In addition, the expression of ACE was also elevated with the increased degree of degeneration of disc (Figures 1(g) and 1(h)). PPAR signaling pathway, alcoholism, and systemic lupus erythematosu (Figures 1(c) and 1(d)). Based on gene counts and gene ratio, we focused on RAS. In addition, AGT (“type”:”entrez-protein”,”attrs”:”text”:”P01019″,”term_id”:”113880″,”term_text”:”P01019″P01019), the origin of Ang II, was found to be expressed higher in severely degenerated group, indicating increased activation of tRAS that may correlate with IDD. Open in a separate window Figure 1 Activation of the tissue renin-angiotensin system (tRAS) in the human degenerated intervertebral disc tissue. (a) The volcano plot of the gene relative expression of NP tissue in the none-degenerated group compared to the severely degenerated group. Pink circles represented upexpressed proteins ( 0.05), whereas black circles suggested proteins without expression differences between these two groups (= 5). (b) The clustering analysis heat map. (c) Melittin Top 20 enriched GO terms. (d) Top ten enriched KEGG pathways. (e) TUNEL assay of the human NP tissue among grades II, III, and IV (= 3). (f) Quantitative results of TUNEL-positive cells. (g) Immunohistochemical analysis of the expression of ACE in the human NP tissue among grades II, III, Melittin and IV (= 3). (h) Quantitative results of ACE-positive cells. (i, j) Western blot results of the protein expression of tRAS components (ACE and AT1) and MMP13 in the human NP tissue (= 3). Scale?bar = 50? 0.05, ?? 0.01, ??? 0.001, ???? 0.0001. We further confirmed the Melittin expression changes of tRAS components in the human NP tissue at the molecular level. Based on Pfirrmann grades on T2-weighed MRI, the human intervertebral disc tissue was divided into three groups: grade II, grade III, and grade IV, respectively (= 3). As shown in Figure 1, the ratio of TUNEL-positive nucleus pulposus increased with Pfirrmann grades (Figures 1(e) and 1(f)). In addition, the expression of ACE was also elevated with the increased degree of degeneration of disc (Figures 1(g) and 1(h)). Further, western blot analysis of the NP tissue of disc confirmed the increased tRAS components Melittin (AT1 and ACE) in degenerated disc (Figures 1(i) and 1(j)). These results above suggested the activation of tRAS in the human degenerated intervertebral disc tissue. 3.2. Inflammatory Cytokines Could Promote the Activation of RAS in Human Nucleus Pulposus Cells In Vitro To further investigate the expression changes of tRAS components in nucleus pulposus, we stimulated human nucleus pulposus with inflammatory cytokines (TNF and IL-1or IL-1secreted higher level of Ang II in supernatant (Figures 2(a) and 2(b) both 0.05). In addition, the results of western blot suggested that inflammatory cytokines could dose-dependently activate the expression of AT1 and ACE in NP cells (Figures 2(c) and 2(d)). RT-qPCR analysis also showed DHRS12 that inflammatory cytokines promoted the activation of tRAS in nucleus pulposus in a time-dependent manner, especially at 12-24?h following inflammatory stimulation (Figures 2(e) and 2(f)). Open in a separate window Figure 2 Inflammatory cytokines promoted the activation of tRAS in human nucleus pulposus cells in vitro. (a, b) Secretion amount of Ang II in supernatant of human NP cells induced by IL-1or TNF was quantified by the ELISA assay (= 5). (c, d) Western blot revealed the relative protein expression of tRAS components (ACE and AT1) in human NP cells induced by IL-1or TNF (= 3). (e, f) RT-qPCR quantified the relative mRNA expression tRAS components (ACE and AT1) in human NP cells induced by IL-1or TNF in a time-change manner (= 3). ? 0.05, ?? 0.01, ??? 0.001, ???? 0.0001. 3.3. Angiotensin II Triggered Human NP Cell Senescence in a Dose-Dependent Manner As shown in Figure 3, Ang II could decrease the cell viability of NP cells in a dose-dependent manner, with the IC50 being 17.63?= 3). (b) Quantitative results of SA-= 3). (c) Immunofluorescence results for the expression of = 3). Scale?bar = 20?= 3). (e) The protein expression.