30 patients with MG were enrolled at designated hospital from May 2019 to July 2020. The blood samples from the patients were collected and immediately stored at 4 °C for further study. Informed consent was obtained from all the participants. The Medical Ethics committee of Beijing Friendship Hospital, Capital Medical University approved (Approve No. KR3234208) the study. All experiments were carried out following standard guidelines and regulations. The diagnosis of MG was made by an experienced neurologist based on clinical presentation, positive response to anticholinesterase medications, presence of autoantibodies, and characteristic electrophysiological findings. Patients with any history of inflammatory or autoimmune diseases such as autoimmune thyroid disease, inflammatory bowel disease, RA, and SLE were excluded from this study.

We built the EAMG model by using the C57BL/6 female mice [20]. Briefly, on day 1, mice were immunized with a synthetic immunogen containing complete Freund’s adjuvant, and on days 30 and 50, mice were immunized with a synthetic immunogen containing incomplete Freund’s adjuvant (SIMGA). After the first injection, we measured the EAMG scores every day; collected the serum; and sacrificed the model mice on the 21st day to conduct other essential experiments. All experiments were performed during the same period of the day (8:00 am to 4:00 pm) to exclude diurnal variations in pharmacological effects. The animals were handled in compliance with the procedures approved by the Animal Resources Centre of Capital Medical University.

We used Light Cycler® 480 SYBR Mix (Roche, Mannheim, Germany) in the LightCycler® 480 real-time PCR system to quantify the genes expression in the PCR system. We utilized the Invitrogen SuperScript double-stranded cDNA synthesis kit for the reverse transcription. NimbleGen’s one-color DNA labeling kit was used to synthesize double-stranded cDNA and then array hybridization was executed by using the NimbleGen hybridization system. In addition, the NimbleGen wash buffer kit was used for washing step. We employed GenePix4000B (Axon, Scottsdale, AZ, USA) and GenePix Pro 6.0 software (Axon, USA) microarray scanner (Molecular Devices) technology to scan the experimental outcomes. The sequences of primers for PTX3 gene used is Forward 5′-CCTGCGATCCTGCTTTGTG-3′, Reverse 5′-GGTGGGATGAAGTCCATTGTC-3′.

Mouse myoblasts (C2C12) was cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) with 10% fetal calf serum supplementation (FCS, Gibco, Carlsbad, CA, USA) in a humid environment of 5% CO${}_2$ at 37 °C. We utilized Lipofectamine 2000 to transfect the PTX3 Plasmids with primer sequence of (5′-GTGCAGGGCTGGGCTGCCCG-3′) into C2C12 cells. After 48 h of transfection, LPS (200 ng) was added genetally to the C2C12 cells for 4 h and later replaced with the fresh medium. The cell line (C2C12 Mouse Myoblasts) used in this study was tested and authenticated using Short Tandem Repeat (STR) profiling, Which confirmed the identity by comparing the obtained profile with a reference databases of known cell lines. Furthermore, the cell line was tasted for Mycoplasma and other contamination using negative and positive controls with validated method. The cell line was free of mycoplasma and other species contamination.

We quantify the level of IL-1$\beta$ (Abcam, Cambridge, UK, ab214025), IL-6 (Abcam, ab178013), INF-$\gamma$ (Abcam, ab289902), IL-17 (Abcam, ab119535), TNF-$\alpha$ (Abcam, ab181421), anti-TAChR IgG/IgG1/IgG2b/IgG2c (Abcam, ab100548) by using an ELISA kit following the manufacturer’s protocol.

Proteins were extracted from the cultured cells using RIPA and PMSF reagents (Beyotime, Beijing, China).We separated the protein lysates according to the molecular weight of respected protein on SDS/PAGE gels and then we transferred them to a Polyvinylidene Fluoride (PVDF, Millipore, Burlington, MA, USA) membrane system. The membranes containing protein were then blocked by using 5% non-fat-milk for 2 hours at room temperature and further, the membranes were placed on shaking incubator at 4 °C and incubated with anti-antibody of PTX3 (Abcam, ab90806), actin (Sigma, St. Louis, MO, USA, SAB4502543), Fl-GSDMD (Abcam, ab210070), N-GSDMD (Abcam, ab215203), NLRP3 (Abcam, ab263899), p-STAT3 (Abcam, ab30647), STAT3 (Abcam, ab68153). After incubation, the membranes were washed with 10% phosphate buffer saline (PBS) for ten minutes and repeated the washing steps three times. After that, membranes were incubated with a secondary antibody for 1 hour at room temperature and again repeated the washing step. The membranes were then visualized by machine.

Microarray data were obtained from NCBI’s Gene Expression Omnibus (GEO) database. All available studies were considered, but only investigations that included a DA/Ao comparison were selected for analysis. One-way ANOVA test was used to analyze contrasts of interest, namely vessel type, to generate lists of DEGs between DA and Ao. Permissive DEG lists (fold change $\ge$1.2) were then separated by increased or decreased DA/Ao expression to generate UP and DOWN lists respectively.

We fixed the cells by using 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 15 minutes, and then the cells slides were blocked using the 5% BSA for 30 minutes at 37 °C. We then treated the cells with primary antibodies at 4 °C overnight. Then, incubated the cells by using the Cy3-conjugated goat anti-rabbit or goat anti-mouse IgG DyLight 488-conjugated secondary antibodies for 2 hours at room temperature. Furthermore, we stained the nuclei of the cells using the DAPI and we utilized a fluorescent illumination microscope (Olympus IX71, Tokyo, Japan) for observing the cells.

DNA was amplified using a methylation-specific primer set, PTX3-MF: 5′-CGTTTGCGGTTAGGAGTATTC-3′ and PTX3-MR: 5′-CAAAACGTCGTCCGTAACTTA-3′, or a non-methylation-specific primer set, PTX3-UF: 5′-TGTGT TTGTGGTTAGGAGTATTTG-3′ and PTX3-UR: 5′-CAA AACATCATCCATAACTTA-3′ [18], in a total volume of 20 µL, using 0.5 units of hot-start Taq-polymerase (Takara, Japan) per reaction. The size of the non-methylated amplicon was 105 bp, and the methylated amplicon was 103 bp.

We employed GraphPad 8.0 Software for analyzing the data and presented the data as mean $\pm$ SD. The statistical threshold was set as the p 0.05. We analyzed the differentiation of two groups by utilizing the Student’s t-test or performing the Tukey post hoc test after applying two-way ANOVA.

To investigate the expression level of the PTX3 gene in MG, we used the PCR assay to identify the PTX3 expression level in patients with MG and mice. Our results revealed that the level of the PTX3 mRNA expression is significantly elevated (Fig. 1A). Serum mRNA expression of PTX3 showed a significant positive correlation with the serum IL-17 levels in patients with MG and the AUC = 0.9236 (Fig. 1B,C). In mice models of MG, we found an elevated level of PTX3 mRNA and protein quantity (Fig. 1D,E).

Fig. 1.

Serum pentaxin 3 (PTX3) expression level in patients with Myasthenia gravis (MG) up-regulated. PTX3 mRNA expression (A), Serum mRNA expression of PTX3 was in negative correlation with serum IL-17 levels (B), AUC analysis results (C), Sensitivity analysis in patients with MG (D), PTX3 mRNA expression (E), while the western blot figure indicate protein expression in mice model. The experiments were performed in triplicate and data is presented an average of these experiments. Normal, normal volunteers’ group; Patients, Patients with MG; Sham, sham control group; MG, mice with MG. **p 0.01 compared with normal volunteers group or sham control group.

Next, we analyzed the correlation between PTX3 expression level and inflammation level in MG models. PTX3 plasmid increased PTX3 mRNA expression and enhanced the inflammatory factors, including IL-1$\beta$, IL-6, IL-17, and TNF-$\alpha$ activity levels in the in-vitro model (Fig. 2A,B). PTX3 up-regulation increased Ccl12, Ccl19, and Ccl13 mRNA expressions in the in-vitro model (Fig. 2C). Furthermore, Sh-PTX3 plasmid reduced PTX3 mRNA expression and decreased inflammatory regulators in the in-vitro model (Fig. 2D,E). Our results also concluded that the down-regulation of PTX3 is associated with the reduction level of Ccl12, Ccl19, and Ccl13 mRNA expressions in the in-vitro model (Fig. 2F).

Fig. 2.

PTX3 promoted inflammation level using in vitro model. PTX3 mRNA expression (A). IL-1$\beta$, IL-6, IL-17, TNF-$\alpha$ activity levels (B). Ccl12, Ccl19, and Ccl13 mRNA expressions (C) in vitro model by over-expression of PTX3. PTX3 mRNA expression (D). IL-1$\beta$, IL-6, IL-17, and TNF-$\alpha$ activity levels (E). Ccl12, Ccl19, and Ccl13 mRNA expressions (F) in vitro model by down-regulation of PTX3. All experiments were performed in triplicate. **p 0.01 compared with the control negative group or sh-nc group.

We next further examined the function of PTX3 in MG using PTX3 recombinant protein. PTX3 recombinant protein increased global clinical score and AChR level and expanded anti-TAChR IgG/IgG1/IgG2b/IgG2c levels, induced Ccl12, Ccl19, and Ccl13 mRNA expressions, while increased the levels of inflammatory regulators, including IL-1$\beta$, IL-6, INF-$\gamma$, IL-17, and TNF-$\alpha$ in the model of MG (Fig. 3).

Fig. 3.

The inhibition of PTX3 presented MG and reduced inflammation levels in the mice model. Global clinical score (A). AChR level (B). anti-TAChR IgG (C)/IgG1 (D)/IgG2b (E)/IgG2c (F) levels. Ccl12 (G), Ccl19 (H) and Ccl13 (I) mRNA expressions. IL-1$\beta$ (J), IL-6 (K), INF-$\gamma$ (L), IL-17 (M), TNF-$\alpha$ (N) activity levels. The experiments were performed in triplicate and data is presented an average of these experiments. **p 0.01 compared with the control negative group or sh-nc group.

Furthermore, we analyzed the regulatory roles of PTX3 on pyroptosis in the in-vitro model of MG. PTX3 up-regulation decreased cell proliferation and increased the activity of LDH in the in-vitro model (Fig. 4A,B). Down-regulation of PTX3 enhanced the cellular proliferative charactertristics and decreased the activities of LDH in the in-vitro model (Fig. 4C,D). Additionally, we discovered in the in-vitro model that downregulating PTX3 decreased the protein level of GSDMD while upregulating PTX3 increased the expression of GSDMD (Fig. 4E,F). Furthermore, in the mice model, PTX3 recombinant protein promoted GSDMD protein expression (Fig. 4G).

Fig. 4.

PTX3 promoted pyroptosis in mice model or in vitro model. Cell growth and LDH activity levels (A and B) in vitro model by over-expression of PTX3. Cell growth and LDH activity levels (C and D) in vitro model by down-regulation of PTX3. GSDMD protein expression in vitro model by over-expression of PTX3 (E). Full-length GSDMD (Fl-GSDMD) and N-terminal domain of GSDMD (N-GSDMD) protein expression in vitro model by down-regulation of PTX3 (F). GSDMD protein expression in mice with MG by sh- PTX3 (G). The experiments were performed in triplicate and data is presented an average of these experiments. **p 0.01 compared with the control negative group or sh-nc group or MG group.

To understand the molecular regulatory role of PTX3 in the MG model, we used microarray technology to perform gene expression profiling (Fig. 5A,B). Our results indicated that PTX3 might regulate STAT3 and NLRP3 levels (Fig. 5B). The recombinant protein product of PTX3 stimulated the expression of p-STAT3/NLRP3 protein in the mice model (Fig. 5C). In the mice model of MG, PTX3 up-regulation induced PTX3 and p-STAT3/NLRP3 protein expressions (Fig. 6A) while, PTX3 down-regulation suppressed PTX3 and p-STAT3/NLRP3 protein expressions in vitro model (Fig. 6B). Confocal showed that PTX3 up-regulation increased PTX3 and p-STAT3 expression levels in vitro model (Fig. 6C). We searched that the gene promoter of PTX3 and STAT3, showing the molecular interaction between PTX3 and STAT3 (Fig. 7A,B). Based on the luciferase reporter assay, the PTX3 WT substantially correlated with the expression of STAT3 (Fig. 7C). Then, the STAT3 inhibitor (2 µM of STAT3-IN-10) downregulated the protein level of p-STAT3 and reduced the protein expression level of GSDMD/NLRP3 in the in-vitro model (Fig. 7D). Moreover, the STAT3 agonist (5 µM of Oleoylcarnitine) induced the protein level of p-STAT3 and stimulated the protein level of GSDMD/NLRP3 in the in-vitro model (Fig. 7E).

Fig. 5.

PTX3 induced STAT3/NLRP3 inflammasome. Heat map (A). Result workflow and analysis (B). Presentation of p-STAT3/NLRP3 protein expression in mice with MG (C). **p 0.01 compared with the MG group.

Fig. 6.

PTX3 promoted gene synthesis of STAT3. PTX3/p-STAT3/NLRP3 protein expression in the in-vitro model by over-expression of PTX3 (A). PTX3/p-STAT3/NLRP3 protein expression in vitro model by down-regulation of PTX3 (B). PTX3/p-STAT3 expression by confocal microscope with the scale bar of 10 μM (C). The data is presented an average of triplicated experiments. **p 0.01 compared with the control negative group or sh-nc group.

Fig. 7.

Gene promoter of PTX3 and STAT3. The gene promoter of PTX3 and STAT3 (A), molecular interaction within PTX3 and PTX3 (B), luciferase reporter (C), p-STAT3/GSDMD/NLRP3 protein expression in vitro model by PTX3+STAT3 inhibitor (D), p-STAT3/GSDMD/NLRP3 protein expression in vitro model by sh-PTX3+STAT3 agonist (E). All experiments were performed in triplicates and the data is presented an average of these experiments. **p 0.01 compared with the control negative group or sh-nc group.

We further examined the mechanism of PTX3 to check the progression of MG. We found that serum mRNA level of PTX3 was positively correlated with the level of METTL3 in MG patients (Fig. 8A). However, PTX3 level (serum mRNA) is not correlated with METTL14 levels in MG patients (Fig. 8B). In the immunoprecipitated fraction, an elevated level of PTX3 was found by using the m6A antibody (Fig. 8C). METTL3 reduced the m6A methylation level of PTX3 (Fig. 8D). Si-METTL3 decreased PTX3 expression levels (Fig. 8E), indicating the stable stimulation of m6A methylation of PTX3. The suspicious methylation modification sites of PTX3 are present at the stop codon region (Fig. 8F). METTL3 reduced PTX3 mRNA levels expression levels (Fig. 8G). In conclusion, these results suggest that METTL3-mediated m6A modification decreases PTX3 stability.

Fig. 8.

METTL3-mediated m6A modification PTX3 stability. Serum mRNA of PTX3 was in negative correlation with METTL3 or METTL14 levels in patients with MG (A and B), fraction immunoprecipitated by m6A antibody (C), m6A methylation level of PTX3 (D), presentation of PTX3 expression levels (E), multiple suspicious methylation modification sites (F), PTX3 mRNA levels expression levels (G). The experiments were performed in triplicate and the data is an average of these expeirments. **p 0.01 compared with the lgG or NC group.