The improvement of modified Si-Miao granule on hepatic insulin resistance and glycogen synthesis in type 2 diabetes mellitus involves the inhibition of TNF-α/JNK1/IRS-2 pathway: network pharmacology, molecular docking, and experimental validation

Abstract

Background: Modified Si-Miao granule (mSMG), a traditional Chinese medicine, is beneficial for T2DM and insulin resistance (IR), but the underlying mechanism remains unknown.

Methods: Using network pharmacology, we screened the compounds of mSMG and identified its targets and pathway on hepatic IR in T2DM. Using molecular docking, we identified the affinity between the compounds and hub target TNF-α. Then these were verified in KK-Ay mice and HepG2 cells.

Results: 50 compounds and 170 targets of mSMG against IR in T2DM were screened, and 9 hub targets such as TNF and MAPK8 were identified. 170 targets were mainly enriched in insulin resistance and TNF pathway, so we speculated that mSMG might act on TNF-α, JNK1 and then regulate insulin signaling to mitigate IR. Experimental validation proved that mSMG ameliorated hyperglycemia, IR, and TNF-α, enhanced glucose consumption and glycogen synthesis, relieved the phosphorylation of JNK1 and IRS-2 (Ser³⁸⁸), and elevated the phosphorylation of Akt (Ser⁴⁷³) and GSK-3β (Ser⁹) and GLUT2 expression in KK-Ay mice. Molecular docking further showed berberine from mSMG had excellent binding capacity with TNF-α. Then, in vitro validation experiments, we found that 20% mSMG-MS or 50 μM berberine had little effect in IR-HepG2 cell viability, but significantly increased glucose consumption and glycogen synthesis and regulated TNF-α/JNK1/IRS-2 pathway.

Conclusion: Network pharmacology and molecular docking help us predict potential mechanism of mSMG and further guide experimental validation. mSMG and its representative compound berberine improve hepatic IR and glycogen synthesis, and its mechanism may be related to the inhibition of TNF-α/JNK1/IRS-2 pathway.

Keywords: Berberine; Insulin resistance; Modified Si-Miao granule; Molecular docking; Network pharmacology; TNF-α/JNK1/IRS-2 pathway; Type 2 diabetes mellitus.

Fig. 1

Workflow of the present study. We first used network pharmacology analysis to predict the compounds, action targets, and important signaling pathways of mSMG on reduction of IR in T2DM, and then combined with animal and cell experiments to verify the mechanism

Fig. 2

Potential action targets, compound-target network, PPI network, and hub targets of mSMG against IR in T2DM. A 170 intersecting targets between IR-associated genes in T2DM and candidate targets of mSMG were considered as the potential action targets of mSMG against IR in T2DM. B Compound-target network. A chartreuse ellipse represents a compound and a red “V” shape represents a target. C PPI network. A node represents a target. The size and the color of the node represents the value of the degree. Blue → yellow → red indicates that the degree value is from low to high, and the bigger the circle, the higher the degree value. D The intersecting 9 targets of the top 15 targets ranked by the 3 topological algorithms (Degree, Closeness, and Betweenness) were identified as the hub targets of mSMG against IR in T2DM. E PPI network of the 9 hub targets. The color of the node represents the value of the degree. Yellow → orange → red indicates that the degree value is from low to high

Fig. 3

GO term and KEGG Pathway enrichment of the potential action targets of mSMG against IR in T2DM. A GO terms, including biological process (BP), cellular component (CC), and molecular function (MF). B KEGG Pathways. C KEGG Pathway map of Insulin resistance in liver cell in T2DM. The arrow (→) indicates promotion, and the T-arrows (⊣) indicates inhibition. The potential action targets of mSMG against IR in T2DM were marked as green color, and the raw map can be viewed at https://www.kegg.jp/kegg/mapper/

Fig. 4

30 representative compounds of mSMG were identified based on UPLC-Q-Orbitrap-MS analysis. A Representative ion chromatogram of mSMG in the negative mode. B Representative ion chromatogram of mSMG in the positive mode

Fig. 5

mSMG ameliorated hyperglycemia, hyperlipidemia, IR and serum TNF-α in KK-Ay mice. A Body weight. B Fasting blood glucose. C 2-h blood glucose of OGTT. D The area under the curve of OGTT. E HbA1c. F HOMA-IR. G Serum total cholesterol. H Serum triglyceride. I Serum low density lipoprotein cholesterol. J Serum tumor necrosis factor-α. K Alanine aminotransferase. L Aaspartate transaminase. M Serum creatinine. N Blood urea nitrogen. Data are expressed as mean ± SD (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 6

mSMG alleviated IR, promoted glycogen synthesis, and inhibited TNF-α/JNK1/IRS-2 pathway in liver of KK-Ay mice. A Representative H&E and Oil Red O staining images of liver sections (magnification 200×). B NAFLD activity score. C Positive area percentage of Oil Red O staining images. D Glycogen content in liver. E Glycogen synthase activity. F–L mSMG reduced the expression of TNF-α protein and the phosphorylation of JNK1 and IRS-2 (Ser³⁸⁸), and promoted the phosphorylation of Akt (Ser⁴⁷³) and GSK-3β (Ser⁹) as well as the expression of GLUT2 protein in liver. Data are expressed as mean ± SD (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 7

Molecular docking between the hub target TNF-α and the top 6 compounds from mSMG ranked by affinity. Molecular docking between TNF-α and A coptisine, B luteolin, C quercetin, D berberine, E palmatine, and F chlorogenic acid, respectively. The label represents the amino acid compositions around the ligand, the yellow line represents the hydrogen bond, and the number next to the yellow line represents the length of the hydrogen bond

Fig. 8

mSMG-MS or berberine alleviated IR and promoted glycogen synthesis in IR-HepG2 cells, which may be related to the inhibition of TNF-α/JNK1/IRS-2 pathway. A Cell viability after treatment with different concentrations of mSMG-MS (0–40%) or berberine (0–250 μM) in IR-HepG2 cells for 4 h. B Glucose consumption of HepG2 cells after treatment with 5 μM insulin and 30 ng/mL TNF-α (IR), or 5 μM insulin and 30 ng/mL TNF-α added with 0.5 mM metformin or mSMG-MS (2.5–20%), or berberine (5–50 μM) for 4 h. 20% mSMG-MS or 50 μM berberine (C–E) improved glucose consumption, glycogen content, and glycogen synthase activity, F–H decreased the phosphorylation of JNK1 and IRS-2 (Ser³⁸⁸), and F, I–K increased phosphorylation of Akt (Ser⁴⁷³) and GSK-3β (Ser⁹) as well as the expression of GLUT2 protein in TNF-α-induced IR HepG2 cells. HepG2 cells were incubated for 4 h in medium containing blank (Control group), or 5 μM insulin and 30 ng/mL TNF-α (IR group), or 5 μM insulin and 30 ng/mL TNF-α added with 20%NS (NS group) or 20%MS (MS group) or 50 μM berberine (BBR group). Data are expressed as mean ± SD (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 9

The biological mechanism of mSMG against hepatic IR in T2DM may involve the inhibition of TNF-α/JNK1/IRS-2 pathway. Elevated TNF-α promotes JNK1 phosphorylation, which phosphorylates serine sites of IRS-2 and then inactivate IRS-2, followed by the suppression of PI3K/Akt pathway. Subsequently, reduced Akt phosphorylation downregulates the GLUT2 levels and the phosphorylation of GSK-3β (Ser⁹), which activates GSK-3β and then leads to inhibition of GS activity, resulting in impaired glucose uptake and glycogen synthesis, known as hepatic IR. mSMG and its representative compound berberine may reverse this process by inhibiting TNF-α/JNK1/IRS-2 pathway