The effects of prophylactic and therapeutic Ori treatment on depressive-like behavior induced by CUMS
In prophylactic treatment, the experimental rats were subjected to CUMS and drug administration simultaneously for three weeks. To determine the potential antidepressive effect of Ori, behavioral analyses were performed. As compared to Control, both CUMS and CUMS + Veh rats showed significantly increased immobility time (respectively, t = 11.71, P < 0.001; t = 18.33, P < 0.001. Figure 2A) and decreased climbing time in the FST (respectively, t = 9.216, P < 0.001; t = 10.44, P < 0.001. Figure 2B). In addition, in comparison with control group, both CUMS and CUMS + Veh also showed fewer percentages of sucrose preference (respectively, t = 13.71, P < 0.001; t = 10.39, P < 0.001. Figure 2C) and lighter body weight (respectively, t = 8.56, P < 0.001; t = 7.058, P < 0.001. Figure 2D). However, both 5 mg/kg and 10 mg/kg of Ori significantly decreased immobility time in CUMS rats compared with CUMS + Veh (respectively, t = 3.918, P < 0.05; t = 9.114, P < 0.001) (Fig. 2A). And only 10 mg/kg of Ori significantly increased climbing time (t = 3.749, P < 0.05. Figure 2B). Similarly, sucrose preference levels were higher in both 5 mg/kg and 10 mg/kg of Ori groups when comparing to CUMS + Veh group at fourth weekend (respectively, t = 2.949, P < 0.05; t = 4.051, P < 0.001. Figure 2C). Meanwhile, no significant differences were detected between 5 or 10 mg/kg and CUMS + Veh in body weight gain measurement (Fig. 2D). Interestingly, the depression-like behaviors in the CUMS rats were not further improved by 20 mg/kg of Ori in the FST and SPT, as well as in body weight gain.
In therapeutic treatment, a visual but insignificant improvement in depression-like behaviors in response to 5 mg/kg of Ori was observed in immobility time and climbing time at sixth weekend (Fig. 2E and 2F)., at which time a significant increase in sucrose preference level compared with CUMS + Veh (t = 2.945, P < 0.05. Figure 2G). Therapeutic treatment with 10 mg/kg of Ori caused substantial inhibition of the depression-like behaviors compared with CUMS + Veh in all test (immobility time, t = 8.384, P < 0.001; climbing time, t = 4.578, P < 0.05; sucrose preference level, t = 5.626, P < 0.001. Figure 2E, 2F and 2G). Same as prophylactic administration, the depression-like behaviors in the CUMS rats were also not improved by 20 mg/kg of Ori therapeutic treatment. In addition, no significant differences were detected among all doses of Ori compared with CUMS + Veh in body weight gain measurement (Fig. 2H). Taken together, these results suggest that both prophylactic and therapeutic application of Ori are able to relieve the depression-like behaviors induced by CUMS, and between the two protocols, the prophylactic treatment was more effective considering the initial effective dose.
Comparison of the anti-depressive effects of Ori, FLX and their combination
Next, we compared the anti-depressive effect of Ori with that of FLX and examined the total effects of these two drugs delivered in the prophylactic treatment. In addition to the usual dose of 10mg/kg, a dose of 18 mg/kg was adopted because this is maximum effective value for the treatment of depression in animal experiments[36, 39, 40]. Compared with CUMS + Veh rats, both prophylactic application of FLX at 10mg/kg and 18mg/kg significantly decreased the immobility time (respectively, q = 7.509, P < 0.001; q = 13.7, P < 0.001. Figure 3A) and increased climbing time (respectively, q = 5.902, P < 0.05; q = 10.68, P < 0.001. Figure 3B) in the FST, meanwhile they increased sucrose preference in the SPT at fourth weekend (respectively, q = 4.589, P < 0.05; q = 9.006, P < 0.001. Figure 3C). The extents of improvement in anti-depressive by FLX at 10mg/kg were significantly weaker compared with those caused by Ori at 10mg/kg in all tests (immobility time, q = 4.617, P < 0.05; climbing time, q = 4.876, P < 0.05; sucrose preference level, q = 4.589, P < 0.05. Figure 3A, 3B and 3C). Ori and FLX were administered in combination to examine the total effects of these two drugs. Our results showed that supplementation with Ori at 5 or 10 mg/kg dose-dependently enhanced the anti-depressive effect of FLX (10mg/kg) alone in all tests, especially 10mg/kg dose of Ori (immobility time, q = 10.81, P < 0.001; climbing time, q = 9.598, P < 0.001; sucrose preference level, q = 8.833, P < 0.001. Figure 3A, 3B and 3C). Interestingly, further observation revealed the anti-depressive effect of Ori (10mg/kg) plus FLX (10mg/kg) was even apparently better than that of FLX at maximum effective dose (18mg/kg) (immobility time, q = 4.617, P < 0.05; climbing time, q = 4.824, P < 0.05; sucrose preference level, q = 4.416, P < 0.05. Figure 3A, 3B and 3C). However, no significant differences were detected among all groups in body weight gain measurement (Fig. 3D). Altogether, these results suggest that the anti-depressive effects of Ori at 10mg/kg are stronger than those of FLX at 10mg/kg. In addition, Ori is a potent adjuvant to increase the effects of FLX.
Ori impeded microglial activation in the prefrontal cortex and hippocampus
Microglial activation in the prefrontal cortex and hippocampus is associated with the development of depression[41, 42]. Therefore, we investigated the change of microglia induced by CUMS and examined the effects of Ori treatment on the prefrontal cortex and hippocampus. According to immunostaining and morphometric analysis, CUMS and CUMS + Veh groups exhibited significantly increased expression levels of Iba-1(a marker of microglia) in the prefrontal cortex compared with Control group (respectively, t = 5.994, P < 0.001; t = 6.114, P < 0.001), suggesting the activation of microglia in the prefrontal cortex of CUMS rats. Ori at 10mg/kg significantly decreased expression levels of Iba-1(t = 3.334, P < 0.05). However, FLX at 10mg/kg could not significantly decrease expression levels of Iba-1(Fig. 4A and 4B). In addition, CUMS and CUMS + Veh groups also exhibited significantly increased expression levels of Iba-1 in the hippocampus compared with Control group (respectively, t = 15.41, P < 0.001; t = 17.36, P < 0.001), while prophylactic application of FLX at 10mg/kg and Ori at 10mg/kg remarkably inhibited the increase of Iba-1 compared with CUMS + Veh group (respectively, t = 4.901, P < 0.05; t = 6.206, P < 0.001) (Fig. 4C and 4D). Above results show that Ori may impede microglial activation in the prefrontal cortex and hippocampus, and FLX also possesses similar function in the latter.
Ori alleviated NLRP3 inflammasome activation in hippocampus and peripheral tissues
To determine the activation of the NLRP3 inflammasome in the hippocampus, we detected its subsequent productions including IL-1β, IL-18 and Caspase-1 (Fig. 5A). As was displayed, relative protein levels of IL-1β, IL-18 and Caspase-1 p10 were significantly upregulated in both CUMS (respectively, t = 5.59, P < 0.001; t = 6.414, P < 0.001; t = 4.914, P < 0.001) and CUMS + Veh groups (respectively, t = 7.216, P < 0.001; t = 6.414, P < 0.001; t = 6.037, P < 0.001) compared with Control group. In addition, both Ori (IL-1β, t = 5.345, P < 0.05; IL-18, t = 3.889, P < 0.05; Caspase-1 p10, t = 3.53, P < 0.05) and FLX (IL-1β, t = 4.025, P < 0.05; Caspase-1 p10, t = 3.354, P < 0.05) hindered those upregulations (Fig. 5B, 5C and 5D). Altogether, Ori impeded NLRP3 inflammasome activation, and FLX also has the ability to inhibit the levels of IL-1β and Caspase-1 p10, except for IL-18.
The ELISA results showed that chronic stress enhanced levels of serum IL-1β, IL-18 and corticosterone, which indicated in the CUMS (respectively, t = 8.736, P < 0.001; t = 6.208, P < 0.001; t = 12, P < 0.001) and CUMS + Veh (respectively, t = 12.94, P < 0.001; t = 5.72, P < 0.001; t = 10.05, P < 0.001) compared with Control. However, injection of Ori normalized serum IL-1β (t = 6.462, P < 0.001), IL-18 (t = 3.646, P < 0.05) and corticosterone (t = 12.48, P < 0.001) when comparing with CUMS + Veh. The function of FLX were similar with Ori in regulating serum IL-1β (t = 3.52, P < 0.05) and corticosterone (t = 9.701, P < 0.001) (Fig. 5E, 5F and 5G).
Ori suppressed CUMS-induced autophagy impairment in the hippocampus
A strong association among autophagy, neuroinflammation and depression has been reported[43–45]. Thus, we investigated autophagy during CUMS-induced neuroinflammation and depressive-like conditions. Western blot results showed that CUMS treatment altered the expression of autophagy-related signaling molecules including Beclin-1, p62, Atg5 and LC3B, while Ori treatment reversed these changes (Fig. 6A). As shown in Fig. 6B, relative protein level of Beclin-1 were significantly downregulated in both CUMS (t = 6.472, P < 0.05) and CUMS + Veh (t = 6.932, P < 0.05) groups compared with Control group, while Ori treatment (t = 3.608, P < 0.05) increased expression level of Beclin-1 compared with CUMS + Veh group. Further, both Ori (t = 6.045, P < 0.05) and FLX (t = 4.207, P < 0.05) treatments upregulated expression levels of P62 compared with CUMS + Veh group respectively (Fig. 6C). As shown in Fig. 6D, relative protein level of Atg5 were significantly downregulated in both CUMS (t = 4.664, P < 0.05) and CUMS + Veh (t = 5.104, P < 0.05) groups compared with Control group, while both Ori (t = 6.108, P < 0.05) and FLX treatments (t = 4.175, P < 0.05) increased expression levels of Atg5 compared with CUMS + Veh group respectively. In addition, the upregulated expression levels of LC3BII observed in the Ori (t = 5.127, P < 0.05) and FLX (t = 4.707, P < 0.05) treatments respectively (Fig. 6E).
Ori dose-dependently blocked the interaction between NLRP3 and NEK7
To explore the molecular mechanisms underlying the effects of Ori, we investigated the interaction between NLRP3 and NEK7 in hippocampus. Firstly, double immunofluorescent staining showed that NEK7 immunoreactivity was mainly double-labeled with Iba-1 (microglia) but not with GFAP (astrocytes) and NeuN (neurons) (Fig. 7A). According to immunoprecipitation and subsequent immunoblotting analyses, Ori treatment dose-dependently blocked the interaction between NLRP3 and NEK7 (Fig. 7B). Co-immunoprecipitation of NEK7 and NLRP3 was then detected (Fig. 7C, 5 mg/kg group, t = 21.86, P < 0.001; 10 mg/kg group, t = 32.2, P < 0.001. Figure 7E, 5 mg/kg group, t = 22.39, P < 0.001; 10 mg/kg group, t = 55.6, P < 0.001). As shown in Fig. 7D and 7F, furthermore, western blotting analysis showed that no alteration in expressions of NEK7 or NLRP3 with Ori treatment, suggesting that Ori could not decrease endogenous levels of NEK7 or NLRP3.
Ori dose-dependently inhibited inflammatory cytokines in Lipopolysaccharide-activated BV2 microglia
The BV2 cells were pretreated with Ori for 12 h followed by incubation with LPS for 24 h (Fig. 8A). Before investigating the effects of Ori on inflammatory response of BV2 cells induced by LPS, we first assayed LPS activity and cytotoxicity by treating BV2 cells with Ori at various concentrations respectively (5, 10, 20, and 40 umol/L). As shown in Fig. 8B, LPS activity decreased in a dose-dependent manner in response to Ori, and the biological activity of LPS was inhibited by about 26% at 40 umol/L (t = 8.362, P < 0.001). Ori was not cytotoxic at concentrations below 20 umol/L, but it decreased cell viability to 93.62% and 92.27% at 40 umol/L in DMSO group and LPS group respectively (t = 3.889, P < 0.05; t = 3.015, P < 0.05. vs. DMSO group) (Fig. 8C). Due to Ori at 40umol/L has certain toxicity to cells, the final concentration of ≦ 20 umol/L was selected for subsequent experiments.
To further investigate whether Ori has any inhibitory effects on the NLRP3 activation in BV2 cells induced by LPS, we assessed the expression levels of IL-1β and IL-18 using ELISA. Our results showed that expression levels of IL-1β and IL-18 were significantly increased following treatment with LPS compared with control (respectively, t = 11.07, P < 0.001; t = 8.586, P < 0.001). In addition, treatment with Ori 10 umol/L decreased the expression levels of IL-1β and IL-18 compared with DMSO group (respectively, t = 7.4, P < 0.05; t = 3.932, P < 0.05). Furthermore, treatment with Ori 20 umol/L has stronger inhibition of IL-1β and IL-18 expressions compared with DMSO group (respectively, t = 11.77, P < 0.001; t = 5.782, P < 0.001) (Fig. 8D and 8E). Above results showed that Ori dose-dependently inhibited inflammatory cytokines in BV2 cells induced by LPS.
Ori suppressed LPS-induced autophagy impairment in Lipopolysaccharide-activated BV2 microglia
Meanwhile, we also investigated autophagy-related proteins in LPS-activated BV2 microglia. As shown in Fig. 9A, LPS treatment altered the expression of autophagy-related proteins including Beclin-1, p62, Atg5 and LC3B, while Ori treatment reversed these changes. According to immunoblotting analyses, expression level of Beclin-1 was significantly decreased in DMSO group (t = 9.61, P < 0.001) compared with Control group, while Ori treatment increased expression level of Beclin-1 compared with DMSO group at 5 umol/L, 10 umol/L and 20 umol/L (respectively, t = 5.093, P < 0.05; t = 3.865, P < 0.05; t = 7.903, P < 0.05) (Fig. 9B). Further, 5 umol/L, 10 umol/L and 20 umol/L Ori treatments increased expression levels of P62 compared with DMSO group respectively (t = 3.973, P < 0.05; t = 5.313, P < 0.05; t = 4.802, P < 0.05) (Fig. 9C). And both 10 umol/L and 20 umol/L Ori treatments increased expression levels of Atg5 compared with DMSO group respectively (t = 4.615, P < 0.05; t = 5.444, P < 0.05) (Fig. 9D). As shown in Fig. 9E, 5 umol/L, 10 umol/L and 20 umol/L Ori treatments increased expression levels of LC3BII compared with DMSO group respectively (t = 3.818, P < 0.05; t = 4.243, P < 0.05; t = 4.069, P < 0.05).