Jianpi Gushen Huayu decoction ameliorated diabetic nephropathy through modulating metabolites in kidney, and inhibiting TLR4/NF-κB/NLRP3 and JNK/P38 pathways

Figure 1 Jianpi Gushen Huayu Decoction administration improved body weight loss, hyperglycemia, and kidney injury in diabetic nephropathy mice. A: Curves of body weight changes; B: Curves of fasting blood glucose changes; C: Creatinine levels; D: Blood urea nitrogen level; E: 24 h urine total protein levels. Control, model, irbesartan, low-dose Jianpi Gushen Huayu Decoction (JPGSL), medium-dose JPGS, and high-dose JPGS groups, n = 10 per group. ^bP < 0.01 as compared to the control group; ^cP < 0.05 as compared to the model group; ^dP < 0.01 as compared to the model group. JPGS: Jianpi Gushen Huayu Decoction; FBG: Fasting blood glucose; Cr: Creatinine; BUN: Blood urea nitrogen; 24h-UTP: 24 h urine total protein; JPGSL: Low-dose Jianpi Gushen Huayu Decoction; JPGSM: Medium-dose Jianpi Gushen Huayu Decoction; JPGSH: High-dose Jianpi Gushen Huayu Decoction; IRBE: Irbesartan.

Figure 2 Pathological changes in kidneys were improved following Jianpi Gushen Huayu Decoction administration. A: Hematoxylin and eosin staining; B: Kidney damage score; C: Periodic acid Schiff staining; D: Glomerulosclerosis index, magnification × 100. ^bP < 0.01 as compared to the control group; ^cP < 0.05 as compared to the model group; ^dP < 0.01 as compared to the model group. JPGSL: Low-dose Jianpi Gushen Huayu Decoction; JPGSM: Medium-dose Jianpi Gushen Huayu Decoction; JPGSH: High-dose Jianpi Gushen Huayu Decoction; IRBE: Irbesartan.

Figure 3 Jianpi Gushen Huayu Decoction reduced oxidative stress and inflammation in kidneys. A: The activities of superoxide dismutase; B: The activities of glutathione peroxidase; C: Malondialdehyde level; D: Interleukin (IL)-6 levels; E: IL-1β levels; F: Tumor necrosis factor-α levels. ^bP < 0.01 as compared to the control group; ^cP < 0.05 as compared to the model group; ^dP < 0.01 as compared to the model group. JPGSL: Low-dose Jianpi Gushen Huayu Decoction; JPGSM: Medium-dose Jianpi Gushen Huayu Decoction; JPGSH: High-dose Jianpi Gushen Huayu Decoction; IRBE: Irbesartan; SOD: Superoxide dismutase; GSH-Px: Glutathione peroxidase; MDA: Malondialdehyde; IL-6: Interleukin 6; IL-1β: Interleukin 1 beta; TNF-α: Tumor necrosis factor alpha.

Figure 4 Jianpi Gushen Huayu Decoction treatment alters the metabolites in kidneys. A: Score plots of PCA among control, model, and high-dose Jianpi Gushen Huayu Decoction (JPGSH) groups; B and C: Score plots and permutation tests of partial least squares discriminant analysis (PLS-DA) between the control and model groups; D and E: Score plots and permutation tests of PLS-DA between the model and JPGSH groups; F: Results of the pathway analysis between the control and model groups; G: Results of the pathway analysis between the model and JPGSH groups. Common pathways are marked in red. The names of the pathways are represented by “a” to “k”: a: Alanine, aspartate and glutamate metabolism; b: Arachidonic acid metabolism; c: Butanoate metabolism; d: Cysteine and methionine metabolism; e: Glycerophospholipid metabolism; f: Histidine metabolism; g: Riboflavin metabolism; h: Steroid hormone biosynthesis; i: Synthesis and degradation of ketone bodies; j: Tryptophan metabolism; k: Tyrosine metabolism. n = 6 per group. JPGSH: High-dose Jianpi Gushen Huayu Decoction.

Figure 5 Jianpi Gushen Huayu Decoction moderated the toll-like receptor 4/nuclear factor-kappa B/NOD-like receptor family pyrin domain containing 3 pathway and ameliorated inflammatory injuries to kidneys. A: Western blotting results; B: Toll-like receptor 4 levels; C: p-P65/P65 levels; D: NOD-like receptor family pyrin domain containing 3 (NLRP3) levels; E: Apoptosis-associated speck-like protein containing a CARD (ASC) levels; F: Cleaved caspase-1/caspase-1 levels; G: Mature interleukin (IL)-1β/IL-1β levels; H: IL-18 levels; I: Nlrp3 levels; J: Asc levels; K: Casp1 levels; L: Il1b levels; M: Il18 levels. ^aP < 0.05 as compared to the control group; ^bP < 0.01 as compared to the control group; ^cP < 0.05 as compared to the model group; ^dP < 0.01 as compared to the model group. JPGSH: High-dose Jianpi Gushen Huayu Decoction; TLR4: toll-like receptor 4; NLRP3: NOD-like receptor family pyrin domain containing 3; ASC: Protein apoptosis-associated speck-like protein containing a CARD; IL: Interleukin.

Figure 6 Jianpi Gushen Huayu Decoction inhibited c-Jun N-terminal kinase/P38 pathway mediated apoptosis and improved kidney injury. A: TUNEL staining; B: Percentage of positive cells in TUNEL staining; C: Western blotting results; D: p-c-Jun N-terminal kinase (JNK)/JNK level; E: p-P38/P38 levels; F: Cleaved-caspase-3/caspase-3 levels; G: Cleaved caspase-9/caspase-9 levels. ^bP < 0.01 as compared to the control group; ^cP < 0.05 as compared to the model group; ^dP < 0.01 as compared to the model group. JPGSH: High-dose Jianpi Gushen Huayu Decoction; JNK: c-Jun N-terminal kinase.

Figure 7 Jianpi Gushen Huayu Decoction could improve kidney inflammatory responses and kidney injury in diabetic nephropathy mice via the toll-like receptor 4/nuclear factor-kappa B/NOD-like receptor family pyrin domain containing 3 pathway and inhibit kidney cell apoptosis in diabetic nephropathy mice via the JNK/P38 pathway. It is possibly related to regulate cysteine and methionine metabolism, alanine, aspartate, and glutamate metabolism, tryptophan metabolism and riboflavin metabolism in kidney. NF-κB: Nuclear factor-kappa B; 5-HTTP: 5-hydroxytryptophan; NAS: N-acetylserotonin; 5-HIAA: 5-hydroxyindole-3-acetic acid; JNK: c-Jun N-terminal kinase; TLR4: toll-like receptor 4; NLRP3: NOD-like receptor family pyrin domain containing 3; ASC: Protein apoptosis-associated speck-like protein containing a CARD; IL: Interleukin.