Tang Luo Ning, a Traditional Chinese Compound Prescription, Ameliorates Schwannopathy of Diabetic Peripheral Neuropathy Rats by Regulating Mitochondrial Dynamics In Vivo and In Vitro

Abstract

Tang Luo Ning (TLN), a traditional Chinese compound prescription, has been used clinically to treat diabetic peripheral neuropathy (DPN) in China. However, the exact mechanisms remain unclear. The objective of this study is to unravel the effects of TLN on mitochondrial dynamics of DPN in streptozotocin-induced rat models and Schwann cells cultured in 150 mM glucose. Mitochondrial function was determined by Ca²⁺ and ATP levels of streptozotocin (STZ)-induced DPN rats and mitochondria structure, mitochondrial membrane potential (MMP), and mtDNA of high glucose incubated SCs. Mitochondrial dynamics protein including mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), optic atrophy 1 (Opa1), and dynamin-related protein 1 (Drp1) were investigated using Western blot or immunofluorescence. Myelin basic protein (MBP), myelin protein zero (MPZ), and sex-determining region Y (SRY)-box 10 (Sox10) were measured to represent schwannopathy. Our results showed that TLN increased ATP levels (0.38 of model, 0.69 of HTLN, 0.61 of LTLN, P＜0.01; 0.52 of 150 mM glucose, 1.00 of 10% TLN, P＜0.01, 0.94 of 1% TLN, P＜0.05), MMP (0.56 of 150 mM glucose, P＜0.01, 0.75 of 10% TLN, P＜0.05, 0.83 of 1% TLN, P＜0.01), and mtDNA (0.32 of 150 mM glucose, 0.43 of 10% TLN, P＜0.01) while decreased Ca²⁺ (1.54 of model, 1.06 of HTLN, 0.96 of LTLN, P＜0.01) to improve mitochondrial function in vivo and in vitro. TLN helps maintain balance of mitochondrial dynamics: it reduces the mitochondria number (1.60 of 150 mM glucose, 1.10 of 10% TLN, P＜0.01) and increases the mitochondria coverage (0.51 of 150 mM glucose, 0.80 of 10% TLN, 0.87 of 1% TLN, P＜0.01), mitochondrial network size (0.51 of 150 mM glucose, 0.95 of 10% TLN, 0.94 of 1% TLN, P＜0.01), and branch length (0.63 of 150 mM glucose, P＜0.01, 0.73 of 10% TLN, P＜0.05, 0.78 of 1% TLN, P＜0.01). Further, mitochondrial dynamics-related Mfn1 (0.47 of model, 0.82 of HTLN, 0.77 of LTLN, P＜0.01; 0.42 of 150 mM glucose, 0.56 of 10% TLN, 0.57 of 1% TLN, P＜0.01), Mfn2 (0.40 of model, 0.84 of HTLN, 0.63 of LTLN, P＜0.01; 0.46 of 150 mM glucose, 1.40 of 10% TLN, 1.40 of 1% TLN, P＜0.01), and Opa1 (0.58 of model, 0.71 of HTLN, 0.90 of LTLN, P＜0.01; 0.69 of 150 mM glucose, 0.96 of 10% TLN, 0.98 of 1% TLN, P＜0.05) were increased, while Drp1 (1.39 of model, 0.96 of HTLN, 1.18 of LTLN, P＜0.01; 1.70 of 150 mM glucose, 1.20 of 10% TLN, 1.10 of 1% TLN, P＜0.05), phosphorylated Drp1 (2.61 of model, 1.44 of HTLN, P＜0.05; 2.80 of 150 mM glucose, 1.50 of 10% TLN, 1.30 of 1% TLN, P＜0.01), and Drp1 located in mitochondria (1.80 of 150 mM glucose, 1.00 of 10% TLN, P＜0.05) were decreased after treatment with TLN. Additionally, TLN improved schwannopathy by increasing MBP (0.50 of model, 1.05 of HTLN, 0.94 of HTLN, P＜0.01; 0.60 of 150 mM glucose, 0.78 of 10% TLN, P＜0.01, 0.72 of 1% TLN, P＜0.05), Sox101 (0.41 of model, 0.99 of LTLN, P＜0.01; 0.48 of 150 mM glucose, 0.65 of 10% TLN, P＜0.05, 0.69 of 1% TLN, P＜0.01), and MPZ (0.48 of model, 0.66 of HTLN, 0.55 of HTLN, P＜0.01; 0.60 of 150 mM glucose, 0.78 of 10% TLN, P＜0.01, 0.75 of 1% TLN, P＜0.05) expressions. In conclusion, our study indicated that TLN's function on DPN may link to the improvement of the mitochondrial dynamics, which provides scientific evidence for the clinical application.

Keywords: Drp1; Mfn1; Mfn2; OPA1; Schwann cells; diabetic peripheral neuropathy; mitochondrial dynamics.

FIGURE 1

TLN treatment on mitochondrial function in DPN rats (A) Serum Ca²⁺ levels of rats were measured. (B) ATP levels in sciatic nerves were measured. n = 6 for each group. ^ΔΔ P< 0.01 vs. control; ^## P < 0.01 vs. model.

FIGURE 2

TLN treatment on mitochondrial dynamics in DPN rats. (A) Representative images of immunofluorescence staining on Mfn1 (green); scale bar, 20 μm. Opa1 (green); scale bar, 75 μm. Drp1 (green); scale bar, 75 μm. Schwann cells are stained with S100β (red). (B-D) Quantifications of indicated proteins. Data were presented as fold change of the control group. (E, F) Western blotting of Mfn2 and phospho-Drp1(Ser616) of sciatic nerves and quantifications of this proteins. n = 6 for each group. ^ΔΔ P< 0.01 vs. control; ^# P< 0.05, ^## P < 0.01 vs. model.

FIGURE 3

TLN serum treatment improved the mitochondrial structure and function of SCs incubated in a high glucose environment. (A) Representative images and quantifications of the mitochondria number and mitochondria coverage of 30 cells. Scale bar, 5 μm. The results were normalized to the values of the 25 mM glucose group. Mitochondria are indicated by red circles. (B) Representative images and quantifications of the mean network size and mean branch length of 20 cells. Scale bar, 5 μm. (C) Quantification of ATP, n = 4 for each group. (D) Representative images of immunofluorescence staining on TMRM (green) and mitochondria (red); scale bar, 5 μm, and quantification of mitochondrial membrane potential, n = 4 for each group. (E) Representative images of immunofluorescence staining on TFAM (green) and mitochondria (red); scale bar, 5 μm, and quantification of mtDNA, n = 4 for each group. ^ΔΔ P< 0.01 vs. 25 mM glucose group; ^## P < 0.01, ^# P< 0.05 vs. 150 mM glucose group.

FIGURE 4

TLN serum treatment increased mitochondrial fusion, while decreased fission of SCs incubated in a high glucose environment. (A)Western blotting of Mfn1, Mfn2, Opa1, Drp1, and phospho-Drp1 (Ser616) of SCs and quantifications of these proteins. The results were normalized to the values of the 25 mM glucose group. β-actin images are reused. (B) Representative images of immunofluorescence staining on Drp1 (green), mitochondria (red), and nucleus (blue). Scale bar, 5 μm, and quantification of Drp1 located on mitochondria, n = 4 for each group. (C) Representative images of immunofluorescence staining on Oma1 (green) and nucleus (blue), magnification 40×, and quantification of Oma, n = 4 for each group. ^ΔΔ P< 0.01, ^Δ P< 0.05 vs. 25 mM glucose group; ^## P < 0.01, ^# P< 0.05 vs. 150 mM glucose group.

FIGURE 5

TLN increased MBP, MPZ, and Sox10 in vivo and in vitro to ameliorate schwannopathy. (A)Western blotting of Sox10 and MBP in sciatic nerves of DPN rats and quantifications of these proteins. The results were normalized to the values of the control group, n = 6 for each group. β-actin images are reused. (B) Representative images of immunofluorescence staining on MPZ (green) and S100β (purple). Scale bar, 20 μm, and quantification of MPZ, n = 6 for each group. ^ΔΔ P< 0.01, ^Δ P< 0.05 vs. control group; ^## P < 0.01, #P< 0.05 vs. model group (C–E) Representative images of immunofluorescence staining on Sox10 (green), MPZ (green), MBP (green), and nucleus (blue). Scale bar, 5 μm, and quantification of Sox10, MPZ, and MBP, n = 4 for each group. ^ΔΔ P< 0.01, ^Δ P< 0.05 vs. 25 mM glucose group; ^## P < 0.01, ^# P< 0.05 vs. 150 mM glucose group.