Figure 4 | Scientific Reports

Figure 4

From: Endogenous Neurosteroid (3α,5α)3-Hydroxypregnan-20-one Inhibits Toll-like-4 Receptor Activation and Pro-inflammatory Signaling in Macrophages and Brain

Figure 4The alternative text for this image may have been generated using AI.

3α,5α-THP inhibits TLR4 signal innately activated in P rat VTA by blocking TLR4/α2 binding and TLR4/MyD88 binding. (A) 3α,5α-THP administration (15 mg/kg) significantly reduced MCP-1 (ELISA; Student’s t(16) = 2.19), TRAF6 (Student’s t(16) = 5.74), and CRF (Student’s t(16) = 3.112) levels compared to vehicle controls, with no effect on TLR4 protein expression. *p < 0.05 compared to control. (B) TLR4 binds α2 in the P rat VTA. Protein extracts from P rat VTA were immunoprecipitated (IP) with the TLR4 or α2 antibodies or normal IgG (control) and the precipitates were reciprocally immunoblotted (IB) with α2 or TLR4 antibodies. Both α2 and TLR4 were seen in the anti-α2 and anti-TLR4 (but not normal IgG) precipitates from P rat VTA, indicative of protein-protein interaction. (C) 3α,5α-THP inhibits TLR4/α2 and the downstream TLR4/MyD88 binding in the P rat VTA. Protein extracts obtained from P rat VTA after 3α,5α-THP (15 mg/kg) or vehicle control administration were immunoprecipitated (IP) with antibody to TLR4. The precipitates were immunoblotted (IB) with α2 antibody. Normal IgG was used as control. α2 co-precipitated with TLR4, but not normal IgG. The levels of α2 co-precipitating with TLR4 were significantly reduced by 3α,5α-THP (62.7 ± 9.2% reduction, p < 0.001). 3α,5α-THP also inhibited the binding of TLR4 to MyD88 (43.5 ± 5.4% inhibition, p < 0.05). HMGB1 bound TLR4, but binding was not altered by 3α,5α-THP. Blots shown were cropped from full length gels for clarity. Original scans of the gels in the composite figures are shown in Supplementary Information Figure S7. Scatter plots show the mean and S.E.M. of values. Box and whisker plots show the median, minimum and maximum values.

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