Oridonin inhibits the progression of atherosclerosis in ApoE−/− mice.
To explore the possible effect of oridonin on the progression of atherosclerosis in vivo, ApoE−/− mice fed with a high-fat diet were intraperitoneally injected with 10 mg/kg (AS + Ori 10 mg/kg group) or 20 mg/kg oridonin (AS + Ori 20 mg/kg group) or normal saline (AS group) every day for 12 weeks. The formation of atherosclerotic lesions was evaluated by oil red O staining in the aortas. The area of the oil red positive lesion was dramatically reduced in the treatment groups compared to the AS group (Fig. 1a and b)., The oil red O staining area of the aortic sinus in the treatment groups was less than that in the AS group, which was consistent with the decrease in overall lesion area (Fig. 1d and e). In accordance with the decrease in lipid content, aortic root lesion size was also decreased in treatment groups (Fig. 1c). In addition, oridonin had no significant effect on overall body weight (data not shown), the levels of circulating total cholesterol (TC) (Fig. 1f) and total triglyceride (TG) (Fig. 1g) in ApoE−/− mice.
To further study the plaque, the aortic sinus sections of mice were stained with immunohistochemistry to evaluate its composition. CD68 (a marker of infiltration macrophages), α-SMA (a marker of vascular smooth muscle cells), and Masson staining (the level of collagen) were detected in the aortic sinus sections. As anticipated, oridonin treatment significantly decreased the number of infiltrated macrophages (Fig. 2a and b). Additionally, a significant increase of α-SMA and Masson staining positive area was observed in the atherosclerotic plaque sections (Fig. 2a, c and d). Taken together, these results revealed that the mice in the oridonin treatment groups had a lower lesion area than mice in AS group, and the plaque was more stable.
Oridonin reduces systemic and arterial inflammation by inhibiting NLRP3 inflammasome in ApoE−/− mice.
Because oridonin is considered to be an effective NLRP3 inhibitor (He et al., 2018), we investigated whether it can reduce inflammation in ApoE−/− mice. Administration of low-dose (10 mg/kg) and high-dose (20 mg/kg) oridonin significantly decreased serum IL-6 and CRP levels (Fig. 3a and b). It suggested that oridonin reduced systemic inflammation levels in ApoE−/− mice. Subsequently, we assessed whether local inflammation levels and the NLRP3 pathway were down-regulated in oridonin-treated aortic tissues. Significantly decreased protein and mRNA levels of NLRP3, Caspase-1, IL-1β and ASC were detected in the arteries of treatment groups (Fig. 3c, d and e). The IL-1β and IL-18 levels in arteries were detected by ELISA and were found to be decreased in oridonin treatment groups (Fig. 3f and g). In addition, immunofluorescence showed that the co-localization of F4/80 (the marker of macrophages) and NLRP3 was significantly decreased in oridonin treatment groups (Fig. 3h). Consistent with CD68 immunohistochemical staining, it showed that oridonin reduced macrophage infiltration and suppressed NLRP3 expression. Collectively, oridonin administration decreased inflammatory cytokines production and NLRP3 activation in the arteries of mice. The level of systemic inflammation also decreased in oridonin treatment groups.
Oridonin reduces oxidative stress by activating Nrf2 pathway in aortic plaques of ApoE−/− mice.
As previously mentioned, inflammatory factors in atherosclerotic plaques lead to the accumulation of ROS, triggering oxidative stress, which is also closely related to the progression of atherosclerosis. Oridonin has been investigated as an Nrf2 activator with antioxidative activities (Du et al., 2008; Yang et al., 2019; Zhao et al., 2022). Therefore, we studied whether oridonin could reduce the accumulation of ROS in the aortas of ApoE−/− mice. Dihydroethidium (DHE) fluorescence staining was used to detect ROS in aortic sinus sections. The results showed that oridonin treatment reduced the level of ROS in aortic plaques (Fig. 4a and b). To further examine the anti-oxidative stress capacity in aortic tissue after oridonin treatment, we examined the protein expression of Nrf2 and HO-1. Compared with the AS group, the protein expression levels of antioxidant genes Nrf2 and HO-1 in the aortas of mice treated with oridonin were significantly increased (Fig. 4c and d). Immunofluorescence staining showed that oridonin decreased the infiltration of macrophages in the plaque while enhancing the expression of Nrf2 in the plaque (Fig. 4e). In general, oridonin improved the ability of aortic tissue to resist oxidant stress by increasing the expression of Nrf2.
Oridonin inhibits NLRP3 activation and upregulates the Nrf2 protein level in peritoneal macrophages in vitro.
To further verify the effect of oridonin on the inflammatory response and antioxidant response in atherosclerotic plaque, we isolated primary peritoneal macrophages from ApoE−/− mice. Then the cells were exposed to ox-LDL (50 µg/mL) for 48 h in the presence or absence of oridonin (2.5 µM and 5 µM) to mimic macrophages in plaques. To evaluate the inhibitory effect of oridonin on ox-LDL-induced inflammation, we examined the expression levels of NLRP3 pathway-related genes and the levels of cytokines in the medium. The protein and mRNA expression levels of NLRP3, Caspase-1, IL-1β and ASC were significantly decreased in oridonin treatment groups (Fig. 5a and b). After oridonin treatment, the IL-1β and IL-18 secretion levels in the medium were detected by ELISA. There was a significant difference between the treatment groups and the control group (Fig. 5c and d). Overall, these results indicate that oridonin can inhibit NLRP3 activation and inflammation cytokines release in ox-LDL induced peritoneal macrophages.
Then we evaluated the antioxidant stress ability of ox-LDL-induced peritoneal macrophages after oridonin treatment. to evaluate ROS production, we utilized DHE fluorescent staining. The macrophages with oridonin treatment showed less red fluorescence than the control group (Fig. 5e). Consistent with the protein expression levels of antioxidant genes in the aortas, oridonin treatment increased the expression of Nrf2 and HO-1 in macrophages (Fig. 5f). To explore how oridonin upregulates the protein expression of Nrf2, we first detected the transcriptional levels of Nrf2 and Hmox1 by qPCR. As shown in Fig. 5g, there was no statistically significant difference in Nrf2 mRNA with treatment of oridonin. The mRNA level of Hmox1 was induced significantly by oridonin. These data showed that oridonin can activate the Nrf2 signaling pathway primarily by increasing the Nrf2 protein level.
Previous studies have shown that Nrf2 activator induces Nrf2 signal pathway mainly by interfering with keap1-dependent ubiquitin coupling mechanism. (Nguyen et al., 2003). Studies have found that oridonin can increase the expression level of Nrf2 in human breast carcinoma cells (Du et al., 2008). We thus evaluated the capacity of oridonin to modulate Nrf2 ubiquitination. Immunoprecipitation showed oridonin suppressed Nrf2 ubiquitination in Raw264.7 cells (Fig. 5h). We also measured the half-life of Nrf2 in Raw264.7 with or without oridonin treatment. Treatment with oridonin enhanced the half-life of the Nrf2 protein in Raw264.7 cells (Fig. 5i). These data revealed that oridonin increased the stability of Nrf2 by blocking Nrf2 ubiquitination in macrophages.
Oridonin inhibits lipid uptake and enhances lipid efflux in macrophages in vivo and in vitro.
Foam cell production is a critical stage in the development of atherosclerotic plaque. Uncontrolled ox-LDL absorption, inordinate cholesterol esterification and blocked cholesterol excretion result in the accumulation of cholesterol ester in the form of lipid droplets, which causes the formation of foam cells (Groenen et al., 2021). As mentioned earlier, the levels of inflammation and oxidative stress affect the lipid metabolism of macrophages. Therefore, we asked whether the inhibition of oridonin on inflammation and oxidative stress could improve the lipid-handling ability of macrophages. Previous studies have reported that selective NLRP3 inflammasome inhibitors decrease foam cell formation via suppression of ox-LDL uptake and enhancement of cholesterol efflux (Chen et al., 2018). Therefore, we speculated whether oridonin treatment could inhibit the formation of foam cells by regulating the “input” and “output” of lipids.
The primary peritoneal macrophages from ApoE−/− mice were exposed to ox-LDL (50 µg/mL) for 48 h with or without oridonin. Oil red O staining showed oridonin decreased ox-LDL-induced lipid deposition in peritoneal macrophages (Fig. 6a). Then, we detected the expression of CD36, the main receptors responsible for the ox-LDL influx, ABCA1 and ABCG1 (ATP Binding Cassette A1 and G1 cholesterol transporters), two transporters mediating cholesterol efflux (Yvan-Charvet et al., 2019). Oridonin treatment group significantly down-regulated CD36 and up-regulated ABCA1 and ABCG1 in ox-LDL induced macrophages (Fig. 6b and c). The expression of lipid flow-related proteins CD36, ABCA1 and ABCG1 was also detected in aortic plaques by Western blot (Fig. 6d). According to the in vitro and in vivo data, oridonin can inhibit intracellular lipid accumulation, and its mechanism may be related to the decrease of CD36 and up-regulation of ABCA1 and ABCG1 expression, inhibition of ox-LDL uptake and promotion of lipid excretion, thus preventing the formation of foam cells.
ABCA1 and ABCG1 expression are positively regulated by the nuclear receptor liver X receptor (LXR), which forms a heterodimer by binding with the retinoid X receptor, and acts as a transcription factor (Yvan-Charvet et al., 2019). Thus, we wondered whether LXRα was upregulated to increase the expression of ABCA1 and ABCG1. As shown in Fig. 6b and d, oridonin treatment up-regulated the protein level of LXRα. These data demonstrated that oridonin alleviates lipid deposition in macrophages by upregulation of LXRα-ABCA1/ABCG1 and downregulation of CD36.