Journal of Traditional Chinese Medicine ›› 2023, Vol. 43 ›› Issue (4): 667-675.DOI: 10.19852/j.cnki.jtcm.20230428.001
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Qizhi Jiangtang capsule (芪蛭降糖胶囊) activates podocyte autophagy in diabetic kidney disease by inhibiting phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathways
GUO Zhaoan1(
), SUN Lina2(), LIU Yingying1, LI Ruifeng3, LIU Chong3, DIAO Ke4, SHI Jing3, SUN Jun3
- 1 Department of Nephrology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250011, China
2 Department of Nephrology, Linyi People’s Hospital, Linyi 276003, China
3 Shandong University of Traditional Chinese Medicine, Jinan 250355, China
4 Shandong GuoxinYiyang Group Zibo Hospital, Zibo 255051, China
-
Received:2022-01-02Accepted:2022-07-27Online:2023-08-15Published:2023-04-28 -
Contact:SUN Lina, Department of Nephrology, Linyi People’s Hospital, Linyi 276003, China. hualuohuakai3@163.com. Telephone: +86-18763794562
GUO Zhaoan, Department of Nephrology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250011, China. gza63@163.com -
Supported by:National Natural Science Foundation of China Project: Experimental Research on Podocyte Autophagy of Diabetic Nephropathy Regulated by Qizhi Jiangtang Capusul(81874440);Natural Science Foundation of Shandong Province Project: Curcumin Ameliorates Diabetic Nephropathy via Regulating the Intestinal Barrier-Inflammation “cross-talk”(ZR2020QH063)
Cite this article
GUO Zhaoan, SUN Lina, LIU Yingying, LI Ruifeng, LIU Chong, DIAO Ke, SHI Jing, SUN Jun. Qizhi Jiangtang capsule (芪蛭降糖胶囊) activates podocyte autophagy in diabetic kidney disease by inhibiting phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathways[J]. Journal of Traditional Chinese Medicine, 2023, 43(4): 667-675.
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Table 1 Effects of QZJT on Urine protein, Scr and BUN in db/db mice ($\bar{x}±s$)
| Group | n | Urine protein (mg/24 h) | Scr(μmol/L) | BUN (mmol/L) |
|---|---|---|---|---|
| db/m | 10 | 80.5±11.9 | 52.3±10.3 | 5.6±1.4 |
| db/db | 10 | 153.7±11.2a | 131.8±13.2a | 12.4±1.2a |
| db/db+RAP | 10 | 126.6±9.5b | 89.4±12.8b | 7.5±1.1b |
| db/db+QZJT | 10 | 129.7±6.1b | 79.2±16.8b | 8.3±1.2b |
Table 1 Effects of QZJT on Urine protein, Scr and BUN in db/db mice ($\bar{x}±s$)
| Group | n | Urine protein (mg/24 h) | Scr(μmol/L) | BUN (mmol/L) |
|---|---|---|---|---|
| db/m | 10 | 80.5±11.9 | 52.3±10.3 | 5.6±1.4 |
| db/db | 10 | 153.7±11.2a | 131.8±13.2a | 12.4±1.2a |
| db/db+RAP | 10 | 126.6±9.5b | 89.4±12.8b | 7.5±1.1b |
| db/db+QZJT | 10 | 129.7±6.1b | 79.2±16.8b | 8.3±1.2b |
Figure 1 QZJT improves pathological damage and podocyte injury in db/db mice Renal tissues were observed by staining with PAS (A1-A4), and Masson (B1-B4) and photographed by a light microscope (× 400 magnification) in db/m, db/db, db/db + RAP, db/db + QZJT group, respectively. C1-C4: representative fields of podocyte foot processes under electron microscopy (scale bars: 5 μm) in db/m, db/db, db/db+RAP, db/db + QZJT group, respectively. D: Western blot of nephrin and podocin expression in the four groups. E: relative band intensities were used in order to quantify nephrin expression levels. F: relative band intensities were used in order to quantify podocin protein expression levels. Eight-week-old db/db mice were treated with or without QZJT or rapamycin. Db/m (control group group, n = 10), db/db (diabetic kidney disease group, n = 10), db/db+RAP (rapamycin (5 mg·kg-1·d-1), n = 10), db/db + QZJT group (QZJT 3.9 g/kg, n = 10). QZJT: Qizhi Jiangtang capsule; RAP: rapamycin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PAS: periodic acid-Schiff; Masson: Masson's trichrome staining; EM: electron microscopy. Student t-test and one-way analysis of variance were used for comparison analysis. aP < 0.01, the db/db group vs the db/m group, bP < 0.05, the db/db group vs the db/db + RAP group or db/db + QZJT group, cP < 0.05, the db/db group vs the db/m group.
Figure 1 QZJT improves pathological damage and podocyte injury in db/db mice Renal tissues were observed by staining with PAS (A1-A4), and Masson (B1-B4) and photographed by a light microscope (× 400 magnification) in db/m, db/db, db/db + RAP, db/db + QZJT group, respectively. C1-C4: representative fields of podocyte foot processes under electron microscopy (scale bars: 5 μm) in db/m, db/db, db/db+RAP, db/db + QZJT group, respectively. D: Western blot of nephrin and podocin expression in the four groups. E: relative band intensities were used in order to quantify nephrin expression levels. F: relative band intensities were used in order to quantify podocin protein expression levels. Eight-week-old db/db mice were treated with or without QZJT or rapamycin. Db/m (control group group, n = 10), db/db (diabetic kidney disease group, n = 10), db/db+RAP (rapamycin (5 mg·kg-1·d-1), n = 10), db/db + QZJT group (QZJT 3.9 g/kg, n = 10). QZJT: Qizhi Jiangtang capsule; RAP: rapamycin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PAS: periodic acid-Schiff; Masson: Masson's trichrome staining; EM: electron microscopy. Student t-test and one-way analysis of variance were used for comparison analysis. aP < 0.01, the db/db group vs the db/m group, bP < 0.05, the db/db group vs the db/db + RAP group or db/db + QZJT group, cP < 0.05, the db/db group vs the db/m group.
Figure 2 QZJT treatment induces podocyte autophagy in db/db mice A: autophagic vesicles (autophagosomes, arrows) of podocytes in the four groups were detected by transmission electron microscopy. Scale bar: 1 μm; B: the number of autophagic vesicles was detected by transmission electron microscopy in 10-15 randomly selected electron micrographs from the four groups; C, D: Western blot of LC3I, LC3II, beclin-1 expression in db/m, db/db, db/db + RAP, db/db + QZJT. E: quantitative analysis of LC3II/LC3I protein levels. Eight-week-old db/db mice were treated with or without QZJT or rapamycin. F: quantitative analysis of beclin-1 protein levels.Eight-week-old db/db mice were treated with or without QZJT or rapamycin. Db/m (control group group, n = 10), db/db (diabetic kidney disease group, n = 10), db/db + RAP (rapamycin (5 mg·kg-1·d-1), n = 10), db/db + QZJT group (QZJT 3.9 g/kg, n = 10). QZJT: Qizhi Jiangtang capsule; HG: high glucose; NG: normal glucose; RAP: rapamycin; LC3: light chain 3; AV: autophagic vacuole. Student t-test and one-way analysis of variance were used for comparison analysis. aP < 0.05, bP < 0.01, the db/db group vs the db/m group; cP < 0.05, the db/db group vs the db/db + RAP group or db/db + QZJT group.
Figure 2 QZJT treatment induces podocyte autophagy in db/db mice A: autophagic vesicles (autophagosomes, arrows) of podocytes in the four groups were detected by transmission electron microscopy. Scale bar: 1 μm; B: the number of autophagic vesicles was detected by transmission electron microscopy in 10-15 randomly selected electron micrographs from the four groups; C, D: Western blot of LC3I, LC3II, beclin-1 expression in db/m, db/db, db/db + RAP, db/db + QZJT. E: quantitative analysis of LC3II/LC3I protein levels. Eight-week-old db/db mice were treated with or without QZJT or rapamycin. F: quantitative analysis of beclin-1 protein levels.Eight-week-old db/db mice were treated with or without QZJT or rapamycin. Db/m (control group group, n = 10), db/db (diabetic kidney disease group, n = 10), db/db + RAP (rapamycin (5 mg·kg-1·d-1), n = 10), db/db + QZJT group (QZJT 3.9 g/kg, n = 10). QZJT: Qizhi Jiangtang capsule; HG: high glucose; NG: normal glucose; RAP: rapamycin; LC3: light chain 3; AV: autophagic vacuole. Student t-test and one-way analysis of variance were used for comparison analysis. aP < 0.05, bP < 0.01, the db/db group vs the db/m group; cP < 0.05, the db/db group vs the db/db + RAP group or db/db + QZJT group.
Figure 3 QZJT treatment plays a protective role in podocytes exposed to HG by regulating autophagy A: representative western blotting images of nephrin and synaptopodin in NG, HG, HG + QZJT, HG + QZJT + 3MA groups; B: representative Western blotting images of LC3 II, LC3 I and beclin-1 in the four groups; C: quantitative analysis of nephrin expression levels; D: quantitative analysis of synaptopodin expression levels; E: quantitative analysis of LC3II/LC3I protein levels; F: quantitative analysis of beclin-1 protein levels; G1: autophagic vesicles (autophagosomes, arrows) of podocytes in NG group were detected by transmission electron microscopy; G2: autophagic vesicles (autophagosomes, arrows) of podocytes in HG group were detected by transmission electron microscopy; G3: autophagic vesicles (autophagosomes, arrows) of podocytes in HG + QZJT group were detected by transmission electron microscopy; G4: autophagic vesicles (autophagosomes, arrows) of podocytes in HG + QZJT + 3MA group were detected by transmission electron microscopy; H: the number of autophagic vesicles was detected by transmission electron microscopy in 10-15 randomly selected electron micrographs from each group. The differentiated podocytes were incubated with normal glucose (NG, 5.5 mM) or high glucose (HG, 35 mM). The cells in the QZJT groups were preconditioned with 10% drug-containing serum for 2 h, then cultured in high glucose for 48 h (HG + QZJT). The cells in the QZJT + 3MA group were pre-treated with 5 mM of 3MA for 1 h before QZJT treatment, then cultured in high glucose for 48 h (HG + QZJT + 3MA). QZJT: Qizhi Jiangtang capsule; HG: high glucose; NG: normal glucose; LC3: light chain 3; AV: autophagic vacuole; 3MA: 3 methyladenine. Student t-test and one-way analysis of variance were used for comparison analysis. aP < 0.01, the HG group vs the NG group; bP < 0.05, cP < 0.01, the HG group vs the HG + QZJT group or HG + QZJT + 3MA group; dP < 0.05, the HG group vs the NG group.
Figure 3 QZJT treatment plays a protective role in podocytes exposed to HG by regulating autophagy A: representative western blotting images of nephrin and synaptopodin in NG, HG, HG + QZJT, HG + QZJT + 3MA groups; B: representative Western blotting images of LC3 II, LC3 I and beclin-1 in the four groups; C: quantitative analysis of nephrin expression levels; D: quantitative analysis of synaptopodin expression levels; E: quantitative analysis of LC3II/LC3I protein levels; F: quantitative analysis of beclin-1 protein levels; G1: autophagic vesicles (autophagosomes, arrows) of podocytes in NG group were detected by transmission electron microscopy; G2: autophagic vesicles (autophagosomes, arrows) of podocytes in HG group were detected by transmission electron microscopy; G3: autophagic vesicles (autophagosomes, arrows) of podocytes in HG + QZJT group were detected by transmission electron microscopy; G4: autophagic vesicles (autophagosomes, arrows) of podocytes in HG + QZJT + 3MA group were detected by transmission electron microscopy; H: the number of autophagic vesicles was detected by transmission electron microscopy in 10-15 randomly selected electron micrographs from each group. The differentiated podocytes were incubated with normal glucose (NG, 5.5 mM) or high glucose (HG, 35 mM). The cells in the QZJT groups were preconditioned with 10% drug-containing serum for 2 h, then cultured in high glucose for 48 h (HG + QZJT). The cells in the QZJT + 3MA group were pre-treated with 5 mM of 3MA for 1 h before QZJT treatment, then cultured in high glucose for 48 h (HG + QZJT + 3MA). QZJT: Qizhi Jiangtang capsule; HG: high glucose; NG: normal glucose; LC3: light chain 3; AV: autophagic vacuole; 3MA: 3 methyladenine. Student t-test and one-way analysis of variance were used for comparison analysis. aP < 0.01, the HG group vs the NG group; bP < 0.05, cP < 0.01, the HG group vs the HG + QZJT group or HG + QZJT + 3MA group; dP < 0.05, the HG group vs the NG group.
Figure 4 Effect of QZJT on PI3K/AKT pathway in db/db mice and HG-induced podocytes A: representative western blotting images of p-PI3K, p-AKT and p-mTOR in the four groups of mice; B: quantitative analysis of p-PI3K expression levels in the four groups of mice; C: quantitative analysis of p-AKT expression levels in the four groups of mice; D: quantitative analysis of p-mTOR expression levels in the four groups of mice. Eight-week-old db/db mice were treated with or without QZJT or rapamycin. Db/m (control group group, n = 10), db/db (diabetic kidney disease group, n = 10), db/db + RAP (rapamycin (5 mg·kg-1·d-1), n = 10), db/db + QZJT group (QZJT 3.9 g/kg, n = 10). QZJT: Qizhi Jiangtang capsule; HG: high glucose; NG: normal glucose; RAP: rapamycin; p-PI3K: phosphorylated phosphatidylinositol 3-kinase; p-AKT: phosphorylated protein kinase B; p-MTOR: phosphorylated mammalian target of rapamycin. Student t-test and one-way analysis of variance were used for comparison analysis. aP < 0.01, the db/db group vs the db/m group, bP < 0.01, cP < 0.05, the db/db group vs the db/db + RAP group or db/db + QZJT group; E: representative western blotting images of p-PI3K, p-AKT and p-mTOR in NG, HG, HG + QZJT, HG + QZJT + 3MA groups. F: relative band intensities were used in order to quantify protein p-PI3K expression levels; G: relative band intensities were used in order to quantify protein p-AKT expression levels; H: relative band intensities were used in order to quantify protein p-mTOR expression levels.The differentiated podocytes were incubated with normal glucose (NG, 5.5 mM) or high glucose (HG, 35 mM). The cells in the QZJT groups were preconditioned with 10% drug-containing serum for 2 h, then cultured in high glucose for 48 h (HG + QZJT). The cells in the QZJT + 3MA group were pre-treated with 5 mM of 3MA for 1 h before QZJT treatment, then cultured in high glucose for 48 h (HG + QZJT + 3MA). 3MA: 3 methyladenine. Student t-test and one-way analysis of variance were used for comparison analysis. dP < 0.01, eP < 0.05, the HG group vs the NG group; fP < 0.01, the HG group vs the HG + QZJT group or HG + QZJT + 3MA group.
Figure 4 Effect of QZJT on PI3K/AKT pathway in db/db mice and HG-induced podocytes A: representative western blotting images of p-PI3K, p-AKT and p-mTOR in the four groups of mice; B: quantitative analysis of p-PI3K expression levels in the four groups of mice; C: quantitative analysis of p-AKT expression levels in the four groups of mice; D: quantitative analysis of p-mTOR expression levels in the four groups of mice. Eight-week-old db/db mice were treated with or without QZJT or rapamycin. Db/m (control group group, n = 10), db/db (diabetic kidney disease group, n = 10), db/db + RAP (rapamycin (5 mg·kg-1·d-1), n = 10), db/db + QZJT group (QZJT 3.9 g/kg, n = 10). QZJT: Qizhi Jiangtang capsule; HG: high glucose; NG: normal glucose; RAP: rapamycin; p-PI3K: phosphorylated phosphatidylinositol 3-kinase; p-AKT: phosphorylated protein kinase B; p-MTOR: phosphorylated mammalian target of rapamycin. Student t-test and one-way analysis of variance were used for comparison analysis. aP < 0.01, the db/db group vs the db/m group, bP < 0.01, cP < 0.05, the db/db group vs the db/db + RAP group or db/db + QZJT group; E: representative western blotting images of p-PI3K, p-AKT and p-mTOR in NG, HG, HG + QZJT, HG + QZJT + 3MA groups. F: relative band intensities were used in order to quantify protein p-PI3K expression levels; G: relative band intensities were used in order to quantify protein p-AKT expression levels; H: relative band intensities were used in order to quantify protein p-mTOR expression levels.The differentiated podocytes were incubated with normal glucose (NG, 5.5 mM) or high glucose (HG, 35 mM). The cells in the QZJT groups were preconditioned with 10% drug-containing serum for 2 h, then cultured in high glucose for 48 h (HG + QZJT). The cells in the QZJT + 3MA group were pre-treated with 5 mM of 3MA for 1 h before QZJT treatment, then cultured in high glucose for 48 h (HG + QZJT + 3MA). 3MA: 3 methyladenine. Student t-test and one-way analysis of variance were used for comparison analysis. dP < 0.01, eP < 0.05, the HG group vs the NG group; fP < 0.01, the HG group vs the HG + QZJT group or HG + QZJT + 3MA group.
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