Fig 6. Inhibition of STAT3 by Stattic alleviates the formation of disc herniations and disc stiffness attributed to loss of ADGRG6 function.
(A-C’) Representative Safranin-O/Fast green staining and (D-F) pSTAT3 IHC in medial-sectioned mouse IVD from placebo-treated Cre (-) control (A-A’, D), placebo-treated Col2Cre;Adgrg6f/f mutant (B-B’, E), and Stattic treated Col2Cre;Adgrg6f/f mutant mice (C-C’, F) by the age of 6 months (A-C’) or 2 months (D-F) (n = 3 mice for each group). Col2Cre;Adgrg6f/f mutant mice display defects of the IVD including lesions and clefts in the CEP and GP (yellow arrowheads, B’) and increased, expression of pSTAT3 in CEP and AF (red arrowheads, E), which is reduced by Stattic treatment (C-C’, F). (G) Quantification of positive pSTAT3 cells in Cre (-) control or in Col2Cre;Adgrg6f/f mutant mice mutant mouse IVDs by the age of two months after two weeks of Stattic treatment. (n = 3 mice for each group, two to five IVDs were scored for each mouse. Dots plot with mean ± SD. ***p≤0.001, ****p≤0.0001, One way ANOVA followed by Tukey HSD test. n.s, not significant.) (H) Heat map to represent contrast-enhanced microCT data from 6-month-old mice from three experimental groups: four placebo-treated Cre (-) controls; three placebo-treated Col2Cre;Adgrg6f/f mutants; and three Stattic-treated Col2Cre;Adgrg6f/f mutants. Plotted by the axial level of the IVD (left axis) and the number of herniations (right axis) observed in each mouse. (**p≤0.01, One way ANOVA followed by Tukey HSD test. n.s, not significant.) (I) Mechanical testing on lumbar discs isolated from the same samples imaged in (H) using 5% strain cyclic loading. (Three to five IVDs were analyzed /mouse. Dots plot with mean ± 95% CI. **p≤0.01, One way ANOVA followed by Tukey HSD test. n.s, not significant.) Scale bars: 200μm in (A-C), 50μm in (A’-C’) and (D-F). AF- annulus fibrosis, CEP- cartilaginous endplate, GP- growth plate, and NP- nucleus pulposus.