The data of normal human cartilage and KOA cartilage were retrieved and downloaded from the Gene Expression Omnibus database (accession no. GSE75181) (19), and the merged gene expression matrix was analyzed using the limma package in R (version 4.0.5) and R Studio (version 4.0.5) (20–22) to screen the differentially expressed genes (DEGs). The DEGs of KOA were obtained by screening using the criteria |log2 (fold change)| ≥1 and P_adj<0.05, and a volcano map was plotted using Hiplot Pro (BGI Group; http://hiplot.com.cn/). The Traditional Chinese Medicine Systems Pharmacology (TCMSP) database (https://old.tcmsp-e.com/tcmsp.php) was used to identify the active ingredients of RJNTF, and then the Search Tool for Interacting Chemicals (http://stitch.embl.de/) database was used to retrieve the potential targets of each active ingredient. Venn diagrams were used to visualize the overlapping targets of RJNTF with the DEGs of KOA. Protein-protein interaction (PPI) analysis was used in the BisoGenet plug-in of Cytoscape software (version 3.8.2) (23), and the results were analyzed based on the topological parameters using the CytoNCA plug-in (http://apps.cytoscape.org/apps/cytonca) to obtain the key targets of RJNTF (24). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on the potential targets of RJNTF that were common DEGs of KOA using the clusterProfiler package in R (version 4.0.5); the potential targets related to apoptosis signaling pathways were obtained, and the key target-signaling pathway map was constructed using Cytoscape (25,26).

RJNTF is comprised of Achyranthes bidentata, Angelica sinensis, Angelica biserrata, Hansenia weberbaueriana, Saposhnikovia divaricata and Glycyrrhiza uralensis at a ratio of 4:2:3:2:2:2. The decoction protocol of RJNTF included adding water at a solid:liquid ratio of 1:10, decocting three times for 1.5 h each time, then filtering the liquid each time to remove the dregs, before combining and mixing, evaporation of the mixed liquid to lyophilize into powder, and storing the lyophilized powder in a vacuum drying oven (preparation method and use of formula authorized by the National Invention Patent; patent nos. ZL201710284659.8 and ZL201810899834.9) (27). The Cell Counting Kit-8 (CCK-8) reagent was purchased from MedChemExpress. DAPI was purchased from Beijing Solarbio Science & Technology Co., Ltd. The Novolink™ Polymer Detection System was purchased from Fuzhou Maixin Biotech Co., Ltd. The Annexin V-FITC Apoptosis Detection Kit was purchased from Nanjing KeyGen Biotech Co., Ltd. L-DMEM was purchased from Shanghai BasalMedia Technologies Co., Ltd. Hydrogen peroxide (H₂O₂) and FBS were purchased from MilliporeSigma. PARP1-knockdown lentiviral vector was constructed by Shanghai GeneChem Co., Ltd. The PCR primers and the Nuclear and Cytoplasmic Protein Extraction Kit used were obtained from Shanghai Biotechnology Co., Ltd. The PARP1 inhibitor PJ34 was purchased from MedChemExpress.

A total of 30 Male Sprague-Dawley (SD) rats (4-week-old; 80±10 g) were purchased from SLAC Laboratory Animal Technology Co., Ltd. [animal license no. SCXK (Hu) 2019-0007] and were used to obtain chondrocytes from the knee articular cartilage, which is relatively easy to obtain. The chondrocytes of 4-week-old rats are in active growth and development, their metabolism is active and the number of chondrocytes is large so they can better adapt to the in vitro culture environment (28,29). Tissues were collected in accordance with the Animal Care and Use Committee of Fujian University of Traditional Chinese Medicine (IACUC issue no. FJTCM IACUC 2022044) and the Declaration of Helsinki. The 4-week-old SPF-grade SD male rats were euthanized by intraperitoneal injection with 100 mg/kg pentobarbital sodium. The cessation of breathing and heart rate were used to confirm the death of the rats. The cartilage of the knee joints was obtained under aseptic conditions. The cartilage was rinsed with PBS three times, and then cut into 1×1×1-mm pieces with a scalpel blade, cleaned with PBS again, and transferred to a 60-mm culture dish; 5 ml 0.2% type II collagenase solution containing 1% penicillin, streptomycin and amphotericin B triple antibiotic solution, which was purchased from Shanghai BasalMedia Technologies Co., Ltd., and placed in a cell culture incubator at 5% CO₂ and 37°C for digestion, collecting the cells every 2 h. The supernatant was aspirated using a pipette and filtered through a 200-mesh nylon sieve. The filtrate was transferred to a 15-ml Eppendorf tube, centrifuged at 1,000 × g for 3 min at room temperature, and the supernatant was discarded; 4 ml complete medium was used to resuspend the cell pellet, and then cultured uniformly in T25 cell culture flasks and incubated for chondrocyte cell culture. Digestion was carried out by adding 5 ml 0.2% collagenase solution containing 1% antibiotic to the original 60-mm petri dish for a total of three times. After 24 h, the culture medium was replaced when the cells had adhered, and the medium was changed every 2 days. Cell growth was observed using an inverted light microscope. When the cell confluence reached ~90%, the chondrocytes were subcultured with 2.5 g/l trypsin (Promega Corporation) and 0.02% EDTA. Chondrocytes at passage two were used for subsequent experiments.

According to the manufacturer's instructions, a CCK-8 kit was used to separately evaluate the effects of the different concentrations of H₂O₂, PJ34 and RJNTF on the viability of the chondrocytes. Second-generation chondrocytes were seeded in 96-well plates (2,000 cells/well), and then exposed to H₂O₂ for 4 h (0, 100, 200, 300, 400, 500, 600, 700 or 1,000 µM), PJ34 for 4.5 h (0.001, 0.01, 0.1, 1, 10, 100 or 1,000 µM), and RJNTF (0, 100, 200, 400, 800, 1,600, 3,200, 6,400 or 12,800 µg/ml) for 24, 48 or 72 h, respectively. Each well was treated with 10% CCK-8 solution and incubated for further 2 h at 37°C. The absorbance was detected at 450 nm using an EnSpire^® multimode plate reader (PerkinElmer, Inc.).

Chondrocytes (2×10⁵ cells/well) were seeded into cell crawls placed in 6-well plates. After 24 h of incubation, the cell crawls were transferred into a new 6-well plate and washed twice with PBS. After fixation with 4% Paraformaldehyde Fix Solution (Beyotime Institute of Biotechnology) for 30 min and rinsing twice with PBS, the chondrocytes were stained with 500 µl DAPI for 10 min at room temperature and then washed twice with PBS for a total of 3 min per wash. The cellular morphology was observed under a Leica DMi8 inverted fluorescence microscope (Leica Microsystems, Inc.).

The Annexin V-FITC/PI apoptosis kit was used according to the manufacturer's protocol. Briefly, chondrocytes were collected and rinsed twice with PBS. Next, chondrocytes were labeled with 500 µl 1× binding buffer with 5 µl PI and 5 µl Annexin V-FITC for 15 min in the dark. Apoptosis rates were measured using a Cyto FLEX flow cytometer (Beckman Coulter Inc.) and CytExpert (version 2.4; Beckman Coulter, Inc.).

Chondrocytes (2×10⁵ cells/well) were seeded into 96-well plates. A short interfering RNA (si)-negative control (NC), si-PARP1 #1 (positive virus 114447), si-PARP1 #2 (positive virus 114448) and si-PARP1 #3 (positive virus 114449) with green fluorescent protein were purchased from GeneChem, Inc. (Table I). After 24 h, the viruses were stored at −80°C and taken out when required, placing on ice to thaw. The four viruses were individually diluted to an multiplicity of infection (MOI) of 100, 50 and 10 using complete medium. The culture media was discarded from cells, and using Nucleic acid transfection kit (Shanghai Genechem, Inc.) with nucleic acid concentration at 1×10⁸ TU/ml and the effective transfection enhancers HiTransG A and HiTransG P (GeneChem, Inc.) were added according to the grouping, and mixed and cultured at 37°C, with 5% CO₂, for 16 h. After transfection, the medium was replaced with 10% FBS DMEM, and cells were incubated at 37°C, with 5% CO₂ for 48 h, and the transfection efficiency was evaluated under a fluorescence microscope. The transfection efficiency was calculated as follows: Transfection efficiency=(number of fluorescent cells/total number of cells) ×100. After singling out viruses with high transfection efficiency, the chondrocyte apoptosis model was replicated.

Chondrocytes (3×10⁶ cells/well) were seeded in 6-well plates and treated with one of the aforementioned different siRNA constructs. Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and the RNA concentration was measured. cDNA was synthesized using an Evo M-MLV RT kit (Hunan Acre Bioengineering Co., Ltd.), according to the manufacturer's protocol, and amplified by qPCR using SYBR Green Pro Tap (Hunan Acre Bioengineering Co., Ltd.) on a Bio-Rad CFX96 amplifier. The thermocycling conditions were 95°C for 3 min; followed by 40 cycles of amplification at 95°C for 10 sec and 60°C for 30 sec. β-actin was used as the internal control, and relative gene expression was normalized to β-actin mRNA expression and analyzed using the 2^−ΔΔCq method (30). The sequences of the primers are listed in Table II.

Cells were washed three times with cold PBS and blotted dry, and then 120 µl RIPA (Beyotime Institute of Biotechnology) buffer supplemented with 1 mM PMSF was added for 30 min on ice; the petri dish was shaken once every 10 min, and the lysate was transferred to a 1.5 ml EP tube which was centrifuged at 4°C for 5 min at 12,000 × g. The supernatant was collected, and a total of 5 µl supernatant was used for BCA protein quantification, and 5× SDS-PAGE protein sampling buffer was added to the remaining supernatant. Nucleus and cytoplasmic proteins were extracted according to the manufacturer's instructions using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology; cat. no. P0028), and the buffer was mixed with the protein supernatant and then heated in a thermostatic metal bath at 99°C for 10 min to denature the proteins sufficiently and stored at −80°C. A total of 30 µg protein sample was loaded per lane and separated by 10% or 15% SDS-PAGE at 30 V for 10 min, 80 V for 30 min, and 110 V for 55 min and subsequently transferred to a 0.45-µm PVDF membrane; 15% PAGE gels were used for caspase-3, cleaved-caspase-3, AIF, MIF, histone and β-actin, while 10% PAGE gels were used for PARP1, cleaved-PARP1, PAR and β-actin. Both the target protein and the corresponding internal reference were run on the same membrane; this was cut into strips and each was probed separately to ensure that every protein can be incubated with a specific antibody. After the membranes were blocked with NcmBlot blocking buffer for 10 min at 25°C, membranes were incubated with primary antibodies against PARP1 (1:5,000; Proteintech Group, Inc.; cat. no. 66520-1-Ig), caspase-3 (1:1,000; Proteintech Group, Inc.; cat. no. 19677-1AP), cleaved PARP1 (1:1,000; Cell Signaling Technology, Inc.; cat. no. 94885S), cleaved caspase-3 (1:1,000; Cell Signaling Technology, Inc.; cat. no. 9661S), PAR (1:200; Enzo Life Sciences, Inc.; cat. no. ALX-804-220-R100), AIF (1:1,000; cat. no. 5318S; Cell Signaling Technology, Inc.), MIF (1:1,000; Abcam; cat. no. Ab175189), β-actin (1:1,000; Cell Signaling Technology, Inc.; cat. no. 8457S), or histone (1:1,000; Abcam; cat. no. Ab1791) at 4°C overnight. The following day, membranes were washed with TBST (TBS with 0.1% Tween-20, 10X) three times and subsequently incubated with corresponding the HRP-conjugated secondary antibodies (1:20,000; Cell Signaling Technology, Inc.; cat. no. 7074S) at 25°C for 1 h. The membranes were rinsed three times with TBST, and signals were visualized with a Bio-Rad ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc.), which using enhanced chemiluminescence western blot substrate. Densitometry analysis was performed using ImageJ (version win64; National Institutes of Health) and normalized to the respective β-actin or histone band.

Diluted antibody (400 µl) against AIF (1:1,000; cat. no. 5318S; Cell Signaling Technology, Inc.), MIF (1:1,000; cat. no. Ab175189; Abcam) and β-actin (1:1,000; cat. no. 8457S; Cell Signaling Technology, Inc.) was aspirated into 50 µl magnetic beads and mixed well, before incubation at 4°C for 2 h. After magnetic separation, the magnetic beads were collected, 500 µl phosphate-buffered saline (Shanghai BasalMedia Technologies Co., Ltd.) was added, the magnetic beads were mixed well and the supernatant was discarded after magnetic separation. Washing was performed four times. A total of 200 µl lysis buffer (Beyotime Institute of Biotechnology) was added, followed by lysis at 4°C for 30 min, with agitation of the cells every 10 min to ensure full contact with the lysis buffer, and then centrifugation at 4°C for 10 min at 12,000 × g. The supernatant was collected and set aside at 4°C. After BCA quantification, 20 µl SDS-PAGE loading buffer was added to the magnetic beads and mixed uniformly, after which the magnetic beads were heated at 99°C for 10 min, the beads were separated and the supernatant was used for western blotting.

Data analysis was performed using GraphPad Prism (version 8.0; Dotmatics). A unpaired two-sample t-test was used to compare differences between two groups. For multiple group comparisons, one-way ANOVA was used, followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The targets of RJNTF for the treatment of KOA were imported into Cytoscape to generate the PPI network, and a total of 1,966 related targets and 48,840 target-to-target interrelationships were obtained; the plugin CytoNCA was further used to filter the network nodes based on their topological attributes using the degree centrality value, after which, 531 relevant targets and 2,218 target-target interrelationships were obtained by excluding irrelevant nodes. Finally, 143 key targets and 3,600 target-target interrelationships were obtained by screening with the betweeness centrality value, including STAT3, PI3K, RAF, RAS, AKT, PARP1 and JALK2, the mechanisms of which include proliferation, inflammation and apoptosis. After layers of screening, PARP1, a special apoptosis target, was found among several targets (Fig. 2A).

The results of KEGG analysis showed that RJNTF could treat KOA by interfering with multiple signaling pathways such as NF-κB, apoptosis, HIF-1 signaling pathway, JAT-STAT signaling pathway and IL-17 signaling pathway (Fig. 2B) (31–34). Based on the results of a large number of previous studies showing that the PARP1/AIF pathway was closely related to apoptosis (35–37), while the results of network pharmacology indicated that the treatment of KOA by RJNTF was related to the PARP1 apoptotic pathway; however, there are no studies assessing the relationship between the PARP1/AIF pathway and apoptosis of chondrocytes. Therefore, the subsequent experiments were designed to ascertain the underlying mechanism.

The transfection efficiency of all three viruses reached >80% when using HiTransG P transfection enhancement solution and an MOI of 100. The effective transfection conditions for all three PARP1-knockdown lentiviruses included MOI=100, the transfection enhancement reagent was HitransG P and the length of time after which the solution was changed following transfection was 16 h (Fig. 5A). The results of RT-qPCR and western blotting showed that PARP1 expression was lowest in cells transfected with si-PARP1 #3 (Fig. 5B-D); thus, this construct was used for all subsequent experiments. The flow cytometry results showed that chondrocyte apoptosis was decreased in the si-PARP1 group compared with that in the si-NC group. Compared with the si-NC group, the rate of apoptosis was increased in the si-NC+ H₂O₂ group. The chondrocyte apoptosis rate was decreased in the si-PARP1+ H₂O₂ group compared with the si-NC+ H₂O₂ group (Fig. 5E and F). Meanwhile, it was found that knockdown of PARP1 resulted in a decrease in PARP1, cleaved PARP1, PAR and nucleus AIF and MIF levels, and an increase in cytoplasmic AIF and MIF expression (P<0.05; Fig. 6A-I). In conclusion, the knockdown of PARP1 effectively inhibited chondrocyte apoptosis.

Apoptosis is closely related to the target PARP1, and although PJ34 is a commonly used PARP1 inhibitor, its inhibitory effect on chondrocytes is unknown. To determine the cytotoxic effect of PJ34, chondrocytes were exposed to PJ34 at different concentrations (0, 0.001, 0.01, 0.1, 1, 10, 100 and 1,000 µM) for 4.5 h. PJ34 had no effect on the activity of chondrocytes at all tested concentrations (Fig. 7A). Based on a previous study (12), 10 µM PJ34 was selected for subsequent experiments. Moreover, apoptosis was reduced when cells were pretreated with PJ34 hydrochloride, suggesting the PARP1-dependent pathway played a pivotal role in chondrocyte apoptosis (Fig. 7B and C). To understand the mechanism of action and determine the impact of PJ34 on chondrocytes, an immunoblot profiling of key factors was performed. Compared with the model group, the protein expression levels of PAR and nucleus AIF and MIF were downregulated, while that of cytoplasmic AIF and MIF was upregulated in the inhibitor group (P<0.05), and the differences in the protein expression levels of caspase-3, cleaved caspase-3, PARP1, and cleaved PARP1 were not statistically significant. The results of Co-IP suggested that AIF interacted with MIF, and that this interaction was reduced by the addition of PJ34. In summary, inhibition of PARP1 effectively reduced chondrocyte apoptosis (Fig. 8A-K).

As it was aforementioned, chondrocyte apoptosis was at least partly mediated by the PARP1/AIF pathway. Using CCK-8 assays, 800, 1,600 and 3,200 µg/ml RJNTF were finally selected to treat cells for 48 h for the subsequent experiments (Fig. 9A). The flow cytometry results showed that compared with the model group, the apoptotic rate of chondrocytes was reduced in the RJNTF low, medium and high dose group, and the apoptotic rate of chondrocytes in the RJNTF high dose group was lower in the PJ34 treated group (P<0.05; Fig. 9B and C). Western blotting showed that compared with the model group, the protein expression levels of cleaved caspase-3, cleaved PARP1, PAR, and nucleus AIF and MIF were downregulated, and PARP1, caspase-3, and cytoplasmic AIF and MIF were upregulated in the RJNTF medium and high dose groups (P<0.05). Compared with the inhibitor group, caspase-3 and PARP1 protein expression levels were upregulated in the RJNTF high dose groups, and cleaved caspase-3, cleaved PARP1 and protein expression levels were downregulated in the RJNTF high dose groups (P<0.05; Fig. 10A-F). AIF and MIF protein expression levels were detected in the precipitates of both the model group and the RJNTF group, indicating that there was an interaction between AIF and MIF, and this interaction was reduced after the addition of RJNTF (Fig. 11A-E). In conclusion, RJNTF could effectively reduce chondrocyte apoptosis by inhibiting the PARP1/AIF pathway.