Fig 2: The Y40C mutant inhibits the interaction between RAC1 and MLK3. (A) Pull-down experiment showing the detection of His-tagged MLK3 proteins with an anti-His antibody. (B) Detection of GST, GST-labeled WT RAC1, and GST-labeled Y40C mutant RAC1 proteins using an anti-GST antibody. (C) Interaction of WT RAC1 and Y40C mutant RAC1 with MLK3 tested with an anti-His antibody. (1) Only His-pcDNA3.1-MLK3 as control. (2) GST- PGEX-4 T-1-RAC1 as prey protein and His-pcDNA3.1-MLK3 as bait protein. (3) GST- PGEX-4 T-1-RAC1 (Y40C) as prey protein and His-pcDNA3.1-MLK3 as bait protein. (D) Graphical abstract. RAC1, activated by hyperglycemia, directly binds to MLK3 through RAC1 Y40, facilitating MLK3-MKK7-JNK signaling module assembly and JNK phosphorylation, then increases levels of NF-?B and cleaved caspase-3, eventually leading to DN.

Fig 3: Inhibition of JNK or CHOP blocks PTH-induced HASMC apoptosis. Apoptosis was detected by flow cytometry. (A) HASMCs were pretreated with SP6000125, a JNK inhibitor, and then treated with 1 × 10-6 mol/L PTH for 3 days. (B) Data are the mean ± SD of three independent experiments. (C) HASMCs were transfected with si-CHOP and then treated with 1 × 10-6 mol/L PTH for three days. (D) Data are the mean ± SD of three independent experiments. HASMCs cultured in normal medium for the same days as control. *p < .05 versus PTH group.

Fig 4: The expression of p-MAPKs in vivo after vehicle administration and 24 h after CFA injection. a–c IF staining of p-MAPKs in rat TG slices in the vehicle and CFA groups. Arrows (solid) show that p-MAPKs co-localized with GS, and arrows (hollow) show that p-MAPKs localized in TG neurons (green: GS; red: p-ERK1/2, p-P38, and p-JNK; blue: DAPI). Scale bars: 50 µm. WB images and graphs in d–f show that the expression of p-ERK1/2, p-P38 and p-JNK in CFA-treated TGs is higher than that in vehicle TGs, and these levels were normalized against their total levels. (n = 6, *P < 0.05 compared with the vehicle group)

Fig 5: Kaempferol suppresses the L. monocytogenes-evoked inflammation response. (A) Western blot analysis of MAPK signaling cascades in MPMs infected with wildtype L. monocytogenes strain EGD and its hly-deficient mutant EGDDhly (MOI, 5) for 5 h in the presence of DMSO vehicle or 32 mg/mL kaempferol. (B) The levels of P-p38, P-ERK and P-JNK relative to ß-actin determined using densitometry are shown. (C) Western blot analysis of NF-?B signaling cascades in MPMs infected with wild-type L. monocytogenes strain EGD and its hly-deficient mutant EGDDhly (MOI, 5) for 5 h in the presence of DMSO vehicle or 32 mg/mL kaempferol. (D) The levels of P-p65 and P-IKK relative to ß-actin determined using densitometry are shown. (E to G) The levels of TNF-a (E), IL-1ß (F), and IL-6 (G) in the coculture supernatants were detected using ELISA. (H to J) The transcriptions of TNF-a (H), IL-1ß (I), and IL-6 (J) were determined by RT-qPCR. All the genes were normalized to the housekeeping gene GAPDH. Similar results were obtained from three independent experiments. Data are presented as means 6 SEM (n = 3). **, P, 0.01 compared to the vehicle-treated group.