0.05, Figure S1A). Compared w vehicle-treated db/db mice, the low muscle fiber cross-sectional areas of skeletal musc were enhanced in both NCTS and Met-treated mice (p < 0.001, Figures 2D and S1B).
Compared to vehicle-treated db/db mice, both NCTS and Met suppressed the e hancement of plasma glucose after the three-week treatment and continued until the e of the experiment (p < 0.01, Figure 3A). Following a 4-h administration of 2 g/kg of gluco the plasma glucose levels of db/db mice were stable and significantly higher than those m/m mice (p < 0.001, Figure 3B). Both NCTS and Met decreased plasma glucose lev from 30 min after glucose administration to 4 h (p < 0.05, Figure 3B). The AUC was sign icantly reduced in both Met-and NCTS-treated mice compared with that in db/db mi (p < 0.01, Figure 3C). Compared with vehicle-treated db/db mice, NCTS and Met signi cantly suppressed the serum levels of fasting plasma glucose (FPG) (p < 0.05, Figure 3 and GHbA1c (p < 0.05, Figure 3E), confirming the effect of NCTS on hyperglycemia.

NCTS-Modulated IR Related to PTP1B in db/db Mice
NCTS and Met administration strongly enhanced the serum levels of insulin (p 0.001, Figure 4A) and significantly decreased the value of HOMA-IR (p < 0.05, Figure 4 in db/db mice, thereby indicating the anti-IR effects of NCTS. In both the liver and muscle of db/db mice, NCTS and Met strongly suppressed t expression of PTP1B (p < 0.05, Figure 4C). Compared with vehicle-treated db/db mi NCTS suppressed the serum levels of (D) fasting plasma glucose (FPG) and (E) glycated hemoglobin A1c (GHbA1c). Data are presented as the mean ± S.D. (n = 6) and analyzed via a one-way ANOVA test followed by post-hoc Tukey's multiple comparison tests. ### p < 0.001 vs. vehicle-treated m/m mice; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. vehicle-treated db/db mice.

NCTS-Modulated IR Related to PTP1B in db/db Mice
NCTS and Met administration strongly enhanced the serum levels of insulin (p < 0.001, Figure 4A) and significantly decreased the value of HOMA-IR (p < 0.05, Figure 4B) in db/db mice, thereby indicating the anti-IR effects of NCTS. NCTS enhanced the expression of P-IRS1 (p < 0.001) and P-AKT (p < 0.01) and reduced t expression levels of PTP1B (p < 0.01) in the liver ( Figure 4D) and muscle ( Figure 4E). Si ilar results were observed in the Met-treated db/db mice. . Quantification data were normalized α/β-actin or the corresponding total protein concentration, and expressed as the percentage of m mice. Data are presented as the mean ± S.D. and analyzed via a one-way ANOVA test followed post-hoc Tukey's multiple comparison tests. # p < 0.05, ## p < 0.01 and ### p < 0.001 vs. vehicle-treat m/m mice; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. vehicle-treated db/db mice.

Discussion
Natural compounds are regarded as a source of great potential in the field of dr development. The natural compounds improving IR derived from plants mainly inclu three categories: flavonoids, terpenes, and phenylpropanoids [26]. In this study, 180 l ands from the TCMID database were docked onto PTP1B. The top six ligands docked both the active and allosteric sites. Among the six compounds, NCTS had the best effe therefore, it can be regarded as a competitive inhibitor that is located at the active site. db/db mice, the hypoglycemic activity of NCTS was analyzed by changes in body weigh food and water intake, OGTT, and the levels of GHbA1c. Weight loss may reduce t degree of IR, which may be mediated by reducing the size of fat cells, and the same resu were noted in NCTS-treated db/db mice. Muscle loss is associated with the degradati of muscle proteins that occurs in diabetes [27], which is accompanied by the occurren of IR. NCTS and Met significantly increased the cross-sectional area of muscle fibers the skeletal muscle of db/db mice. Therefore, the anti-IR effects of NCTS were analyz based on the levels of insulin and the expression of PTP1B and its related proteins.
A typical symptom of diabetes in mice is a change in drinking and urinating pattern The six-week administration of NCTS significantly reduced the water intake of db/ . Quantification data were normalized to α/β-actin or the corresponding total protein concentration, and expressed as the percentage of m/m mice. Data are presented as the mean ± S.D. and analyzed via a one-way ANOVA test followed by post-hoc Tukey's multiple comparison tests. # p < 0.05, ## p < 0.01 and ### p < 0.001 vs. vehicle-treated m/m mice; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. vehicle-treated db/db mice.
In both the liver and muscle of db/db mice, NCTS and Met strongly suppressed the expression of PTP1B (p < 0.05, Figure 4C). Compared with vehicle-treated db/db mice, NCTS enhanced the expression of P-IRS1 (p < 0.001) and P-AKT (p < 0.01) and reduced the expression levels of PTP1B (p < 0.01) in the liver ( Figure 4D) and muscle ( Figure 4E). Similar results were observed in the Met-treated db/db mice.

Discussion
Natural compounds are regarded as a source of great potential in the field of drug development. The natural compounds improving IR derived from plants mainly include three categories: flavonoids, terpenes, and phenylpropanoids [26]. In this study, 180 ligands from the TCMID database were docked onto PTP1B. The top six ligands docked to both the active and allosteric sites. Among the six compounds, NCTS had the best effect; therefore, it can be regarded as a competitive inhibitor that is located at the active site. In db/db mice, the hypoglycemic activity of NCTS was analyzed by changes in body weights, food and water intake, OGTT, and the levels of GHbA1c. Weight loss may reduce the degree of IR, which may be mediated by reducing the size of fat cells, and the same results were noted in NCTS-treated db/db mice. Muscle loss is associated with the degradation of muscle proteins that occurs in diabetes [27], which is accompanied by the occurrence of IR. NCTS and Met significantly increased the cross-sectional area of muscle fibers in the skeletal muscle of db/db mice. Therefore, the anti-IR effects of NCTS were analyzed based on the levels of insulin and the expression of PTP1B and its related proteins.
A typical symptom of diabetes in mice is a change in drinking and urinating patterns. The six-week administration of NCTS significantly reduced the water intake of db/db mice, suggesting an improvement in hyperglycemia symptoms. The OGTT is an important criterion for diagnosing diabetes. NCTS significantly suppressed plasma glucose levels in the OGTT, suggesting an association between NCTS and glucose tolerance. Glucose tolerance can be enhanced by reducing IR [28], and the knockout of PTP1B helps to improve high-fat, diet-induced poor glucose tolerance [29]. FPG can be used to evaluate the plasma glucose regulation process, and elevated FPG levels indicate the failure of glycemic regulation [30]. GHbA1c is the product of a non-enzymatic reaction of plasma glucose and hemoglobin in the body [31]. Since this process is irreversible, GHbA1c can objectively reflect the plasma glucose concentration in mice. NCTS showed hypoglycemic activity related to its anti-IR. Insulin, which is secreted by pancreatic β-cells, plays a crucial role in the occurrence and development of diabetes. Insufficient insulin secreted by pancreatic β cells cannot compensate for IR, which easily leads to the occurrence of T2DM [32]. IR can be directly assessed by HOMA-IR values [33,34], and a high HOMA-IR value can reflect a lower insulin sensitivity or a high level of IR [35]. NCTS remarkably enhanced the serum levels of insulin, consequently reducing the value of HOMA-IR in db/db mice and confirming its anti-IR activity.
PTP1B is a specific non-receptor protein tyrosine phosphatase (PTPase) with a unique structure that can promote the dephosphorylation of related proteins such as insulin receptor and IRS during insulin action, thus acting as a negative regulator of insulin receptor signaling [36]. PTP1B inhibitors can effectively reduce the value of HOMA-IR, ultimately improving glucose intolerance and hyperglycemia [37]. NCTS reduced the expression levels of PTP1B in the liver and muscle of db/db mice.
Insulin plays a central role in the maintenance of glucose homeostasis. Insulin receptors are phosphorylated and activated after binding to insulin. IRS1 acts upstream of AKT [38], and the phosphorylation of IRS1 subsequently activates phosphatidylinositol 3-kinase (PI3K)/AKT [39], thereby activating the insulin signaling pathway, enhancing glucose uptake, lowering plasma glucose levels, and increasing glycogen and protein synthesis [40]. The phosphorylation level of IRS1 can be increased by inhibiting the expression of PTP1B [41]. In the presence of inhibition of PTP1B expression, the phosphorylation of IRS1 increases, leading to the interaction of IRS1 with the p85 regulatory subunit of PI3K and resulting in the subsequent activation of AKT [40].
At present, the research on NCTS is still in the preliminary stage. NCTS can achieve antiinflammatory effects by inhibiting NF-κB and iNOS signaling pathways in lipopolysaccharideinduced RAW264.7. Our study proves the hypoglycemic efficacy of NCTS. Comparatively, chronic and high-dose administration with Met may show side effects on the gastrointestinal tract [42], and its high hydrophilicity leads to a reduced degree of metabolic absorption and insufficient bioavailability [43,44]. However, the security of NCTS needs further investigation. On the other hand, IR is reported to be related to the dysfunction of the transport of glucose transporter 4 (GLUT4) [26]. However, based on our present study, the effects of NCTS on GLUT4 failed to be detected, which still needs further investigation. Furthermore, the roles of PTP1B and IRS1 in NCTS-mediated hypoglycemic effects should be further confirmed in in vitro and in vivo experiments.

Conclusions
In conclusion, we successfully explored the hypoglycemic effect of NCTS in db/db mice related to the expression of PTP1B. This study provides a theoretical method for the rapid screening of natural compounds with hypoglycemic effects. This research is bound to play a beneficial role in the establishment of natural product libraries with improved insulin resistance and the screening of clinical drugs for human T2DM.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nu14153031/s1, Figure S1: (A) The relative size of lipid droplets and (B) quantification of the muscle fiber cross-sectional areas, according to representative H&E staining results in Figure 2D (n = 3). Data are presented as the mean ± S.D. and analyzed via a one-way ANOVA test followed by post-hoc Tukey's multiple comparison tests. ### p < 0.001 vs. vehicle-treated m/m mice; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. vehicle-treated db/db mice.  Institutional Review Board Statement: All animal experimental procedures in this study were approved by the Experimental Animal Center of Jilin University (NO. SY202105006) and ARRIVE guidelines.