Effects of the Tangningtongluo formula as an alternative strategy for diabetics via upregulation of insulin receptor substrate-1

Abstract

Tangningtongluo (TNTL), a traditional Chinese medicine, has been widely used in clinics for decades in southwest China. Its pharmacological properties and underlying molecular mechanisms remain unclear. The main goal of ethnopharmacology is to identify novel bioactive compounds derived from plants for use in indigenous medical practice. This knowledge can be used to develop novel pharmaceuticals. In the present study, hyperglycemic C57BL/KsJ‑db/db (db/db) mice were used to test the effect of TNTL on microvasculature of the retina and hypoglycemia. Metformin (Met) was selected as a positive control. 26‑week‑old mice were randomly assigned to receive either the anti‑diabetic agent Met [140 mg/kg body weight (BW)], 1.8, 0.9 or 0.45 g/kg BW TNTL, or a placebo. The fasting blood glucose, serum insulin and glycated hemoglobin levels were measured. Histopathologic examination of the pancreas was performed to confirm the hypoglycemic effect. Fluorescein angiography was applied to detect diabetes‑induced retinal angioma in the db/db mice. TNTL intake significantly decreased the fasting blood glucose level in a dose‑dependent manner. Additionally, TNTL intervention resulted in a significant decrease in the insulin resistance index. Notably, TNTL treatment markedly reduced the speed of retinal degeneration and mildly reversed microvascular caliber degeneration. Western blot analysis indicated that upregulation of phosphorylated insulin receptor substrate‑1 (p‑IRS‑1) by the administration of TNTL may be strongly involved in the improvement of insulin resistance. In conclusion, TNTL exerted a strong hypoglycemic effect and reversed retinal degeneration via upregulation of ISR‑1. The present findings provide important scientific evidence supporting TNTL as an effective alternative approach for the management of Type 2 diabetes mellitus.

Figure 1.

Fasting blood glucose levels from weeks 1 to 18. Sustained TNTL treatment exerts a time-dependent hypoglycemic effect. Data are presented as the mean ± standard deviation. **P<0.01 vs. Normal mice, ^#P<0.05 vs. other 4 treatment groups (Met, TNTL-m, TNTL-h and TNTL-l), ^##P<0.01 vs. other 4 treatment groups (Met, TNTL-m, TNTL-h and TNTL-l). Normal, C57BL mice; DM, untreated C57BLdb/db mice; Met, db/db mice treated with metformin; TNTL-h, TNTL-m and TNTL-l, C57BLdb/db mice treated with 1.8, 0.9 and 0.45 g/kg body weight Tangningtongluo, respectively.

Figure 2.

HbA1c in each group at week 18. Data are presented as the mean ± standard deviation. *P<0.05 vs. DM group; **P<0.01 vs. DM group. Normal, C57BL mice; DM, untreated C57BLdb/db mice; Met, C57BLdb/db mice treated with metformin; TNTL-h, TNTL-m and TNTL-l, C57BLdb/db mice treated with 1.8, 0.9 and 0.45 g/kg body weight Tangningtongluo, respectively; HbA1c, glycated hemoglobin A1c.

Figure 3.

HOMA-IR in each group at week 18. Data are presented as the mean ± standard deviation. *P<0.05 vs. DM group; **P<0.01 vs. DM group. Normal, C57BL mice; DM, untreated C57BLdb/db mice; Met, C57BLdb/db mice treated with metformin; TNTL-h, TNTL-m and TNTL-l, C57BLdb/db mice treated with 1.8, 0.9 and 0.45 g/kg body weight Tangningtongluo, respectively; HOMA-IR, homeostatic model assessment of insulin resistance.

Figure 4.

Retinal imaging using FA in each group. (A) Results of the fundus examination using FA prior to drug intervention in the (a) normal, (b), DM, (c), Met, (d), TNTL-h, (e) TNTL-m and (f) TNTL-1 groups. (B) Results of the fundus examination using FA after the 18-week drug intervention in the (a) normal, (b), DM, (c), Met, (d), TNTL-h, (e) TNTL-m and (f) TNTL-1 groups. Magnification, ×40. Normal, C57BL mice; DM, untreated C57BLdb/db mice; Met, C57BLdb/db mice treated with metformin; TNTL-h, TNTL-m and TNTL-l, C57BLdb/db mice treated with 1.8, 0.9 and 0.45 g/kg body weight Tangningtongluo, respectively; FA, fluorescein angiography.

Figure 5.

Quantification of retinal imaging in each group. Density of vascular calibers in the fundus oculi in each group. TNTL significantly decreased the number of small-caliber vessels. The metformin showed no significant alleviation. Data are presented as the mean ± standard deviation. *P<0.05 vs. model group. Low, middle and high dose, 0.45, 0.9 and 0.18 g/kg body weight Tangningtongluo treatment.

Figure 6.

Histopathological alterations in the pancreas in each group. Representative images of mice in the (A) normal control, (B) diabetic control, (C) metformin and (D) 1.8, (E) 0.9 and (F) 0.45 g/kg body weight Tangningtongluo-treated groups.

Figure 7.

Protein expression levels of IRS-1 and p-IRS-1 in each group. Representative western blot images of (A) liver and (B) muscle tissue, and (C) quantification of IRS-1 and p-IRS-1 protein expression levels in each group. β-actin served as an internal control. Met, metformin; IRS, insulin receptor substrate-1; p, phosphorylated; DM, untreated C57BLdb/db mice; TNTL-h, TNTL-m and TNTL-l, C57BLdb/db mice treated with 1.8, 0.9 and 0.45 g/kg body weight Tangningtongluo.