2.1 Target prediction and protein–protein interaction (PPI) network construction

The targets of palmatine were predicted by TCM systematic pharmacology database (TCMSP, https://tcmspw.com/tcmsp.php), Pharmmapper database (http://www.lilab-ecust.cn/pharmmapper/) and Swiss target prediction database (http://swisstargetprediction.ch/). All gene targets related to CIAKI were obtained from Genecards database (https://www.genecards.org/), OMIM database (https://www.omim.org/) and Disgenet database (https://www.disgenet.org/). Standardized targets were obtained from the UniProt database(https://www.uniprot.org/). The overlapping genes and Venn diagram of palmatine and CIAKI targets were obtained from the online Venn map platform (https://bioinfogp.cnb.csic.es/tools/venny/https://bioinfogp.cnb.csic.es/tools/venny/). The gene targets were further imported into STRING database (https://string-db.org/) to explore the interactions between the known and predicted proteins. The topological parameters in the PPI network were analyzed using Cytoscape 3.9.0 software.

2.2 Enrichment of gene ontology (GO) terms and kyoto encyclopedia of genes and genomes (KEGG) pathways

We imported the obtained potential targets of palmatine for CIAKI treatment into the target gene name list through the DAVID database (https://david.ncifcrf.gov/home.jsp), restricting the species to human. GO enrichment analysis and KEGG pathway annotation analysis were performed on the potential targets to screen the important signaling pathways of palmatine in CIAKI.

2.3 Cell culture and evaluation of cell viability (MTT assay)

Plamatine and cisplatin were purchased from MACKLIN and MCE (Shanghai, China), respectively. Human embryonic kidney 293 T and human colon cancer HCT116 were provided by the Peking Union Cell Center. Human non-small cell lung cancer H460 was provided by Professor Zhi Yao (Tianjin Medical University, Tianjin, China). 293 T, H460 and HCT116 cells were cultured in DMEM, 1640 and IMDM media supplemented with 10 % (v/v) fetal bovine serum (FBS), 100 IU/mL penicillin and 100 IU/mL streptomycin, respectively. All cells were maintained in a humidified air/CO₂ incubator (5 % v/v) at 37 °C.

The MTT assay was used to determine the cell viability. Cells were inoculated into 96-well plates at a density of 5 × 10^-4 for 24 h and treated with cisplatin (12.5 μM) and/or palmatine (0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, 10000, 100000 nM) for a further 48 h. Then, DMSO was added for 10 min, after which MTT (5 mg/mL) was added for 4 h. Absorbance was measured using a microplate reader (Tecan, Austria) at 492 nm/630 nm.

2.4 Cell apoptosis assay and intracellular reactive oxygen species (ROS) assay

The FITC-Annexin V/PI double staining kit (Sanjian Biotechnology Co., LTD., Tianjin) was used to examine cell apoptosis. After palmatine and cisplatin treatment, 293 T cells were collected after trypsinization and centrifugation. Before adding PI solution (5 μL), the cells were incubated with Annexin V-FITC (5 μL) for 10 min at room temperature in the dark. After co-incubation for 10 min, apoptosis was detected by flow cytometry (Accuri, USA).

The peroxide-sensitive fluorescent probe 2, 7-dichlorofluorescein diacetate (DCFH-DA) (Solebo Technology Co., LTD., Beijing) was used to measure the level of intracellular ROS. After exposure to palmatine and cisplatin, cells were harvested and cultured in serum-free medium containing DCFH-DA (10 μM) for 30 min, followed by detection using flow cytometry (Accuri, USA).

2.5 CIAKI animal experiments

Male C57BL/6J mice (6–8 weeks) were purchased from Sikebeisi Biotechnology Co. Ltd (Henan, China; permit number SYXK (Tianjin), 2020–0005). All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Tianjin University of Science and Technology and approved by the Animal Ethics Committee of Tianjin University of Science and Technology. All mice were randomly divided into four groups (n = 8 ∼ 10) including normal, model, low dose of palmatine (PAL-L), high dose of palmatine (PAL-H). Normal and model mice received 5 % glucose (10 mL/kg) by intraperitoneal injection for 7 days. PAL-L and PAL-H mice were injected intraperitoneally with palmatine at 5 mg/kg and 10 mg/kg, respectively, for 7 days. Except for the normal group, all other groups were injected with a single dose of cisplatin (20 mg/kg) on day 4. Normal mice received 0.9 % NaCl (10 mL/kg) on day 4.

On day 7, all mice were sacrificed. Blood samples were collected and centrifuged at 3000 rpm for 10 min to obtain serum, which was stored at −80 °C. Kidneys were weighed and pathological examination was performed by HE staining. The renal tubular injury score was calculated based on the degree of lesion: no obvious lesion, 0 points; slight or very little, 0.5 points; mild or small, 1 point; moderate or more, 2 points; severe or more quantity, 3 points; extremely severe or a lot, 4 points (Li et al., 2019). The concentrations of creatinine (CRE), blood urea nitrogen (BUN), superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) were determined using commercial assay kits purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). The levels of TNF-α, IL-6 and IL-1β were determined by enzyme-linked immunoassay (ELISA) purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China).

2.6 Western blot

Cell or kidney samples were rapidly lysed with protein lyase. Protein samples were obtained after centrifugation and determined by the BSA method. Protein was separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with Bax (41162S, 1:1000), Bcl-2 (15071S, 1:1000), p65 (8242S, 1:1000); p-p65 (3033S, 1:1000); p-ERK (4370S, 1:2000), ERK (4695S, 1:2000), p-p38 (4511S, 1:1000), p38 (8690S, 1:1000), p-Akt (4060S, 1:2000), Akt (7292S, 1:2000), p-mTOR (5536S, 1:1000), mTOR (2893S, 1:1000) or α-Tubulin (2148S, 1:1000) overnight. Subsequently, the membranes were incubated with the appropriate secondary antibodies (Invitrogen-Thermo Fisher Scientific, USA) for 1 h. α-Tubulin was used as an internal reference protein. All antibodies were purchased from Cell Signaling Technology (USA). The grey intensity of the bands was analyzed and quantified using ImageJ software.

2.7 Statistical analysis

All data were represented as mean ± SD. One-way analysis of variance (ANOVA) was used for statistical analysis. p < 0.05 was considered statistically significant.

3.1 Network pharmacological analysis of the relationship between palmatine and CIAKI

A total of 245 palmatine-related target genes were obtained by searching the TCMSP, Swiss target prediction and Pharmmapper database. In addition, a total of 570 CIAKI targets were obtained from Genecards, OMIM and the Disgenet database. After discarding duplicate data, 61 potential palmatine targets against CIAKI were obtained (Fig. 1A). The overlapping targets were imported into the String database to achieve PPI network with a confidence score > 0.4, and the network of core proteins were constructed by Cytoscape software (Fig. 1B). Among the core proteins, the top ten proteins of palmatine against CIAKI were acquired based on the degree values and other topological properties (Table 1).

Close

Fig. 1

Prediction of potential core targets, construction of PPI networks and enrichment analysis of palmatine against CIAKI. (A) Venn diagram of potential anti-CIAKI targets. (B) PPI network of proteins. (C) Top 5 Gene Ontology biological processes, cellular components and molecular functions. (D) Top 10 KEGG pathways. The abscissa represents the gene proportion, the ordinate represents pathway name, the bubble size represents the number of targets in the pathway and the color represents the p value.

Export to PPT

To comprehensively describe the mechanism of palmatine against CIAKI, we used the DAVID database to perform GO and KEGG enrichment analysis. A total of 767 GO enrichment items related to CIAKI were obtained, of which 89 were related to cellular component, 412 were related to biological process, and 266 were related to molecular function. KEGG pathway enrichment analysis showed that the potential targets were mapped to 140 pathways. As shown in Fig. 1C-D, only the top 5 related functions were shown in the bubble diagrams. The key targets of the interaction between palmatine and CIAKI were mainly involved in the negative regulation of apoptotic process, positive regulation of kinase activity, PI3K signaling and other biological processes. The signaling pathways revealed by palmatine against CIAKI were mainly involved in oxidative stress, FoxO, PI3K-Akt and MAPK.

3.2 Palmatine inhibited cisplatin-induced renal cell injury and improved cisplatin cytotoxicity to cancer cells

The concentrations of palmatine within 1 μM were safe for 293 T cells (Fig. 2A). As shown in Fig. 2B, cell viability was decreased when exposed to cisplatin (12.5 μM) alone, while it was significantly increased in the presence of palmatine. The data suggested palmatine attenuated cisplatin-induced toxicity in 293 T cells.

Close

Fig. 2

The effect of palmatine (PAL) on cisplatin induced damage to 293 T cells. Palmatine could increase the cell viability within the concentration of 100 nM, but significantly decreased the cell viability at 10000–100000 nM (A). Cisplatin (12.5 μM) significantly decreased cell viability at 12.5 μM, while palmatine significantly improved cell viability (B). Palmatine had little effect on H460 (C) and HCT116 cells (D), while cisplatin was cytotoxic to both cancer cells. Palmatine showed a slight ameliorating effect on the cytotoxicity of cisplatin. #p < 0.05 vs control and * p < 0.05 vs cisplatin.

Export to PPT

The cytotoxicity activity of cisplatin with or without palmatine was performed on H460 and HCT116 cells. As shown in Fig. 2C-D, palmatine had little cytotoxicity to the cancer cell lines. Cisplatin (12.5 μM) could reduce the cell viability of H460 and HCT116 cells. Meanwhile, palmatine could improve the cytotoxicity of cisplatin to H460 and HCT116 cells. This means that when cisplatin exerted anti-tumor effects, it was toxic to normal kidney cells, and palmatine could alleviate this toxicity while improving the anti-tumor effect of cisplatin.

3.3 Palmatine inhibited cisplatin-induced renal cell apoptosis and intracellular ROS accumulation

We further investigated the effect of palmatine on cisplatin-induced apoptosis and ROS accumulation in 293 T cells. Cisplatin-induced apoptosis of 293 T cells was significantly inhibited by pretreatment with palmatine in a dose-dependent manner (Fig. 3A and B). Cisplatin caused the accumulation of endogenous ROS, which contributed to cell death, while palmatine alone had almost no effect on ROS. However, the cellular ROS accumulation induced by cisplatin was reduced after palmatine treatment (Fig. 3C and D). The results revealed that palmatine attenuated the ability of cisplatin to induce apoptosis and ROS production in kidney cells.

Close

Fig. 3

Effects of palmatine (PAL) on cisplatin-induced apoptosis and ROS in 293 T cells. Cells were treated with palmatine (0.001, 1, 1000 nM) and cisplatin (12.5 μM) for 48 h. The cells were stained with Annexin V-FITC/PI fluorescence and detected by flow cytometry to test the apoptosis (A). Palmatine alone had little effect on cell apoptosis, while it could significantly decrease cell apoptosis induced by cisplatin (B). The cells were stained with DCFH-DA fluorescence and detected by flow cytometry to evaluate ROS (C). Cisplatin caused the accumulation of ROS in 293 T cells, which was significantly reduced by palmatine (D). #p < 0.05 vs control and *p < 0.05 vs cisplatin-treated group.

Export to PPT

3.4 Palmatine attenuated cisplatin-induced renal cell injury by suppressing NF-κB/MAPK and activating PI3K/Akt pathways

Apoptosis related proteins were detected. In 293 T cells exposed to cisplatin, palmatine markedly blocked increased Bax and significantly increased decreased Bcl-2 (Fig. 4A). The results suggested that palmatine could inhibit cisplatin-induced apoptosis in 293 T cells.

Close

Fig. 4

Proteins regulation by palmatine (PAL) on cisplatin-induced injury of 293 T cells. The ratio of Bax/Bcl-2 was significantly increased after cisplatin, while palmatine reversed the increase (A). Cisplatin significantly decreased the levels of Akt and mTOR, which were restored to normal levels by palmatine (B). In the MAPK pathways, the levels of p65, ERK and p38 were significantly increased by cisplatin and decreased after treatment with palmatine (C). #p < 0.05 vs control, *p < 0.05 vs cisplatin group.

Export to PPT

Through network pharmacological enrichment analysis, the mechanism of palmatine against CIAKI was related to PI3K/Akt and MAPK pathways. We detected the protein expressions in the Akt and MAPK pathways. As shown in Fig. 4B, the levels of p-Akt and p-mTOR were decreased after cisplatin treatment, while they were significantly increased by the addition of palmatine. The MAPK pathway was significantly associated with the inflammatory response and played an important role in the regulation of apoptosis. The expressions of p-p65, p-p38 and p-ERK were activated after cisplatin treatment in 293 T cells, but they were obviously suppressed by palmatine (Fig. 4C).

3.5 Palmatine prevented CIAKI in animal model

The CIAKI animal model was established by a single injection of cisplatin (20 mg/kg) on the fourth day (Fig. 5A). The body weight of mice in the model group was obviously decreased, which was significantly increased by palmatine (Fig. 5B). The kidney index was elevated in the model, while it was reduced in palmatine (Fig. 5C). The high serum BUN and CRE levels indicated impaired renal function. We found that palmatine decreased the levels of BUN and CRE, especially in the high dose group (Fig. 5D and E).

Close

Fig. 5

Effect of palmatine (PAL) on the CIAKI mice model. Flow chart of drug administration (A) and body weight of mice (B) during the experiment. Kidney index (C), serum levels of CRE (D) and BUN (E) were significantly increased in the CIAKI model, while they were restored to normal after palmatine treatment. #p＜0.05 vs normal group, *p＜0.05 vs model group.

Export to PPT

3.6 Palmatine inhibited cisplatin-induced oxidative damage and inflammation

HE staining showed that CIAKI mice exhibited pathological changes, including proximal tubular cell necrosis, inflammatory cell infiltration, tissue vacuolation and luminal enlargement. In contrast, palmatine significantly alleviated the above symptoms especially in the high dose group (Fig. 6A-B). The tubular injury score of mice was significantly increased after cisplatin treatment, while it was reversed after palmatine treatment (Fig. 6C). To evaluate the oxidative stress in the mice, the levels of MDA, SOD and GSH in kidney tissue were determined. As shown in Fig. 6D-F, cisplatin induced an increase in MDA and a decrease in SOD and GSH, which was reversed by palmatine. Interestingly, palmatine restored MDA, SOD and GSH to normal expression levels. Moreover, we measured the concentrations of pro-inflammatory factors in the mice. The levels of TNF-α, IL-6 and IL-1β were significantly increased in the cisplatin group, while palmatine significantly decreased the expressions of these cytokines (Fig. 6G-I). The results indicated that palmatine prevented CIAKI by inhibiting oxidative stress and inflammation.

Close

Fig. 6

Effect of palmatine (PAL) on renal tissue pathology, oxidative stress indicators and inflammatory factors in the CIAKI mice model. The renal appearance of palmatine was similar to that of the normal group (A). HE staining showed significant changes in the renal tissue structure of the model group, while palmatine alleviated these changes (scale bar, 20 μm, B). The renal tubular injury score in HE was quantitatively analyzed according to the lesion (C). Indicators of oxidative stress including GSH (D), SOD (E) and MDA (F) in the kidney were apparently regulated in the model group, but were restored to normal levels by palmatine treatment. The concentrations of TNF-α (G), IL-6 (H) and IL-1β (I) in kidney were significantly increased in the model group, while palmatine restored them to normal levels. #p < 0.05 vs normal group, *p < 0.05 vs model group.

Export to PPT

3.7 Palmatine prevented CIAKI by regulating Akt and NF-κB/MAPK pathways

Consistent with the results in 293 T cells, the ratio of Bax to Bcl-2 was remarkably increased in the CIAKI model, which was significantly reversed by palmatine (Fig. 7A). Cisplatin decreased the phosphorylation of Akt and increased the phosphorylation of p65, p38 and ERK. After palmatine treatment, the levels of Akt, p65, p38 and ERK returned to normal expression (Fig. 7B and C). The results demonstrated that palmatine prevented CIAKI by activating the Akt pathway and suppressing the NF-κB/MAPK pathway.

Close

Fig. 7

Effects of palmatine (PAL) on kidney related protein levels in the CIAKI mice model. The ratio of Bax to Bcl-2 was significantly increased in the CIAKI model, but returned to normal after palmatine treatment (A). Akt (B) and MAPK (C) pathway proteins were affected by cisplatin and restored by palmatine. #p < 0.05 vs normal group, *p < 0.05 vs model group.

Export to PPT