Platelet-rich plasma attenuates intervertebral disc degeneration via delivering miR-141-3p-containing exosomes

Figures & data

Figure 1. Cell pyroptosis and inflammation involved in regulating IVD degeneration. The mice IVD tissues were collected, and (a, b) Western Blot analysis was performed to examine NLRP3, cleaved Gasdermin D and caspase-1 expressions. The (c) mRNA levels in IVD tissues and (d-g) secretion of the pro-inflammatory cytokines in mice serum were detected by Real-Time qPCR and ELISA. The NP cells were subjected to H₂O₂ treatment, and (h, i) NLRP3, cleaved Gasdermin D and caspase-1 were examined by Western Blot, and (j) Real-Time qPCR and (k-n) ELISA were respectively used to measure the generation and secretion of the pro-inflammatory cytokines. (o, p) The NP cells were double-stained with Annexin V-FITC and PI, and FCM was performed to examine cell apoptosis ratio. *P < 0.05 was deemed as statistical significance

Figure 2. The reversal effects PRP-exo and NAC on H₂O₂ treated NP cells. (a) The PRP-exo was observed by electron microscope, which were subsequently identified by Western Blot analysis for its biomarkers TSG101 and CD81. (b) The NP cells were stained with DCFH-DA probes, and the fluorescent microscope was used to examine ROS density. (c) The GSH/GSSG ratio was determined in NP cells to reflect oxidative stress. (d, e) The expression status of NLRP3, cleaved Gasdermin D and caspase-1 were measured by Western Blot analysis. (f) Cytokines generation and (g-j) secretion were quantified by Real-Time qPCR and ELISA. (k, l) Annexin V-FITC/PI double staining method was performed to evaluate cell apoptosis. *P < 0.05 was deemed as statistical significance

Figure 3. The regulating effects of PRP-exo on the Keap1-Nrf2 pathway in NP cells. (a-c) The expression levels of Keap1 and Nrf2, and (d-f) Nrf2 translocation from cytoplasm to nucleus were measured by using the Western Blot analysis. *P < 0.05 was deemed as statistical significance

Figure 4. PRP-exo exerted its protective effects on H₂O₂-induced NP cell death in a Nrf2-dependent manner. (a, b) Cell proliferation and (c, d) viability were respectively examined by MTT assay and trypan blue staining assay. (e, f) The FCM was performed to measure the Annexin V-FITC- or PI-positive apoptotic cells. (g, h) Quantification of NLRP3, cleaved Gasdermin D and caspase-1 by Western Blot analysis. (i) Real-Time qPCR was used to examine mRNA levels of the pro-inflammatory cytokines generation. (j-m) The protein levels of the inflammation associated cytokines in the supernatants were measured by ELISA. *P < 0.05 was deemed as statistical significance

Figure 5. PRP-exo delivered exosomal miR-141-3p to modulate the Keap1/Nrf2 signaling pathway in NP cells. Real-Time qPCR was used to examine the expression levels of the upstream miRNAs for Keap1 in (a) PRP-exo, and the expression status of miR-141-3p in NP cells were also determined. (c) The targeting sites of miR-141-3p and 3'UTR of Keap1 mRNA were predicted, which were further verified by (d) dual-luciferase reporter gene system assay and (e) RNA pull-down assay. (f) The mRNA and (g, h) protein levels of Keap1 were respectively examined. (i-l) Western Blot analysis was used to analyze the Nrf2 translocation in NP cells. *P < 0.05 was deemed as statistical significance

Figure 6. Overexpression of miR-141-3p attenuated H₂O₂-induced cell death in NP cells. (a) MTT assay and (b) trypan blue staining assay were used to examine cell proliferation and viability, respectively. (c, d) Cell apoptosis ratio in the NP cells was examined by FCM assay. (e, f) The expression levels of NLRP3, cleaved Gasdermin D and caspase-1 were examined by Western Blot. The expression status of the pro-inflammatory cytokines were examined by (g) Real-Time qPCR and (h-k) ELISA, respectively. *P < 0.05 was deemed as statistical significance

Availability of data and material

All the data had been included in the manuscript, and the raw data for this work could be obtained from the corresponding author upon reasonable request.