Statistics of clinical samples

The patient information (including age, gender, liver injury level, and whether or not they received acupuncture treatment) was collected from the electronic medical records of cancer patients treated at the First Affiliated Hospital of Henan University of Traditional Chinese Medicine from March 24, 2016 to May 24, 2018. Then, the biochemical indexes of the cancer patients having platinum chemotherapy treatment who did not receive acupuncture versus those who received acupuncture (acupuncture point prescriptions including Dazhui GV14, Ganshu BL18, Shenshu BL23, and Zusanli ST36) before and after acupuncture were statistically analyzed, and these indexes include aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), γ-glutamyltranspeptidase (GGT), total bilirubin (T-Bil), alkaline phosphatase (ALP), serum creatinine (Scre), platelet ratio (Platelet), AST to platelet ratio index (APRI), and fibrosis in the liver index (FIB-4). ALT had normal upper limit (40 IU / L).

Referring to the method of Tomoyoshi et al. (Miyamoto and Domoto et al., 2022), APRI and FIB-4 can be used to evaluate the degree of hepatic fibrosis with the following formulas:

$$}=\frac(\text)\text(\text)} \, (\times }^\text)}\times 100$$

The albumin-bilirubin (ALBI) scoring system was used to grade the liver function impairment of patients in the study, with Grade 1 with score ≤ − 2.60, Grade 2 with score = > − 2.60 to ≤ − 1.39, and Grade 3 with score > − 1.39. The following scoring formula is used (Demirtas and Alessio et al., 2021): ALBI score = − 0.085 × Albumin (g/L) + 0.66 × lgTotal Bilirubin (μmol/L).

In our study, the patient inclusion criteria are as follows:

1.

In terms of pathology (cytology or histology), the patients had confirmed diagnosis of gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, esophageal cancer, hepatic cancer, lymphatic cancer, or bladder cancer for the first time.

2.

Patients receiving one or more platinum-based chemotherapies

Exclusion criteria:

1.

Hepatitis virus-positive patients and patients with underlying substantial liver diseases;

2.

Patients not using platinum-based drugs in any treatment group

This case control study complies with the ethical principles of the Declaration of Helsinki and was approved by the Ethics Committee of The First Affiliated Hospital of Henan University of Chinese Medicine Hospital (approval number: H20160219). Once the patient was included into our study, a written informed consent was provided, and the patient information was only analyzed after getting the patient’s consent. Moreover, the patients were allowed to withdraw at any time, and if the patient withdrew from our study, the information of this patient would be not identifiable by the team analyzing the data anymore.

Experimental animals

A total of 40 SPF male C57/B16 mice of 5 weeks old were used in our study, weighing 30 ± 2 g, and they were purchased from Aksomics. After adaptive feeding for 3 consecutive days, the mice were randomly classified into 4 groups by weight: the blank group, the model group, the acupuncture group, and the blocker-acupuncture group. The mice in the model group, acupuncture group and blocker-acupuncture group were injected intraperitoneally with 0.01 mg/g dose of cisplatin (DDP) according to their body weights, and the mice in the blank group were injected intraperitoneally with the same dose of 0.9% Nacl solution. The modeling was successfully completed after 24 h. According to the sequence of the modeling time, acupuncture treatment was performed 24 h after the completion of modeling. The needle insertion was about 3 mm, and the needles were retained for 6 min; the acupuncture was performed once a day for 5 consecutive days. The acupuncture point prescriptions including Dazhui GV14, Ganshu BL18, Shenshu BL23, and Zusanli ST36 (Guo, 2016). In the blocker-acupuncture group, the mice were injected intraperitoneally with 0.01 mg/g of DAPT 30 min before the acupuncture treatment every day. After the treatment was completed on the 5th day, the mice were fasted and not hydrated, and were killed by dislocation of cervical vertebrae after blood sampling from the eyeballs on the 6th day. The intact liver lobes were quickly removed after dissection. After weighing the wet weight of the liver, the surrounding connective tissues were removed, and the left lateral lobe of the liver was isolated for preservation and preparation for examination. All animal studies were conducted in strict accordance with international ethical guidelines and the Guidelines for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People’s Republic of China. The study was approved by the Henan University of Chinese Medicine Animal Ethics Committee (approval number: DWLL2018030013).

Selection of acupuncture points

During acupuncture treatment of mice, the following acupuncture points were selected: “Dazhui” (GV14), “Ganshu” (BL18), “Shenshu” (BL23), and “Zusanli” (ST36). The acupuncture points are distributed as follows: “Dazhui” os located in the depression between the seventh cervical vertebra and the first thoracic vertebra, in the middle of the back; “Ganshu” is located on both sides of the lower ninth thoracic vertebra, with one acupuncture point on the left side and one on the right side; “Shenshu” is located on both sides of the intercostal space, with one acupuncture point on each side; “Zusanli” is located in the hind limb knee joints behind the lateral side of the fibula of the mouse, about 5 mm below the tip of the fibula, with one point on the left side and one point on the right side.

RNA-Seq

Sequencing was performed using the Illumina HiSeq platform to obtain clean reads, and the Bowtie2 software was used to match the clean reads to the reference gene sequences. Then, the RSEM software was used to calculate the gene expression levels of each specimen. The DEGseq software was utilized for differential gene detection. Based on the MA-plot statistical analysis, the P-values were calculated based on normal distribution. Then, P-values were corrected to Q-values by two strategies proposed by Benjamini and Hochberg (1990) and Storey and Tibshirani (2003) [27, 28]. The genes with a multiplicity of difference of more than twofold and a Q-value ≤ 0.001 were defined as significantly differentially expressed genes.

GO and KEGG enrichment analysis

In the GO functional significance enrichment analysis, all candidate genes were mapped to individual terms in the Gene Ontology (http://www.geneontology.org/) database first, the number of genes in each term was calculated, and then the hypergeometric test was performed to identify GO terms of significant enrichment in the candidate genes by comparing them to all background genes in the species. We calculated the P-value using the basis function phyper of R (https://stat.ethz.ch/R-manual/R-manual/R-devel/library/stats/html/Hypergeometric.html). Then, multiple tests and corrections were performed on the P-value. The correction package was qvalue (https://bioconductor.org/packages/release/bioc/html/qvalue.html). Finally, with Q-value (corrected P-value) ≤ 0.05 as the threshold, the GO terms that satisfied this condition were defined as GO terms with significant enrichment in the candidate genes.

The Pathway significance enrichment analysis was performed in terms of KEGG Pathway, and the hypergeometric test was carried out to find the pathways with significant enrichment in candidate genes by comparing them to the whole genomic background. Finally, the pathways with Q-value ≤ 0.05 were defined as the pathways with significant enrichment in differentially expressed genes.

RT-PCR

The mRNA was extracted from the mouse liver tissues using the Trizol (TAKARA, Japan) method, and reverse transcribed into cDNA using All-in-One™ First-Strand cDNA Synthesis Kits (Thermo Scientific, USA). The sequences of the target genes in the Gene Bank database were used as the reference sequences, and the Primer Express Software v2.0 (ABI, USA) were used to design the primers, which were synthesized by BGI. On a ViiA 7 PCR (ABI, American) instrument, the Ct values in the reaction were collected using a corrected threshold setting, and with GAPDH as the internal reference gene, the 2−ΔΔCt method was used for relative quantification. The primer information is shown in Table 1 in the Appendix.

Western blot

The liver tissues were homogenized in the RIPA lysis buffer, the supernatant was obtained by centrifugation, and after preparing the BCA working solution, the protein concentration was determined in a Multiskan FC zymograph (ThermoFisher, America). Then, the protein electrophoresis was performed on denaturing polyacrylamide discontinuous gel (SDS-PAGE), after which, the specimen was transferred to a PVDF membrane for closure and incubated with primary antibody at 4 °C. The membrane was washed after incubation with the secondary antibody, and finally, the ECL chemiluminescence development was performed. Grey analysis of the blot area was performed using the ImageJ image analysis software (http://imagej.nih.gov/ij/). The antibody information is presented in Table 2 in the Appendix.

Histopathological analysis

HE staining: the mouse liver tissues were fixed and embedded in paraffin to make wax slices, the slices were deparaffinized and hydrated in xylene and descending alcohols, then, the hematoxylin–eosin staining was performed, and the specimen was sealed in neutral tree resin after dehydration.

Massion staining: the early treatment was the same as the HE staining, the staining was performed using Trichrome Stain Kit-Methylene Blue (Masson’s Trichrome Stain Kit, Solarbio, China), and then, the specimen was dehydrated and covered with Entellan®.

Platelet count

Blood was sampled from the eyeballs of mice in the blank group, model group and acupuncture group, and the platelet ratio was detected using the microscopic method. The blood cell counting plate was calibrated before counting, and two experimental staff completed the PLT count independently according to the counting rules, and the average value was taken.

Biochemical indexes

Blood was taken from the venous plexus of the inner canthus of the eyeballs of mice in each group, and the serum was separated. The serum ALT and AST levels were measured according to the requirements of the kit (ab282882/ab263882, Abcam) and the automatic biochemical analyzer (Biobase, China).

Data analysis

A database was established using Excel, and the SPSS 21.0 statistical analysis software was used to analyze the relevant data. The measurement data were expressed as mean (x) ± standard deviation (s), and the test standard was α = 0.05. First of all, we determined whether the samples conformed to the normal distribution. If they were normally distributed, the one-way ANOVA was employed to conduct the homogeneity test of variance, and if the variance was homogeneous, the LSD method was used to compare two samples; if the variance was not homogeneous, the Tamhane method was used to compare two samples; if the samples did not conform to normal distribution, the non-parametric test was employed.