Journal of Traditional Chinese Medicine ›› 2024, Vol. 44 ›› Issue (3): 458-467.DOI: 10.19852/j.cnki.jtcm.20240423.005
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Huoxue Jiedu Huayu recipe (活血解毒化瘀方) inhibits macrophage-secreted vascular endothelial growth factor-a on angiogenesis and alleviates renal fibrosis in the contralateral kidneys of unilateral ureteral obstruction rats
GAO Xiaomeng1, QIANG Panpan1, CHANG Jingyue1, FAN Lili1, YANG Fan2(
), XU Qingyou2()
- 1 Graduate School, Hebei University of Chinese Medicine; Hebei Key Laboratory of Integrative Medicine on Liver-Kidney Patterns, Hebei University of Chinese Medicine, Shijiazhuang 050091, China
2 Graduate School, Hebei University of Chinese Medicine; Hebei Key Laboratory of Integrative Medicine on Liver-Kidney Patterns, Hebei University of Chinese Medicine; Institute of Integrative Medicine, Hebei University of Chinese Medicine, Shijiazhuang 050091, China
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Received:2023-02-10Accepted:2023-07-07Online:2024-06-15Published:2024-04-30 -
Contact:YANG Fan,XU Qingyou -
About author:Prof. YANG Fan, Institute of Integrative Medicine, Hebei University of Chinese Medicine, Shijiazhuang 050091, China. yangfanwy@hebcm.edu.cn Telephone: +86-15732190326
Prof. XU Qingyou, Institute of Integrative Medicine, Hebei University of Chinese Medicine, Shijiazhuang 050091, China. qingyouxu@hebcm.edu.cn;
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Supported by:Natural Science Foundation-funded Project: Effect of Macrophage-to-Myofibroblast Transition in Contralateral Kidney of Unilateral Ureteral Obstruction Rats Through the Aldosterone/MR/SGK1 Pathway and Inhibition of Chinese Herbs(81873251);Natural Science Foundation-funded Project: Aldosterone Stimulates MR Activation to Induce Lymphangiogenesis in the Contralateral Kidney of UUO Rats and the Protective Effect of Yiqi Jiedu Huayu Herbs(82174317);Hebei Provincial Postgraduate Innovative Ability Cultivation Funding Project: Aldosterone Induced Macrophages to Secrete VEGFA to Participate in Renal Angiogenesis and the Protective Effect of Huoxue Jiedu Huayu Recipe(CXZZBS2023140);Construction Program of New Research and Development Platform and Institution, Hebei Province Innovation Ability Promotion Plan under Grant(20567624H)
Cite this article
GAO Xiaomeng, QIANG Panpan, CHANG Jingyue, FAN Lili, YANG Fan, XU Qingyou. Huoxue Jiedu Huayu recipe (活血解毒化瘀方) inhibits macrophage-secreted vascular endothelial growth factor-a on angiogenesis and alleviates renal fibrosis in the contralateral kidneys of unilateral ureteral obstruction rats[J]. Journal of Traditional Chinese Medicine, 2024, 44(3): 458-467.
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Figure 1 Effects of UUO, HJHR and EPL treatment on renal histology and fibrosis in the contralateral kidneys A: kidney sections from all groups were stained with HE to assess changes in morphology. Scale bars, 100 μm. A1: Sham group; A2: UUO group; A3: EPL group; A4: HJHR group. B: expression of CD68 was measured by immunohistochemical staining. Scale bars, 50 μm. B1: Sham group; B2: UUO group; B3: EPL group; B4: HJHR group. C: Masson's trichrome (Masson) staining was used to assess fibrosis. Scale bars, 100 μm. C1: Sham group; C2: UUO group; C3: EPL group; C4: HJHR group. D: serum creatinine was evaluated for renal function. E: blood urea nitrogen was evaluated for renal function. F: serum aldosterone level was measured. Sham group (the left ureter was exposed but not ligated, 180 d); UUO group (the left ureter was ligated, 180 d); EPL group (EPL, 100 mg·kg-1·d-1, 180 d); HJHR group (HJHR, 11.7 g·kg-1·d-1, 180 d). UUO: unilateral ureteral obstruction; EPL: eplerenone; HJHR: Huoxue Jiedu Huayu recipe; HE: hematoxylin and eosin. Quantification data are presented as the mean ± standard deviation, n = 6. aP < 0.05 vs the sham group; bP < 0.05 vs the UUO group.
Figure 1 Effects of UUO, HJHR and EPL treatment on renal histology and fibrosis in the contralateral kidneys A: kidney sections from all groups were stained with HE to assess changes in morphology. Scale bars, 100 μm. A1: Sham group; A2: UUO group; A3: EPL group; A4: HJHR group. B: expression of CD68 was measured by immunohistochemical staining. Scale bars, 50 μm. B1: Sham group; B2: UUO group; B3: EPL group; B4: HJHR group. C: Masson's trichrome (Masson) staining was used to assess fibrosis. Scale bars, 100 μm. C1: Sham group; C2: UUO group; C3: EPL group; C4: HJHR group. D: serum creatinine was evaluated for renal function. E: blood urea nitrogen was evaluated for renal function. F: serum aldosterone level was measured. Sham group (the left ureter was exposed but not ligated, 180 d); UUO group (the left ureter was ligated, 180 d); EPL group (EPL, 100 mg·kg-1·d-1, 180 d); HJHR group (HJHR, 11.7 g·kg-1·d-1, 180 d). UUO: unilateral ureteral obstruction; EPL: eplerenone; HJHR: Huoxue Jiedu Huayu recipe; HE: hematoxylin and eosin. Quantification data are presented as the mean ± standard deviation, n = 6. aP < 0.05 vs the sham group; bP < 0.05 vs the UUO group.
Figure 2 Effects of UUO, HJHR and EPL on angiogenesis in the contralateral kidney tissue of UUO rats A: expression of CD34 was examined by immunohistochemical staining. Scale bars, 50 μm. A1: Sham group; A2: UUO group; A3: EPL group; A4: HJHR group. B: expression of CD105 was examined by immunohistochemical staining. Scale bars, 50 μm. B1: Sham group; B2: UUO group; B3: EPL group; B4: HJHR group. C: expression of NGAL and SGK-1 protein in the contralateral kidney tissue of UUO rats was examined by Western blotting. D: protein ratio of NGAL/GAPDH; E: protein ratio of SGK-1/GAPDH; 1: Sham group; 2: UUO group; 3: EPL group; 4: HJHR group. Sham group (the left ureter was exposed but not ligated, 180 d); UUO group (the left ureter was ligated, 180 d); EPL group (EPL, 100 mg·kg-1·d-1, 180 d); HJHR group (HJHR, 11.7 g·kg-1·d-1, 180 d). UUO: unilateral ureteral obstruction; EPL: eplerenone; HJHR: Huoxue Jiedu Huayu recipe; NGAL: neutropil gelatinase-associated lipocalin; SGK-1: serum/glucocorticoid regulated kinase 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase. The data are presented as the mean ± standard deviation, n = 3. aP < 0.05 vs the sham group, bP < 0.05 vs the UUO group.
Figure 2 Effects of UUO, HJHR and EPL on angiogenesis in the contralateral kidney tissue of UUO rats A: expression of CD34 was examined by immunohistochemical staining. Scale bars, 50 μm. A1: Sham group; A2: UUO group; A3: EPL group; A4: HJHR group. B: expression of CD105 was examined by immunohistochemical staining. Scale bars, 50 μm. B1: Sham group; B2: UUO group; B3: EPL group; B4: HJHR group. C: expression of NGAL and SGK-1 protein in the contralateral kidney tissue of UUO rats was examined by Western blotting. D: protein ratio of NGAL/GAPDH; E: protein ratio of SGK-1/GAPDH; 1: Sham group; 2: UUO group; 3: EPL group; 4: HJHR group. Sham group (the left ureter was exposed but not ligated, 180 d); UUO group (the left ureter was ligated, 180 d); EPL group (EPL, 100 mg·kg-1·d-1, 180 d); HJHR group (HJHR, 11.7 g·kg-1·d-1, 180 d). UUO: unilateral ureteral obstruction; EPL: eplerenone; HJHR: Huoxue Jiedu Huayu recipe; NGAL: neutropil gelatinase-associated lipocalin; SGK-1: serum/glucocorticoid regulated kinase 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase. The data are presented as the mean ± standard deviation, n = 3. aP < 0.05 vs the sham group, bP < 0.05 vs the UUO group.
Figure 3 EndMT in the contralateral kidneys of UUO rats A: expression of α-SMA and vimentin was detected by immunohistochemistry (scale bars, 100 μm) and real-time PCR to examine renal infiltration of myofibroblasts. A1-A4: immunohistochemical staining using antibodies against α-SMA. A1: Sham group; A2: UUO group; A3: EPL group; A4: HJHR group. A5: expression of α-SMA mRNA in the contralateral kidney tissue of UUO rats was examined by real-time PCR. A6-A9: immunohistochemical staining using antibodies against vimentin. A6: Sham group; A7: UUO group; A8: EPL group; A9: HJHR group. A10: expression of vimentin mRNA in the contralateral kidney tissue of UUO rats was examined by real-time PCR. B: expression of collagen I was detected by immunohistochemistry (scale bars, 100 μm) and real-time PCR. B1-B4: immunohistochemical staining using antibodies against collagen I. B1: Sham group; B2: UUO group; B3: EPL group; B4: HJHR group. B5: expression of collagen I mRNA in the contralateral kidney tissue of UUO rats was examined by real-time PCR. C: coexpression (Merged, yellow) of vascular endothelial cell (FITC, CD34, green) and myofibroblast (TRITC, α-SMA, red) markers in the UUO group was examined by two-color immunofluorescence staining, and nuclei were stained with DAPI (blue). Scale bars, 50 μm. D: coexpression (Merged, yellow) of vascular endothelial cell (FITC, CD105, green) and myofibroblast (TRITC, α-SMA, red) markers in the UUO group was examined by two-color immunofluorescence staining, and nuclei were stained with DAPI (blue). Scale bars, 50 μm. Sham group (the left ureter was exposed but not ligated, 180 d); UUO group (the left ureter was ligated, 180 d); EPL group (EPL, 100 mg·kg-1·d-1, 180 d); HJHR group (HJHR, 11.7 g·kg-1·d-1, 180 d). UUO: unilateral ureteral obstruction; EPL: eplerenone; HJHR: Huoxue Jiedu Huayu recipe; EndMT: endothelial-to-mesenchymal transition; α-SMA: α-smooth muscle actin. DAPI: 4',6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate; TRITC: tetraethyl rhodamine isothiocyanate. The data are presented as the mean ± standard deviation, n = 3. aP < 0.05 vs the sham group, bP < 0.05 vs the UUO group.
Figure 3 EndMT in the contralateral kidneys of UUO rats A: expression of α-SMA and vimentin was detected by immunohistochemistry (scale bars, 100 μm) and real-time PCR to examine renal infiltration of myofibroblasts. A1-A4: immunohistochemical staining using antibodies against α-SMA. A1: Sham group; A2: UUO group; A3: EPL group; A4: HJHR group. A5: expression of α-SMA mRNA in the contralateral kidney tissue of UUO rats was examined by real-time PCR. A6-A9: immunohistochemical staining using antibodies against vimentin. A6: Sham group; A7: UUO group; A8: EPL group; A9: HJHR group. A10: expression of vimentin mRNA in the contralateral kidney tissue of UUO rats was examined by real-time PCR. B: expression of collagen I was detected by immunohistochemistry (scale bars, 100 μm) and real-time PCR. B1-B4: immunohistochemical staining using antibodies against collagen I. B1: Sham group; B2: UUO group; B3: EPL group; B4: HJHR group. B5: expression of collagen I mRNA in the contralateral kidney tissue of UUO rats was examined by real-time PCR. C: coexpression (Merged, yellow) of vascular endothelial cell (FITC, CD34, green) and myofibroblast (TRITC, α-SMA, red) markers in the UUO group was examined by two-color immunofluorescence staining, and nuclei were stained with DAPI (blue). Scale bars, 50 μm. D: coexpression (Merged, yellow) of vascular endothelial cell (FITC, CD105, green) and myofibroblast (TRITC, α-SMA, red) markers in the UUO group was examined by two-color immunofluorescence staining, and nuclei were stained with DAPI (blue). Scale bars, 50 μm. Sham group (the left ureter was exposed but not ligated, 180 d); UUO group (the left ureter was ligated, 180 d); EPL group (EPL, 100 mg·kg-1·d-1, 180 d); HJHR group (HJHR, 11.7 g·kg-1·d-1, 180 d). UUO: unilateral ureteral obstruction; EPL: eplerenone; HJHR: Huoxue Jiedu Huayu recipe; EndMT: endothelial-to-mesenchymal transition; α-SMA: α-smooth muscle actin. DAPI: 4',6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate; TRITC: tetraethyl rhodamine isothiocyanate. The data are presented as the mean ± standard deviation, n = 3. aP < 0.05 vs the sham group, bP < 0.05 vs the UUO group.
Figure 4 Effects of VEGFA produced by macrophages on angiogenesis in the contralateral kidneys of UUO rats A: coexpression (Merged, yellow) of vascular endothelial cell (FITC, VEGFA, green) and macrophage (TRITC, CD68, red) markers was examined by two-color immunofluorescence staining, and nuclei were stained with DAPI (blue). Scale bars, 50 μm. A1-A4: Sham group; A5-A8: UUO group; A9-A12: EPL group; A13-A16: HJHR group. B: expression of VEGFA and VEGFR-2 protein in the contralateral kidney tissue of UUO rats was examined by Western blotting. C: protein ratio of VEGFA/GAPDH; D: protein ratio of VEGFR-2/β-Tubulin; 1: Sham group; 2: UUO group; 3: EPL group; 4: HJHR group. E: expression of VEGFA mRNA in the contralateral kidney tissue of UUO rats was examined by real-time PCR. Sham group (the left ureter was exposed but not ligated, 180 d); UUO group (the left ureter was ligated, 180 d); EPL group (EPL, 100 mg·kg-1·d-1, 180 d); HJHR group (HJHR, 11.7 g·kg-1·d-1, 180 d). UUO: unilateral ureteral obstruction; EPL: eplerenone; HJHR: Huoxue Jiedu Huayu recipe; DAPI: 4',6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate; TRITC: tetraethyl rhodamine isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; VEGFA: vascular endothelial growth factor-A; VEGFR-2: vascular endothelial growth factor receptor-2. The data are presented as the mean ± standard deviation, n = 3. aP < 0.05 vs the sham group, bP < 0.05 vs the UUO group.
Figure 4 Effects of VEGFA produced by macrophages on angiogenesis in the contralateral kidneys of UUO rats A: coexpression (Merged, yellow) of vascular endothelial cell (FITC, VEGFA, green) and macrophage (TRITC, CD68, red) markers was examined by two-color immunofluorescence staining, and nuclei were stained with DAPI (blue). Scale bars, 50 μm. A1-A4: Sham group; A5-A8: UUO group; A9-A12: EPL group; A13-A16: HJHR group. B: expression of VEGFA and VEGFR-2 protein in the contralateral kidney tissue of UUO rats was examined by Western blotting. C: protein ratio of VEGFA/GAPDH; D: protein ratio of VEGFR-2/β-Tubulin; 1: Sham group; 2: UUO group; 3: EPL group; 4: HJHR group. E: expression of VEGFA mRNA in the contralateral kidney tissue of UUO rats was examined by real-time PCR. Sham group (the left ureter was exposed but not ligated, 180 d); UUO group (the left ureter was ligated, 180 d); EPL group (EPL, 100 mg·kg-1·d-1, 180 d); HJHR group (HJHR, 11.7 g·kg-1·d-1, 180 d). UUO: unilateral ureteral obstruction; EPL: eplerenone; HJHR: Huoxue Jiedu Huayu recipe; DAPI: 4',6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate; TRITC: tetraethyl rhodamine isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; VEGFA: vascular endothelial growth factor-A; VEGFR-2: vascular endothelial growth factor receptor-2. The data are presented as the mean ± standard deviation, n = 3. aP < 0.05 vs the sham group, bP < 0.05 vs the UUO group.
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