Modified Huangqi Chifeng decoction inhibits excessive autophagy to protect against Doxorubicin-induced nephrotic syndrome in rats via the PI3K/mTOR signaling pathway

Abstract

The aim of the present study was to investigate whether modified Huangqi Chifeng decoction (MHCD) could be an effective treatment against Doxorubicin-induced nephrosis in rats and whether it regulates autophagy via the phosphoinositide-3 kinase/mammalian target of rapamycin (PI3K/mTOR) signaling pathway. A total of 40 male Sprague-Dawley rats were randomly divided into blank, model, telmisartan and MHCD groups. The rat model of nephrosis was induced by intragastric administration of Doxorubicin for 8 weeks. Rats were housed in metabolic cages and urine was collected once every 2 weeks to measure 24-h protein levels. Blood samples were obtained from the abdominal aorta and levels of albumin (ALB), total cholesterol (TCH), triacylglyceride (TG) and serum creatinine (Scr) were assessed. Renal pathological changes were examined using hematoxylin-eosin, Masson's trichome and periodic acid-Schiff staining. Podocytes and autophagosomes were observed using an electron microscope. The expression and distribution of microtubule-associated proteins 1A/1B light chain 3B (LC3), LC3-I, LC3-II, beclin-1, PI3K and mTOR were determined using immunohistochemistry and western blotting. At weeks 6 and 8, 24-h proteinuria significantly decreased in the MHCD group compared with the model group (P<0.05). Compared with the model group, the MHCD group exhibited significantly reduced levels of TG, TCH and Scr, as well as significantly increased ALB levels (P<0.05). MHCD was demonstrated to prevent glomerular and podocyte injury. The number of autophagosomes was significantly decreased and the expression of beclin-1, LC3, LC3-I and LC3-II was inhibited following MHCD treatment compared with the model group (P<0.05). MHCD treatment significantly increased the expression of PI3K and mTOR in Doxorubicin nephrotic rats compared with the model group (P<0.05). In conclusion, MHCD was demonstrated to ameliorate proteinuria and protect against glomerular and podocyte injury by inhibiting excessive autophagy via the PI3K/mTOR signaling pathway.

Keywords: Chinese herbal medicine; Doxorubicin-induced nephrotic rats; autophagy; mammalian target of rapamycin; phosphoinositide-3 kinase.

Figure 1.

MHCD ameliorates proteinuria over time. Data are expressed as the mean ± standard deviation (n=10). *P<0.05 and **P<0.01 vs. the blank group; #P<0.05 vs. the model group. MHCD, modified Huangqi Chifeng decoction.

Figure 2.

MHCD ameliorates Doxorubicin-induced changes in (A) ALB, (B) Scr, (C) TG and TCH levels. Data are expressed the mean ± standard deviation (n=10). *P<0.05 vs. the blank group and #P<0.05 vs. the model group. MHCD, modified Huangqi Chifeng decoction; ALB, albumin; Scr, serum creatinine; TCH, TG, triacylglyceride; total cholesterol.

Figure 3.

MHCD prevents glomerular injury in rats with Doxorubicin-induced nephrosis. Renal pathological changes in each group were observed using light microscopy (magnification, ×400). MHCD, modified Huangqi Chifeng decoction; HE, hematoxylin-eosin; PAS, periodic acid Schiff.

Figure 4.

MHCD prevents podocyte injury in rats with Doxorubicin-induced nephrosis. Changes in podocyte morphology in each group were observed using transmission electron microscopy (magnification, ×8,000). MHCD, modified Huangqi Chifeng decoction.

Figure 5.

MHCD inhibits the formation of excess autophagosomes in podocytes. Podocytes were observed using transmission electron microscopy (magnification, ×20,500). Black arrows indicate autophagosomes. MHCD, modified Huangqi Chifeng decoction.

Figure 6.

MHCD ameliorates Doxorubicin-induced increases in autophagy by inhibiting LC3 and beclin-1 protein levels. (A) The expression of LC3 and beclin-1 in the glomeruli of Doxorubicin-induced nephrotic rats was assessed using immunohistochemistry (magnification, ×400). Quantification of (B) beclin-1 and (C) LC3. Data are expressed the mean ± standard deviation (n=10). *P<0.05 vs. the blank group; #P<0.05 vs. the model group. MHCD, modified Huangqi Chifeng decoction; LC3, microtubule-associated proteins 1A/1B light chain 3B.

Figure 7.

MHCD decreases the expression of autophagy signaling pathway proteins in rats with Doxorubicin-induced nephrosis. LC3-I, LC3II, beclin-1, PI3K and mTOR expression was (A) measured using western blotting and (B) quantified. Data are expressed as the mean ± standard deviation. *P<0.05 vs. the blank group; #P<0.05 vs. the model group. MHCD, modified Huangqi Chifeng decoction; LC3, microtubule-associated proteins 1A/1B light chain 3B; PI3K, phosphoinositide-3 kinase; mTOR, mammalian target of rapamycin.

Figure 8.

MHCD activates the autophagy signaling pathway. (A) PI3K and mTOR expression in the glomeruli of Doxorubicin-induced nephrotic rats was assessed using immunohistochemistry (magnification, ×400). Quantification of (B) PI3K and (C) mTOR expression. Data are expressed the mean ± standard deviation. *P<0.05 vs. the blank group; #P<0.05 vs. the model group. MHCD, modified Huangqi Chifeng decoction; LC3, microtubule-associated proteins 1A/1B light chain 3B; PI3K, phosphoinositide-3 kinase; mTOR, mammalian target of rapamycin.