Huangqi decoction (黄芪汤) attenuates renal interstitial fibrosis via transforming growth factor-β1/mitogen-activated protein kinase signaling pathways in 5/6 nephrectomy mice

doi:10.19852/j.cnki.jtcm.2022.05.006

Abstract

Abstract:

OBJECTIVE: To investigate the effect of Huangqi decoction (黄芪汤) on renal interstitial fibrosis and its association with the transforming growth factor-β1 (TGF-β1) / mitogen-activated protein kinase (MAPK) signaling pathway.

METHODS: 120 C57/BL mice were randomly divided into six groups: sham group, Enalapril (20 mg/kg) group, 5/6 nephrectomy model group, and 5/6 nephrectomy model plus Huangqicoction (0.12, 0.36 and 1.08 g/kg respectively) groups. Detecting 24hours urinary protein, blood pressure, serum creatinine, urea nitrogen content changes. Periodic Acid-Schiff stain (PAS) and Masson's trichrome staining was used to observe the renal tissue pathological changes. Protein expression of TGF-β1, Phosphorylated P38 mitogen activated protein kinases (P-P38), Phosphorylated c-jun N-terminal kinase (P-JNK), Phosphorylated extracellular regulated proteinhnase (P-ERK), Fibroblast-specific protein-1 (FSP-1), Alpha smooth muscle actin (α-SMA), Type III collagen (Collagen III), Connective tissue growth factor (CTGF), Bcl-2 Assaciated X protein (Bax) and B cell lymphoma 2 (Bcl-2) were measured with western blot and immunohistochemical.

RESULTS: Both Huangqi decoction and Enalapril improved the kidney function, 24 h urinary protein and the fibrosis in 5/6 nephrectomy mice, Huangqi decoction downregulated the expressions of TGF-β1, FSP-1, α-SMA, Collagen III and CTGF in a dose-dependent manner, and it has a significant difference (P < 0.01) compared with model group.Huangqi decoction downregulated the expressions of P-P38, P-JNK, P-ERK and Bcl-2 in a dose-dependent manner, while upregulated the expression of Bax.

CONCLUSIONS: The protective effect of Huangqi decoction for renal interstitial fibrosis in 5/6 nep-hrectomized mice via the inhibition of Epithelial-Mesenchymal Transitions and downregulating the TGF-β1/ MAPK signaling pathway.

Key words: nephrectomy, transforming growth factor beta1, mitogen-activated protein kinases, Signal transduction, renal interstitial fibrosis, Huangqi decoction

ZHAO Jie, WANG Li, CAO Ai-li, WANG Yun-man, CHI Yang-feng, WANG Yi, WANG Hao, PENG Wen. Huangqi decoction (黄芪汤) attenuates renal interstitial fibrosis via transforming growth factor-β1/mitogen-activated protein kinase signaling pathways in 5/6 nephrectomy mice[J]. Journal of Traditional Chinese Medicine, 2022, 42(5): 723-731.

Figures/Tables 8

Table 1 Effect of Huangqi decotion on the blood pressure of the mice (mean, n = 6)

Group	Week 1	Week 4	Week 8	Week 12	Week 16
Sh	116.9±6.4	119.8±6.8	125.8±7.6	125.3±7.1	125.7±2.5
Nx	112.8±7.0	129.9±7.5	150.5±2.3^a	151.9±6.5^a	155.0±4.8^a
En	110.4±6.4	121.4±6.5	130.0±5.2^b	121.8±4.0^b	122.9±3.9^c
Hl	114.0±5.3	139.9±8.9	140.8±6.4	141.5±7.4	147.9±7.1
Hm	118.3±6.6	136.0±10.0	143.4±7.9	142.6±5.5	147.7±3.3
Hh	115.8±6.8	128.3±9.3	138.9±10.1	142.0±7.2	145.3±8.6

Figure 1 Effect of Huangqi decotion on weight,kidney index and blood pressure changes. A: the body weight during the first week; B: the body weight at the end of week17; C: the body weight during the whole process; D: the weight of the remanent kidney; E: the kidney index of the mice. Sh group was the control group; Nx group was the 5/6 nephrectomy model group; En group was the control group by Enalapri; Hl group was the Low dosing of Huangqi decotion group (Hl, 0.12g /kg, n = 20); Hm group was the Medium dosing of Huangqi decotion group (Hm, 0.36g/kg, n = 20); HH group was the High dosing of Huangqi decotion group (HH, 1.08g/kg, n = 20). Sh: Sham group; En: Enalapri group; Nx: 5/6 nephrectomy model group; Hh: High dosing of Huangqi decotion group; Hm: Medium dosing of Huangqi decotion group; Hl: Low dosing of Huangqi decotion group. aP < 0.05, compared with Sham group; bP < 0.01 or cP < 0.05, compared with Nx group, n = 8-11.

Table 2 Effect of Huangqi decotion on 24 h urinary protein (s, n = 6)

Group	Week 1	Week 4	Week 8	Week 12	Week 16
Sh	4.0±0.4	3.8±0.4	3.5±0.2	3.4±0.3	3.9±0.3
Nx	4.0±0.4	1.3±0.2^a	1.3±0.2^a	16.9±0.7^a	16.8±0.6^a
En	3.9±0.3	1.9±0.3	1.4±0.1	10.6±0.6^b	9.4±0.2^c
Hl	4.0±0.4	1.0±0.2	1.4±0.2	17.2±0.7	16.4±0.6
Hm	3.9±0.3	1.0±0.1	2.5±0.2^b	16.4±0.6	15.8±0.5
Hh	3.9±0.2	1.6±0.2	2.8±0.2^c	16.8±0.5	13.8±0.5^b

Figure 2 Effect of Huangqi decotion on renal function. A: effect of Huangqi decotion on serum creatinine; B: effect of Huangqi decotion on blood urea nitrogen. Sh group was the control group; Nx group was the 5/6 nephrectomy model group; En group was the control group by Enalapri; Hl group was the Low dosing of Huangqi decotion group (Hl, 0.12g /kg, n = 20); Hm group was the Medium dosing of Huangqi decotion group (Hm, 0.36g/kg, n = 20); HH group was the High dosing of Huangqi decotion group (HH, 1.08g/kg, n = 20). Sh: Sham group; En: Enalapri group; Nx: 5/6 nephrectomy model group; Hh: High dosing of Huangqi decotion group; Hm: Medium dosing of Huangqi decotion group; Hl: Low dosing of Huangqi decotion group. aP < 0.05, compared with Sham group; bP < 0.01 or cP < 0.05, compared with Nx group, n = 8-11.

Figure 3 Huangqi treatment attenuates renal damage in a dosage-dependent manner in Nx mice A: representative PAS-stained kidney sections from Sham, Nx, enalapril, Hl, Hm, Hh groups; B: histopathological scores and inflammatory cells count within the tubulointerstitium from sections similar to those shown in (A); C: representative Masson's modified trichrome stained kidney sections from Sham, Nx, enalapril, Hl, Hm, Hh groups; D: quantitative analysis of the extent of renal interstitial expansion (area %) from sections similar to those shown in. Sh group was the control group; Nx group was the 5/6 nephrectomy model group; En group was the control group by Enalapri; Hl group was the Low dosing of Huangqi decotion group (Hl, 0.12g /kg, n = 20); Hm group was the Medium dosing of Huangqi decotion group (Hm, 0.36g/kg, n = 20); HH group was the High dosing of Huangqi decotion group (HH, 1.08g/kg, n = 20). Sh: Sham group; En: Enalapri group; Nx: 5/6 nephrectomy model group; Hh: High dosing of Huangqi decotion group; Hm: Medium dosing of Huangqi decotion group; Hl: Low dosing of Huangqi decotion group. aP < 0.05, compared with Sham group; bP < 0.01 or cP < 0.05, compared with Nx group, n = 8-11. Magnification: (× 400).

Figure 4 Huangqi decoction decreases the Nx kidneys' CTGF and Collagen III expression by immunohistochemistry A, C: representative photomicrographs of CTGF and Collagen III immunohistochemistry on kidney sections from the Sham, Nx, enalapril, Hl, Hm, Hh groups; B, D: quantitative analysis of CTGF and Collagen III expression from sections similar to those shown in A, C. Sh group was the control group; Nx group was the 5/6 nephrectomy model group; En group was the control group by Enalapri; Hl group was the Low dosing of Huangqi decotion group (Hl, 0.12g /kg, n = 20); Hm group was the Medium dosing of Huangqi decotion group (Hm, 0.36g/kg, n = 20); HH group was the High dosing of Huangqi decotion group (HH, 1.08g/kg, n = 20). Sh: Sham group; En: Enalapri group; Nx: 5/6 nephrectomy model group; Hh: High dosing of Huangqi decotion group; Hm: Medium dosing of Huangqi decotion group; Hl: Low dosing of Huangqi decotion group. aP < 0.05, compared with Sham group; bP < 0.01 or cP < 0.05, compared with Nx group, n = 8-11. Magnification: (× 400).

Figure 5 Huangqi decotion alleviates matrix accumulation in Nx kidneys A: protein expressions of TGF-β1, FSP-1 and α-SMA by Western Blot; B-D: statistical analyses versus A. E: representative photomicrographs of FSP-1by immunohistochemistry on kidney sections from the Sham, Nx, enalapril, Hl, Hm, Hh groups; F: quantitative analysis of FSP-1 expression from sections similar to those shown in E. Sh group was the control group; Nx group was the 5/6 nephrectomy model group; En group was the control group by Enalapri; Hl group was the Low dosing of Huangqi decotion group (Hl, 0.12g /kg, n = 20); Hm group was the Medium dosing of Huangqi decotion group (Hm, 0.36g/kg, n = 20); HH group was the High dosing of Huangqi decotion group (HH, 1.08g/kg, n = 20).Sh: Sham group; En: Enalapri group; Nx: 5/6 nephrectomy model group; Hh: High dosing of Huangqi decotion group; Hm: Medium dosing of Huangqi decotion group; Hl: Low dosing of Huangqi decotion group. aP < 0.05, compared with Sham grou;. bP < 0.01 or cP < 0.05, compared with Nx group, n = 8-11. Magnification: (× 400).

Figure 6 Huangqi decotion downregulats MAPK signaling pathway on kidney sections A: protein expressions of P-P38, P-JNK, P-ERK, Bax and Bcl-2 by Western Blot; B-F: statistical analyses versus A. Sh group was the control group; Nx group was the 5/6 nephrectomy model group; En group was the control group by Enalapri; Hl group was the Low dosing of Huangqi decotion group (Hl, 0.12g /kg, n = 20); Hm group was the Medium dosing of Huangqi decotion group (Hm, 0.36g/kg, n = 20); HH group was the High dosing of Huangqi decotion group (HH, 1.08g/kg, n = 20).Sh: Sham group; En: Enalapri group; Nx: 5/6 nephrectomy model group; Hh: High dosing of Huangqi decotion group; Hm: Medium dosing of Huangqi decotion group; Hl: Low dosing of Huangqi decotion group. aP < 0.05, compared with Sham group. bP < 0.01 or cP < 0.05, compared with Nx group, n = 8-11.

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