Shipi Shugan Decoction Protected against Sequela of Pelvic Inflammatory Disease via Inhibiting SIRT1/NLRP3 Signaling Pathway in Pelvic Inflammatory Disease Rats

Figures

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  Figure 1. SSD dose-dependently attenuated inflammation of uterine and fallopian tube in SPID rats. 35 female rats were used for these experiments: Sham group (n = 8), SPID group (n = 6), high-dose SSD group (n = 8), low-dose SSD group (n = 6), and Fuke Qianjin Capsules (FQC) group (n = 7). After the SPID model was established, the rats in the high-dose SSD group and low-dose SSD group were intragastrically administered with 6.64 g/kg • day and 1.66 g/kg • day SSD, respectively. The rats in the FQC group were intragastrically administered with 0.22 g/kg • day after the suspension of FQC. The rats in the sham group and SPID group were given the same amount of normal saline. The contents of TNF-α (a) IL-1β (b) IL-6 (c) and IL-18 (d) in serum samples of rats in each group were tested by enzyme-linked immunosorbent assay (ELISA). (e) The numbers of WBCs and lymphocytes were counted under a microscope. (f) H&E stain was used to observe the pathological changes in the fallopian tube and uterine tissues. The magnification is 200× and 400×. Yellow arrow: vasodilation and hyperemia; green arrow: degeneration and necrosis of epithelial cells; blue arrow: lymphocyte; black arrow: endometrial lamina…

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  Figure 2. SSD dose-dependently inhibited mitochondrial dysfunction and oxidative stress of SPID rats. (a) The compromised mitochondria were tested by TEM. Scale, 1 μm. Yellow arrow: normal mitochondria; green arrow: pyknotic mitochondria; red arrow: swollen mitochondria. (b and c) Flow cytometry was used to detect mitochondrial membrane potential. (d) The content of ROS in serum samples of rats in each group was tested by enzyme-linked immunosorbent assay (ELISA). The generation of MDA (e) GSH-Px (f) and SOD (g) in serum samples was analyzed by corresponding commercial kits. SPID: sequela of pelvic inflammatory disease; SSD: Shipi Shugan Decoction; FQC: Fuke Qianjin Capsules; TEM: transmission electron microscopy. ∗P < 0.05 (versus sham), ∗∗P < 0.01 (versus sham), #P < 0.05 (versus SPID), ##P < 0.01 (versus SPID), and ###P < 0.001 (versus SPID). Data represent the mean ± SEM.

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  Figure 3. Modulation of SSD on SIRT1/NLRP3 signaling. (a and e) The protein expression levels of SIRT1, NLRP3, and caspase-1 were determined by Western blot analysis. β-actin was used as a loading control. (b, c, d, f, g, h) Quantitative analysis of western blot analysis normalized to SIRT1, NLRP3, and caspase-1.(versus sham),(versus SPID), and(versus SSD). Data represent the mean ± SEM.

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  Figure 4. SSD against inflammation and pathological damage of upper genital tract by inhibiting SIRT1/NLRP3 signaling. 33 rats were used for these experiments: sham group (n = 8), SPID group (n = 6), SSD group (n = 6), SIRT1 inhibitor (EX527) group (n = 6), and SSD + EX527 group (n = 7). After the SPID model was established, the rats in the EX527 group were intragastrically administered with a 5 mg/kg/day SIRT1 inhibitor (Sigma, Munich, Germany; No. E7034). The rats in SSD + EX527 group were intragastrically administered with 5 mg/kg/day and 6.64 g/kg • day SSD. The rats in the sham group and SPID group were given the same amount of normal saline. The content of TNF-α (a) IL-1β (b) IL-6 (c) and IL-18 (d) in serum samples of rats in each group was detected by enzyme-linked immunosorbent assay (ELISA). (e) The numbers of WBCs and lymphocytes were counted under a microscope. (f) H&E stain was used to observe the pathological changes in the fallopian tube and uterine tissues. The magnification is 200× and 400×. Yellow arrow: vasodilation and hyperemia; blue arrow: lymphocyte; black arrow: endometrial lamina propria edema; H&E: hematoxylin and eosin; WBCs: white blood cells; TNF: tumor necrosis…

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  Figure 5. SSD against mitochondrial dysfunction and oxidative stress by inhibiting SIRT1/NLRP3 signaling. (a) The compromised mitochondria were measured using TEM. Scale, 1 μm. Yellow arrow: normal mitochondria; red arrow: swollen mitochondria. (b and c) Flow cytometry was used to analyze mitochondrial membrane potential. (d) The content of ROS in serum samples of rats in each group was tested by enzyme-linked immunosorbent assay (ELISA). The generation of MDA (e) GSH-Px (f) and SOD (g) in serum samples was assayed by corresponding commercial kits. SPID: sequela of pelvic inflammatory disease; SSD: Shipi Shugan Decoction; FQC: Fuke Qianjin Capsules; TEM: transmission electron microscopy. ∗∗∗P < 0.001 (versus sham), ##P < 0.01 (versus SPID), ###P < 0.001 (versus SPID), &&P < 0.05 (versus SSD), and &&P < 0.01 (versus SSD). Data represent the Mean ± SEM.