Fig 2: MiR-122-5p overexpression attenuates the relaxation function of vaginal smooth muscle cells (SMCs). (A) Morphology of rat vaginal SMCs under an inverted microscope. (B) Length of rat SMCs. (C) Free Ca2+ content in rat vaginal SMCs. (D) The proliferation rate of rat vaginal SMCs measured with a Cell counting kit-8 (CCK8) assay. (E) The apoptosis rate of rat vaginal SMCs with flow cytometry and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay. (F) Cyclic adenosine monophosphate (cAMP) concentration and protein kinase A (PKA) activity. (G) mRNA expression levels of vasoactive intestinal peptide receptor 1 (VIPR1), stimulatory G protein (Gs), adenylate cyclase (AC), and PKA. (H) VIPR1, Gs, AC, and PKA protein expression determined by western blotting. Vaginal SMCs were isolated from one rat. All indicators extracted from SMCs were detected three times except for cellular morphology. The results are the mean ± standard error of the mean (SEM) and were analyzed by t-tests. *: P-value<0.05; **: P-value<0.01.

Fig 3: MiR-122-5p silencing results in increased relaxation function of rat vaginal smooth muscle cells (SMCs). (A) Morphology of rat vaginal SMCs under an inverted microscope. (B) Length of rat SMCs. (C) Free Ca2+ content in rat vaginal SMCs. (D) The proliferation rate of rat vaginal SMCs detected with a Cell counting kit-8 (CCK8) assay. (E) The apoptosis rate of rat vaginal SMCs with flow cytometry and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay. (F) Cyclic adenosine monophosphate (cAMP) concentration and protein kinase A (PKA) activity. (G) mRNA expression levels of vasoactive intestinal peptide receptor 1 (VIPR1), stimulatory G protein (Gs), adenylate cyclase (AC), and PKA. (H) VIPR1, Gs, AC, and PKA protein expression determined by western blotting. Vaginal SMCs were isolated from one rat. All indicators extracted from SMCs were detected three times except for cellular morphology. The results are the mean ± standard error of the mean (SEM) and were analyzed by t-tests. *: P-value<0.05; **: P-value<0.01.

Fig 4: VIP and PACAP regulate pro-inflammatory and anti-inflammatory cytokine expression in nerve explants. (A) 24 h treatment with VIP or PACAP inhibit TNFα, IL6, ILα, ILβ, and MCP-1 expression in cultured sciatic nerve explants. Sciatic nerve explants were cultured from uninjured mouse sciatic nerve. (B) 24 h treatment with VPAC1 or VPAC2 receptor-specific agonist treatment inhibits TNFα, IL6, ILα, ILβ, and MCP-1 expression in cultured sciatic nerve explants. In contrast, PAC1 receptor specific agonist treatment induces IL6 and ILβ expression. Sciatic nerve explants were cultured from uninjured mouse sciatic nerve. (C) 24 h treatment with VIP, PACAP or receptor specific agonists induce anti-inflammatory cytokines IL4, IL10, and IL13 expression in nerve explants. Nerve explants were cultured from the distal nerve stump at 10 days post-transection injury. All samples were normalized to GAPDH and control samples were made relative to 1. n = 3. ∗∗P < 0.01, ∗∗∗P < 0.001. 27 mice were used in (A,B) for nerve explant culture, and 54 mice were used in (C) for nerve explant culture.

Fig 5: Up-regulation of VPAC1, VPAC2, and PAC1 following injury in the mouse distal sciatic nerve. (A) RT-PCR showing the presence of VPAC1, VPAC2, and PAC1 mRNAs in the intact (Con) mouse sciatic nerve and in the distal sciatic nerve 7 days after transection injury. (B) qRT-PCR showing VPAC1, VPAC2, and PAC1 mRNA up-regulation in the mouse distal sciatic nerve at 4, 7, 10 and 14 days following transection injury, n = 3. (C) Western blot showing VPAC1, VPAC2, and PAC1 protein expression in control uninjured (C), proximal (P), and distal (D) mouse sciatic nerve at 7, 10, and 14 days following transection injury. (D–F) Quantification of VPAC1 (D), VPAC2 (E), and PAC1 (F) protein levels from three independent western blot results showing VPAC1, VPAC2, and PAC1 protein up-regulation in the distal nerve stump. All samples were normalized to GAPDH and control samples were normalized to 1. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (G–I) Double staining of Vasoactive Intestinal Peptide (VIP) with the neuronal marker NeuN showing that sensory neurons in the DRG express VIP at 7 days after sciatic nerve transection injury. (J–L) Double staining of Pituitary Adenylyl Cyclase Activating Peptide (PACAP) with the neuronal marker NeuN showing that sensory neurons in the DRG express PACAP 7 days after sciatic nerve transection injury. (M–O) Double staining of PACAP with neurofilament (NF) showing that PACAP is present in leading regenerating axons in the nerve bridge 7 days after sciatic nerve transection injury. Scale bars in I and L 20 μm. Scale bar in O 40 μm. Thirty six mice were used in A and B for RT-PCR and qPCR experiments. Twenty seven mice were used in C-F for western blot experiments. Three mice were used in (G–O) for immunostaining and three sections from each mouse were used for each staining.