Journal of Traditional Chinese Medicine ›› 2024, Vol. 44 ›› Issue (3): 458-467.DOI: 10.19852/j.cnki.jtcm.20240423.005
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GAO Xiaomeng1, QIANG Panpan1, CHANG Jingyue1, FAN Lili1, YANG Fan2(), XU Qingyou2()
Received:
2023-02-10
Accepted:
2023-07-07
Online:
2024-06-15
Published:
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: +86-15732190326Supported by:
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.
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 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.
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