Tween-80, span-80, and octyldodecanol were supplied by Evonik Industries AG (Germany). Caprylic acid capric triglyceride (GTCC) was purchased from BASF SE (Germany). Butylated hydroxytoluene (BHT) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). PPG-26-Buteth-26 was obtained from GATTEFOSSE (Shanghai, China). Cell-Counting Kit-8 (CCK8) and D-Hanks were obtained from Solarbio (Beijing, China). Carbomer 940 and 4-phenyl butyric acid (4-PBA) were purchased from RHAWN (Shanghai, China). Medical breathable adhesive tape was purchased from Hongsheng Medical Technology Co., Ltd. (Shanghai, China). HE, Masson, and Sirius red staining kits were obtained from Servicebio (Wuhan, China). Antibodies for P-PERK, P-IRE1α and CHOP were purchased from Proteintech Group (Rosemont, IL, United States). Primary antibodies for TXNIP and goat anti-rabbit IgG H&L (HRP) were purchased from Affinity Biosciences (Cincinnati, OH, United States), NLRP3 and caspase-1 antibodies were obtained from SAB (Maryland, United States). CultureSure Freezing Medium was purchased from FUJIFILM Wako Pure Chemical Corperation (Guangzhou, China). Type I collagenase, LPS, and monoiodoacetic acid (MIA) were obtained from Sigma-Aldrich (Sigma, St. Louis, MO, United States). 5×HiScript II qrt SuperMix (Reverse transcription reagent) and TRIzol were obtained from Vazyme (Nanjing, China). Hieff qPCR SYBR Green Master Mix (Low Rox Plus) was obtained from Yeasen (Shanghai, China). RIPA Lysis and Extraction Buffer, DMEM, and FBS were purchased from Gibco (Rockville, United States). Besides, ELISA kits for IL-1β and IL-18 were supplied by Jinyibai Biotechnology Co., Ltd. (Nanjing, China). The primers were supplied by Sangon Biotech (Shanghai, China).

“Sanse Powder” (batch No. 2010020) was obtained from Preparation Department, Affiliated Hospital of Nanjing University of Traditional Chinese Medicine (Jiangsu pharmaceutical system number Z04000566). The herbal medicine (Sanse Powder) is composed of twenty individual herbs (detailed information are shown in Table 1). In clinical use, the above doses of herbs are finely powdered and sterilized with cobalt 60. To obtain the essential oil extract, 150 g Sanse Powder was extracted in water distillation method using a PTHW type thermoregulated electric heating set device (Nanjing Nan’ao Technology Co., Ltd.) attached to a round bottom flask (5 L) with 3,000 ml of deionized water (5 h) at moderate temperature. The essential oil was dehydrated by adding a certain amount of anhydrous sodium sulfate and stored at 4°C away from light until it was used for nanomaterial analysis and biological assays.

The chemical composition of Sanse Powder and NEs-SP-EO were analyzed on an Agilent 7890A-5975C gas chromatograph-mass spectrometer (GC-MS). We separated essential oil on an HP-5 MS column with specifications: 30 m length, 0.25 mm i. d., and 0.25 μm film (Thermo Fisher Scientific, Waltham, MA, United States). We operated the spectrometer in the electron ionization mode at 70 eV and set the scan range at (m/z) 30-500. The ion source temperature was 230°C and the inlet temperature was 250°C. We injected 1 µL of the samples at a shunt ratio of 1:20. We used the following programmed operating conditions: 45°C for 2 min, 10°C/min to 100°C for 5 min, 5°C/min to 200°C for 5 min, total running time 52 min. Helium carrier gas at 1 ml/min. We identified the resultant peaks using Agilent Mass Hunter Qualitative Analysis B.06.00 software (www.agilent.com) and each component detected in the total ion chromatogram was searched through the NIST11 database and PubChem database (>80% match). The chromatographic peaks were calculated by the area normalization method to obtain the percentage of each component.

In this study, the process of formulating nanoemulsion is a two-step procedure. Briefly, firstly, the screened emulsifiers (tween-80, span-80), co-emulsifiers (octyldodecanol, PPG-26-Buteth-26), and BHT were mixed and sonicated by using a JY92-l probe ultrasonicator (Xinzhi Biotechnology Co., Ltd., Ningbo, China) at 40 kHz amplitude ultrasound for 5 min as the A phase. Then, GTCC was heated and stirred until molten and the appropriate amount of essential oil was added as the B phase. The isothermal phase A is then slowly added to phase B and stirred to form an emulsion mixture. Secondly, the emulsion mixture was homogenized three times at 120 Pa in a JN-02HC high-pressure homogenizer (Juneng Biotechnology Co., Ltd., Guangzhou, China). After cooling, NEs-SP-EO was obtained by filtration through a 0.45 μm microporous filter membrane. The NEs-SP-EO formulation ratios are shown in Table 2.

The morphology and structure of NEs-SP-EO were observed by a JEM1230 transmission electron microscope (JEOL, Tokyo, Japan), with an accelerating voltage of 120 kV. The NEs-SP-EO were diluted in ddH₂O water (1:20 v/v) and then the mixture was dropped onto the copper mesh. After 10 min, the liquid was blotted up with filter paper and dried at room temperature. Finally, the images were taken at 30,000 times magnification. The particle size values, PDI, and ZP of NEs-SP-EO were calculated in triplicate by dynamic light scattering, using a NanoZS90 Zeta Potential Analyzer (Malvern Instrument Inc., Malvern, UK). An ultraviolet-visible absorption spectrum was obtained using a UV-2401 spectrophotometer (Shimadzu, Tokyo, Japan).

Topical administration of NEs-SP-EO to the knee joint was performed on the rat model following the Guidelines for Care and Use of Experimental Animals of Nanjing University of Chinese Medicine, approved project number: 013034003002. Animals were purchased from Nanjing Qinglongshan Animal Farm (License number: SCXK-SU-201-0001). 8–10 weeks male SD rats, weighing 185–205 g, were housed in an SPF-grade environment with controlled temperature and humidity and an alternating 12/12 h light/dark cycle. After 1 week of environmental adaptation, rats were randomly divided into four groups: Normal group (n = 10), KOA group (n = 10), NEs-SP-EO group (n = 10), and 4-PBA group (n = 10). The KOA model was induced by intra-articular injection of MIA (50 µL) at a concentration of 40 mg/mL. As a control, rats in the other group were injected with 50 µL of sterilized saline. Next, 4-PBA solution of 80 mg/kg was prepared (b.w. i. p. in 37°C salines) and the pH was set to 7.4 using sodium bicarbonate (Jain et al., 2016). The animals were administrated intraperitoneally with 4-PBA for 28 days after injection of MIA. Besides, studies have suggested that doses of 20–120 mg/kg/day of 4-PBA are reliable for use in vivo models (Qi et al., 2004; Jain et al., 2016). At the same time, The NEs-SP-EO group received topical application of NEs-SP-EO hydrogels (carbomer coated with 2 mg SP-EO) while the other three groups received carbomer hydrogel only, for 8 h each day. After 28 days, we sacrificed the animals by 3% pentobarbital sodium (b.w. i. p.90 mg/kg) to harvest the synovial tissue and plasma.

HE staining, Sirius red staining, and Masson staining of synovial tissue were performed according to the instructions of the kit. In brief, synovial tissue underwent 4% paraformaldehyde fixation, paraffin embedding, sectioning, specific staining, cleaning, and other steps. Finally, the synovial tissue was observed by a DMI3000B microscope (Leica, Germany) system, with the use of a bright field.

The method of extraction and culture of FLSs is the same as previously stated (Liao et al., 2020). In brief, first, five rat knee synovial tissue was extracted in a sterile environment and washed three times with PBS. After sufficient shearing of the synovial membrane, it was digested for 2 h by adding collagenase type I at a concentration of 0.2%. The cells were then filtered through a 70 μm cell sieve and centrifuged at 1,000 rpm for 5 min 5–10 ml of medium containing 10% FBS and 1% penicillin-streptomycin solution was used to resuspend the cell precipitate and the culture medium was refreshed in the next day and then was changed every 2 days. After about a week, the cells grew all along the wall. 3rd to 6th generation FLSs were used in this assay and cell identification was carried out as described in our previous study (Zhao et al., 2018).

The cell experiments were divided into four groups: Normal group, LPS group, NEs-SP-EO group, and 4-PBA group. The FLSs were inoculated in the 6-well plate and grew to about 80% adherent. In the following experiment, 1% serum DMEM medium should be replaced. The treatment of NEs-SP-EO and 4-PBA was summarized as follows. At first, 4-PBA (5 mM), a classical endoplasmic reticulum (ER) stress inhibitor, was added to FLSs 12 h in advance. Then, LPS (5 μg/ml) was added to FLSs for 24 h, followed by ATP (4 mmol/L) for 4 h to imitate the inflammatory environment of KOA. Finally, NEs-SP-EO (1 μg/ml) was administered for 24 h in further experiments.

The CCK8 kit detected the survival of synovial cells. FLSs were incubated in 96-well plates overnight and then treated with NEs-SP-EO (1, 2, 4, 8, 10, and 20 μg/ml) for 24 h. Next, 10 µL CCK-8 solution was added for 4 h at 37°C and the absorbance was recorded at 450 nm on a microplate spectrophotometer (EnSpire).

Total protein of synovial tissue and cultured cells was extracted by RIPA lysate in a container filled with ice. The protein concentration was determined by a BCA protein detection kit (Beyotime, Nanjing, China) to calculate the sample loading amount of each group. 5x SDS-PAGE buffer was added to the protein supernatant and denatured at 99°C for 10 min. SDS-page gel electrophoresis was then performed in two stages (80 v, 30 min; 120 v, 1 h). PVDF membranes were selected for transfer of protein gels with different molecular weights. 5% BSA was used to block non-specific proteins and TBST was applied to wash thrice for 10 min. PVDF membranes were incubated in primary antibody (1:1,000) overnight at 4°C and secondary antibody (1:5,000) for 2 h. Finally, bands were visualized by exposure to ECL method and ImageJ 7.04 Software was applied to evaluate the overall gray value of protein bands.

First, total RNA was extracted from synovial cells and tissues using the TRIzol. In order to ensure the purity of RNA, the OD value ratio (260/280 nm) should be between 1.8—2.0. Next, 4 µL of 5 × HiScript II qRT Super Mix was added to the mixture solution and the RNA was reverse transcribed into cDNA in two steps (50°C, 15 min; 85°C, 5 s). The corresponding gene sequences were searched in the GenBank database and primers were designed by using oligo v6.6 software. The primer sequences were shown in Table 3. PCR was performed by using Hieff qPCR SYBR Green Master Mix (Low Rox Plus) according to the manufacturer’s instructions. β-actin was served as a control gene, and each gene was repeated thrice in this experiment. The 2^−ΔΔCT data analysis method was used to calculate relative gene expression levels.

The expression levels of IL-1β and IL-18 in the cell culture supernatant were detected by ELISA. The gradient dilution of the standards was performed at first, followed by adding 10 µL of sample and 40 µL of sample dilution to each well at 37°C for 30 min. After washing for five times, HRP-conjugated antibody working solution, substrate working solution, and termination solution were added. OD values were determined at 450 nm, and the concentration of samples in each group was calculated according to the standard curve.

The experimental data were performed independently at least thrice. Graphpad 7 (San Diego, CA, United States) was applied for graphs and SPSS 19.0 software was used for statistical analysis of the data. Shapiro-Wilk test and homogeneity of variances test were used to examine the normality and homogeneity of variances of the data, respectively. Data conforming to normal distribution were expressed as mean ± standard deviation (SD), where the least significant difference (LSD) test was used for those with homogeneous variances and Dunnett’s T3 test was used for those with non-homogeneous variances. p < 0.05 was considered a statistically significant.

Firstly, we analyzed the components of SP-EO and NEs-SP-EO by GC-MS and obtained the total ion flow diagrams of SP-EO and NEs-SP-EO (Figures 1A,B). After software data processing and database query, the chemical composition tables of SP-EO and NEs-SP-EO (Table 4 and Supplementary Material) were summarized and analyzed. After identification, a total of 30 chemical components (matching degree >80%) were identified from the SP-EO, of which the top five were: ar-turmerone, (17.5739%), cadina-1 (10), (15.4345%), turmerone, (11.5095%), curlone, (7.9284%), tau-cadinol, (7.9144%). By comparing the TCMSP database (www.tcmspw.com), we found that the total content of components belonging to Curcuma longa L. amounted to 40.9616% (ar-turmerone, turmerone, curlone, alpha-curcumene, and beta-Turmerone). On the other hand, 11 components of NEs-SP-EO were identified, among which the total content of Curcuma longa L. was as high as 45.5339%. They were turmerone (27.2945%), ar-turmerone (10.5669%) and curlone (7.6721%). The main ingredients of NEs-SP-EO were consistent with SP-EO, which indicated that components belonging to Curcuma longa L. were detected most frequently in the SP-EO and NEs-SP-EO.

Figure 2 showed that images of the NEs-SP-EO at a magnification of ×30,000 (Figure 2A). The micrographs of the NEs-SP-EO showed a spherical circular structure. What’s more, the images of NEs-SP-EO showed possible SP-EO droplets (The red arrow indicates the location) inside the spherical circular structures. The particle size range of NEs-SP-EO was about 18.1 nm, which was confirmed by transmission electron microscopy. From the ultraviolet-visible spectroscopy, we found that SP-EO and NEs-SP-EO have similar absorption peaks (Figure 2B), and the absorption value of NEs-SP-EO was lower than SP-EO. This further confirmed the stability of the NEs-SP-EO. The formulation stability of NEs-SP-EO was assessed for 2 weeks at room temperature (Figure 2C). No signs of turbidity, foaming or phase separation were detected on the 1st, 7th, and 14th days (Figure 2D). The average particle size of NEs-SP-EO was 18.1 ± 0.896 nm on the first day, and there was no significant change in the particle size on the 7th and 14th days, which showed that the process reproducibility of NEs-SP-EO was satisfactory. The PDI of NEs-SP-EO on the 1st, 7th, and 14th days were all less than 0.3, which showed that NEs-SP-EO was distributed uniformly. Besides, the absolute values of ZP were −8.6 ± 0.344, −8.8 ± 0.045, and −8.7 ± 0.026 mv on the 1st, 7th, and 14th days, respectively, with small variations, indicating that NEs-SP-EO was stable. The results showed that the formulation exhibited suitable particle size, negative charge, and stable system.

Nextly, the CCK8 method was applied to explore the appropriate concentration of NEs-SP-EO, and no significant decrease in survival was observed in FLSs treated with NEs-SP-EO (0–4 μg/ml) compared to the control group, determined that 1 μg/ml was non-cytotoxic administration concentration (Figure 3A). In the following experiment, 1% serum DMEM medium should be replaced. At first, 4-PBA (5 mM), a classical endoplasmic reticulum stress inhibitor, was added to FLSs 12 h in advance. Then, LPS (5 μg/ml) was added to FLSs for 24 h, followed by ATP (4 mmol/L) for 4 h to imitate the inflammatory environment of KOA. Finally, NEs-SP-EO (1 μg/ml) was administered for 24 h in further experiments. Next, to determine the interventional effect of NEs-SP-EO on ERS/TXNIP/NLRP3 signaling axis in the LPS-induced model, the expression of relevant proteins and genes in FLSs were measured by western blot (Figures 3B,C) and qRT-PCR (Figure 3D). Compared with the normal group, proteins expression of P-PERK, P-IER1α, CHOP, TXNIP, NLRP3, and caspase-1 in the LPS group were significantly upregulated (p < 0.05), On the other hand, genes expression of CHOP, TXNIP, NLRP3, and caspase-1 in the LPS group were significantly upregulated (p < 0.05). In contrast, NEs-SP-EO and 4-PBA groups suppressed this effect. Moreover, ELISA dates demonstrated that the expression of the pro-inflammatory factors (IL-1β and IL-18) was decreased in the supernatant upon treatment with NEs-SP-EO or 4-PBA (Figure 3E).

MIA (40 mg/ml) was used for modeling and 4-PBA solution (80 mg/kg) or NEs-SP-EO was applied for drug administration (Figure 4A). In order to further verify the therapeutic effect of this new formulation in vivo, NEs-SP-EO hydrogel (carbomer coated with 2 mg SP-EO) was applied topically to the knee joint of rats (Figure 4B). Carbomer gel changed from transparent to milky white after loading NEs-SP-EO, and the content of NEs-SP-EO in carbomer gel with SP-EO was about 2 mg. Then, it was firmly attached to the lateral side of the knee joint for 8 h every day until the end of 28 days (Figure 4C). Next, HE, Masson, and Sirius red staining methods were used to evaluate the histopathologically ameliorative effect of NEs-SP-EO or 4-PBA on synovial inflammation. In HE staining (Figure 4D), the normal group showed no thickening of cells in the synovial lining layer, neatly arranged cells, the subintimal layer was dominated by adipocytes, and almost no inflammatory cell infiltration were seen. The KOA group showed four to five layers of cells in the lining layer, disorganized arrangement, increased density and proliferation of synovial cells, and massive inflammatory cell infiltration. The rats that received NEs-SP-EO or 4-PBA exhibited less formation of lining cell layers, less resident cell hyperplasia, and inflammatory cell infiltration compared with the KOA group. Krenn’s scores were consistent with synovial pathology. (p < 0.05, Figure 4E). Masson staining and collagen volume fraction (CVF) were often used to evaluate synovial fibrosis (Figures 4F,G). NEs-SP-EO and 4-PBA groups had less synovial fibrosis than the KOA group (blue color indicates fibrosis), and the 4-PBA group had a better fibrosis reduction effect than NEs-SP-EO. Under polarized light microscopy, type I collagen appears as orange or bright red coarse fibers, and type III collagen appears as green fine fibers. When synovial inflammation or fibrosis occurred, the orange or bright red type I collagen fibers were abnormally increased. NEs-SP-EO and 4-PBA groups decreased the deposition of type I collagen fibers compared to the KOA group, and the 4-PBA group had a better-alleviating effect than NEs-SP-EO group (Figures 4H,I).

Finally, The animals were administrated intraperitoneally with 4-PBA for 28 days after knee injection of MIA. At the same time, the NEs-SP-EO group received topical application of NEs-SP-EO hydrogels (carbomer coated with SP-EO, 2 mg SP-EO) while the other three groups received carbomer hydrogel only, for 8 h each day, a total of 28 days. Next, examination of ERS and NLRP3 inflammasome related proteins and genes in MIA-induced KOA model by western blot (Figures 5A,B) and qRT-PCR (Figure 5C). The results showed that the significant inhibition of proteins expression of P-PERK, P-IER1α, CHOP, TXNIP, NLRP3, caspase-1, and mRNA expression of CHOP, TXNIP, NLRP3, caspase-1 at the doses of NEs-SP-EO (NEs-SP-EO or 4-PBA) in comparison to KOA group. Besides, we found that pro-inflammatory cytokines increased in the KOA group, while NEs-SP-EO or 4-PBA inhibited this upregulation (Figure 5D).