Animals
Thirty-two male Wistar rats (200 ± 30 g) were kept in a 12-h light/dark cycle with ad libitum access to water and food. The current protocol was approved by the Animal Care and Use Committee of Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine. All experimental procedures were conducted strictly in accordance with the Guide for Care and Use of Laboratory Animals.
Drugs and chemicals
PTX was purchased from Shanghai Tauto Biotech Co., Ltd. (Shanghai, China). Crude drugs, including Astragali Radix (AR), Cinnamomi Ramulus (CR), Paeonia Radix Alba (PRA), Jujubae Fructus (JF), and Zingiberis Rhizoma (ZR) were provided by Jiangsu Hospital of Integrated Traditional Chinese and Western Medicine, and authenticated based on the instructions recorded in the Chinese Pharmacopoeia (2015 edition). The voucher specimens were deposited in Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine.
HGWD preparation
HGWD was prepared in the following procedure: AR (140 g), CR (70 g), PRA (70 g), JF (70 g), and ZR (70 g) were immersed in distilled water (10 times the total weight) for 0.5 h and then gently refluxed two times, each time for 1 h. The two liquid extracts were combined and concentrated to 2 g/ml under reduced pressure and then stored at -80°C before use.
Experimental design and treatment protocol
A schematic of the experimental design is shown in Fig. 2a. Rats were randomly divided into four groups (n = 8). PTX (2 mg/kg, prepared in cremophor EL/ethanol at a ratio of 1:1) was injected intraperitoneally (i.p.) for four alternate days (days 1, 3, 5, and 7) to induce PIPN, as described previously [13]. HGWD 10 or 20 g/kg was administered orally once a day for 3 weeks from the first dose of PTX injection (1 h prior to each PTX injection, if coinciding with chemotherapy treatment). Behavioural tests were performed before and on days 6, 8, 11, 14, and 21 after the first PTX withdrawal. Blood and tissues were collected within 24 h after the final dose.
Mechanical allodynia (von Frey test)
Mechanical allodynia was evaluated using von Frey hair unit (IITC, Life Sciences, USA) according to a previously reported method [13]. Stimuli were applied to the hind paw of rats, and the minimum force required to elicit a clear paw withdrawal or shaking was recorded. Measurements were repeated three times at 5-min intervals, and the mean value was calculated.
Thermal hyperalgesia (hot-plate test)
Thermal hyperalgesia was assessed by hot plate at 52°C. The latency to jump or hind-paw lick was recorded. A cut-off time of 30 s was applied to avoid tissue damage.
Histological analysis of dorsal root ganglia (DRG) and sciatic nerve tissue
The dissected lumbar 4–6 (L4-6) DRG was fixed in 10% formalin solution, embedded in paraffin, and then sliced into 4–5 µm-thick sections. The sections were then stained with Nissl staining solution and examined microscopically. Sciatic nerve samples were harvested, fixed in 4% glutaraldehyde, and post-fixed in 1% osmic acid at 25°C for 2 h. Samples were then dehydrated with graded ethanol series and embedded in Epon 812. Ultrathin sections (80 nm) were prepared, double-stained with uranium acetate and lead citrate, and then observed with an HT7700 transmission electron microscope (Hitachi, Tokyo, Japan).
Quantification of IENF density
For quantification of IENFs, hind-paw intra-plantar skins of rats were collected for immunohistochemical studies. The antibodies used were anti-protein gene product 9.5 (PGP9.5) primary Ab (ab108986, 1:200; Abcam, Cambridge, MA, USA) and Cy3-conjugated goat anti-rabbit IgG (ab6939, 1:300; Abcam). PGP9.5-positive IENFs were counted and expressed as the numbers of fibres/length of epidermis (IENFs/mm). The scorer was completely blinded to the experimental setup.
Measurement of nerve growth factor (NGF) and oxidative stress markers
Serum levels of NGF and markers of oxidative stress, such as superoxide dismutase (SOD), malondialdehyde (MDA), and 8-isoprostane F2α, were measured. MDA and SOD levels were examined by colorimetric kits (Beyotime Biotechnology Co.Ltd, Shanghai, China). NGF and 8-isoprostane F2α levels were measured using a rat beta-NGF ELISA Kit (Invitrogen, Grand Island, NY, USA) and 8-iso-PGF2α ELISA kit (Enzo Life Sciences, New York, USA), respectively.
Construction of the chemical component database and screening of active compounds with its targets
All chemical ingredients in HGWD were collected from the TCMSP and TCMID databases. The TCMSP database is a systematic pharmacology platform that reveals the mechanisms of Tradition Chinese Medicine by identifying a drug-target network, covering pharmacokinetic properties including absorption, distribution, metabolism, and excretion (ADME). In the present study, components with oral bioavailability (OB) ≥ 30%, drug likeness (DL) ≥ 0.18, and drug half-life (HL) ≥ 4 were selected as active ingredients possessing biological activity for further study. The TCMID database was applied to replenish HGWD ingredients according to target proteins that were not recorded in the TCMSP database. Next, to reveal the pharmacological mechanism of the active ingredients of HGWD, the potential targets of the candidate active compounds were predicted using the QSAR model in TargetNet. The target information collected from TCMSP, TCMID, and DrugBank databases and literatures were integrated to supplement the target database.
Construction of “HGWD-PIPN” common targets database
The main targets of PIPN-related diseases were obtained from the following databases: OMIM, GeneCards, NCBI-gene, and DisGENET. In this study, “paclitaxel-induced peripheral neuropathy” was used as the main keyword to screen disease-related targets in the above bioinformatics websites. Next, we mapped the prediction targets of the HGWD with PIPN-related disease targets. Both the PIPN-related targets and HGWD-related targets obtained were imported to draw a Venn diagram, and the overlapping part showed the common targets of diseases and compounds. In addition, the common targets network was established and inspected using the Cytoscape 3.7.2 software.
Gene Ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG), and target proteins distribution enrichment analyses
GO and KEGG pathway enrichment analyses were carried out using the KOBAS website. Enrichment analysis for cell and tissue distribution of the target proteins of HGWD was conducted on the Metascape website. To elucidate whether HGWD exerts neuroprotective effect in chemotherapy, we subjected the candidate targets to enrichment analysis of biological processes, cellular components, molecular functions, and signalling pathways, with P-value < 0.05 considered as statistically significant. The results were mainly visualised using the ggplot2 package in R 3.6.0. Information of Public databases and its website involved in the study are listed in Supplementary Table S1.
Network construction
Protein-protein interaction (PPI) predictions were used to predict the outcome of pairs or groups of protein interactions. In this study, PPI network was generated by importing the common targets into the String database, with the species restricted to “Homo sapiens” and the highest confidence core > 0.9. Finally, the obtained results were exported in the TSV format and imported into the Cytoscape software for visualisation. To analyse and visualise the relationship between effective active compounds, targets, and pathways, the compound-target (C-T) network and target-pathway (T-P) network were established by Cytoscape 3.7.2 software.
Western blotting
DRG tissues were harvested, washed, homogenised, and extracted using RIPA lysis buffer (Beyotime Biotechnology Co., Ltd.) containing protease inhibitor. Protein concentrations of lysates were measured by the bicinchoninic acid method (Beyotime Biotechnology Co., Ltd.). Protein samples were electrophoresed in a 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk and incubated with the following primary antibodies overnight at 4°C: TLR4 (1:1000; Affinity Biosciences, Ltd., Cincinnati, OH, USA); MyD88, Keap1 (1:1000; Cell Signalling Technology, USA); NF-κB p65, p-NF-κB (1:1000; Absin Biotechnology Co., Ltd); PI3K, Akt, phospho-Akt(p-Akt) (1:1000; from Cell Signaling Technology, USA); Nrf2 (1:1000; Abcam); HO-1(1:1000, ABclonal Biotechnology Co., Ltd); and β-tubulin (1:3000; Affinity Biosciences, Ltd., Cincinnati, OH, USA). Afterwards, the membranes were washed with PBST, incubated with HRP-conjugated secondary antibodies (Affinity Biosciences, Ltd.), and visualised by enhanced chemiluminescence (Beyotime Biotechnology Co. Ltd.). The results were quantified using the NIH ImageJ software.
Inflammatory factors measurement
The serum levels of inflammatory cytokines, including interleukin (IL)-1β, IL-6, tumour necrosis factor-α (TNF-α), and IL-10 were tested using ELISA kits (Multi Sciences (Lianke) Biotech Co., Ltd., Hangzhou, China). IL-1β, IL-6, TNF-α, and IL-10 mRNA in DRG tissue was measured by real-time polymerase chain reaction (RT-PCR). The gene-specific primers for rat IL-1β, IL-6, TNF-α, and IL-10 are listed in Supplementary Table S2.
MTT assay
Cancer cell lines, including MDA-MB-231 and CFPAC-1 cells, were purchased from the Bank of Cell, Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were maintained in DMEM (MDA-MB-231 cells) or IMEM (CFPAC-1 cells) plus 10% foetal bovine serum at 37°C in 5% CO2. The effect of HGWD on the tumour cell-killing ability of PTX was evaluated via an enzymatic MTT assay.
Tumour xenograft model
CFPAC-1 cells (4× 106) were injected subcutaneously into the right flank of nude BALB/c mice (aged 3–4 weeks). When the tumours had grown to approximately 150–300 mm3, the mice were randomised into three groups (n = 7 per group): vehicle (normal saline), PTX (10 mg/kg in normal saline, i.p., q.d (days 1, 3, and 5)), and PTX (10 mg/kg) combined HGWD (30 g/kg, i.g., q.d (days 1–22)). Tumour size was measured every 3 days and calculated according to the following formula: tumour volume = length × width2/2. At the end of the experiment, the mice were sacrificed, and tumour xenografts were excised and weighed.
Statistical analysis
The difference between two different groups was analysed via unpaired Student’s t-test using the GraphPad Prism 8 software. A one-way analysis of variance (ANOVA) followed by Bonferroni analysis was used for in vivo comparisons among multiple groups. P < 0.05 was regarded as statistically significant.