Oridonin relieves depressive-like behaviors by inhibiting neuroinflammation and autophagy impairment in rats subjected to chronic unpredictable mild stress

Major depressive disorder (MDD) is a severe life-threatening disorder with increasing prevalence. However, the mechanistic interplay between depression, neuroinflammation, and autophagy is yet to be demonstrated. This study investigated the effect of Oridonin on CUMS-induced depression, neuroinflammation, and autophagy impairment. Male 4-week-old Sprague – Dawley rats were subjected to chronic unpredictable mild stress (CUMS), some of which were injected with Oridonin, fluoxetine (FLX), or their combination at different durations of CUMS. CUMS significantly increased the levels of cytokines (IL-1 β , IL-18, and caspase-1), reduced autophagy-related protein levels (Beclin-1, p62, Atg5, and LC3B), and caused microglia cells activation. Oridonin prevented and reversed the depressive-like behavior. Furthermore, it has a stronger and longer-lasting antidepressant effect than FLX. And the antidepressant effect of Oridonin in combination with fluoxetine was greater than that of high-dose fluoxetine alone. In addition, Oridonin significantly normalized autophagy-related protein levels, and reduced levels of cytokines by blocking the interaction between NLRP3 and NEK7. Similarly, Oridonin abolished levels of cytokines and reversed autophagy impairment in LPS-activated BV2 cells. All these results supported our hypothesis that Oridonin possesses potent anti-depressive action, which might be mediated via inhibition of neuroinflammation and autophagy impairment by blocking the interaction between NLRP3 and NEK7.

(interleukin 1β) and , were positively associated with depressive symptoms (Rosa et al., 2004;Wu et al., 2020). And, several studies showed that elevation in brain IL-1β level is both necessary and sufficient for producing a high incidence of depression, and reducing brain IL-1 levels may have potent anti-depressive actions (Ellul, Boyer, Groc, Leboyer, & Fond, 2016;Goshen et al., 2008). The NLRP3 inflammasome is one multiprotein complex that serves as platform for the activation of caspase-1, leading to the processing and secretion of IL-1β and IL-18 (Gross, Thomas, Guarda, & Tschopp, 2011). Therefore, depressive-like behaviors required a functional NLRP3 inflammasome (Alcocer-Gomez et al., 2016;Zhang et al., 2015). Microglia in the central nerve system (CNS) act as the first line of broader immune response to pathogen-associated molecular patterns. When stimulated by external factors, microglia can be activated via TLR-like or NOD-like receptors, and then released immune molecules, including proinflammatory cytokines, chemokines, and reactive oxygen species (Walsh, Muruve, & Power, 2014), which leads to the development of depressive symptoms (Wong et al., 2016).
Autophagy is a catabolic process that degrades the cytosolic constituents through autophagosome formation. It regulates cytokine production and secretion, inflammasome activation, and clearance of accumulating as well as invading pathogens (Deretic & Levine, 2018).
In the last decade, numerous studies have further indicated that autophagy can regulate NLRP3 inflammasome activation through various mechanisms (Saitoh & Akira, 2016). On the contrary, a few studies have also shown that NLRP3-deficient mice have increased autophagy levels at baseline and under stress conditions (Kim et al., 2018;Zhang et al., 2014). In addition, caspase-1 was also reported to regulate the autophagic process through cleavage of other substrates (Jabir et al., 2014;Yu et al., 2014). Studies have evidenced the activation/ inhibition of autophagy and excessive activation of microglia to have a close relationship with depression, but the role of autophagy in depression pathogenesis is ambiguous (Shih et al., 2019;Tan et al., 2018). Therefore, scholars across the world postulated that future procedures targeting the NLRP3 inflammasome may have promising effects in the prevention and treatment of depression (Kaufmann et al., 2017;Xu et al., 2016).
Oridonin (Ori), a diterpenoid isolated from Rabdosia rubescens, has multiple biological properties, especially anti-inflammatory and neuroregulatory activities (Lin et al., 2019;Xu, Li, Zhang, Schluesener, & Zhang, 2019). Although it has been suggested that Ori may be involved in antidepressant effects through PPAR-γ/AMPA receptor signaling pathway, its specific targeting mechanism has not been clarified (Liu & Du, 2020). Recently, one finding suggested that Ori can be used to treat a variety of inflammatory diseases by blocking the interaction between NLRP3 and NEK7 (He et al., 2018). NIMA-related kinase 7 (NEK7), which plays a crucial role in mitosis entry, cell cycle progression, cell division, and mitotic processes, has been proven to be a vital mediator during inflammasome activation in macrophages (Shi et al., 2016). In our previous study, Ori effectively reduced insulin resistance in dysglycemia comorbid depression model by inhibiting peripheral inflammation (Liang, Zheng, Xie, Xiao, & Wang, 2021).
However, the molecular mechanism of Ori in the treatment of neuroinflammation-induced autophagy impairment and depressivelike behaviors is yet to be elucidated. Therefore, we investigated whether Ori can attenuate depressive-like behaviors by inhibiting neuroinflammation and autophagy impairment. For better appraisal, we also compared the anti-depressive effects of Ori with those of fluoxetine (FLX, as classic antidepressants). In addition, in order to verify whether be used as a complementary treatment for antidepressants, we tested the total effects of the combined application of these two drugs.

| Chronic unpredictable mild stress (CUMS) protocols
The procedure used for the chronic unpredicted mild stress (CUMS) was adapted from our past research (H. Wang, Xiao, Wang, & Wang, 2020). Rats in the CUMS group were subjected to various mild stressors for 4 and 6 weeks, respectively. The stressors applied included the following: cage tilting (45 ) for 24 h, damp sawdust (200 mL of water in a cage) for 24 h, swimming in water at 45 C for 5 min, swimming in water at 5 C for 5 min, tails clamped for 1 min, 24 h of water deprivation, 12 h of overnight illumination, cage shaking for 10 min, making noise for 10 min and 24 h of food deprivation.
During the stress procedure, two different stressors were applied each day, and the sequence of stressors changed every 3 days. Control rats were housed under the condition of the same breed but with no exposure to the above stressors.

| Drug administration and experiment design
Ori (purity ≥98%, CAS: 28957-04-2) purchased from Alfabiotech company (Chengdu, China) was dissolved in dimethylsulfoxide (DMSO, Sigma, St. Louis, MO) and then diluted in saline to a concentration of 20 mg/mL. The structure of the Ori is shown in Figure 1a. FLX (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in phosphate-buffered saline to obtain a stock solution of 5 mg/mL (PBS, pH 7.4).
The doses of Ori administered intraperitoneally were adopted from previous studies of Alzheimer's disease (Wang et al., 2014;Wang et al., 2016). All rats were randomly selected and given intraperitoneal injection of Ori and FLX.
Two patterns of drug application were employed in this study: prophylactic and therapeutic treatment. According to our preliminary experimental results (Figure 1b-e), the rats subjected to mild stressors developed depressive symptoms from the third week. Thus, in the prophylactic treatment, drugs were given after rats were subjected to mild stressors at first day until third weekend. In this paradigm, the rats were treated with Ori at 5, 10, or 20 mg/kg and FLX at 10 or 18 mg/kg once per day for 3 weeks. In addition, FLX at 10 mg/kg together with varied doses of Ori in the drug combination study. The FST was performed at the fourth weekend, SPT at third and fourth weekend and body weight measurement once per week. For the therapeutic treatment, the rats received daily treatment with Ori at 5, 10, or 20 mg/kg once per day from the fourth weekend to the sixth weekend during CUMS procedure. The FST, SPT, and body weight measurements were performed at the sixth weekend ( Figure 1f).

| Behavioral tests
All behavioral tests, including sucrose preference test and forced swim test, were all carried out in the dark phase (19:00-22:00 p.m.).

| Sucrose preference test
The sucrose preference test (SPT) is a widely used method of evaluating depressive-like behavior in animals (Willner, 2017). According to this literature, food and water were deprived 20 h before SPT. During the test, the rats were allowed to drink 1% sucrose solution (250 ml) and the same volume of water for 2 h. The one bottle of sucrose solution was changed into water on the second day. Fluid consumptions were checked after the 2 h test. Prior to the CUMS, baseline preference for sucrose was recorded, and only rats that showed a sucrose preference >65% were included in this study. Sucrose preference proportion = sucrose solution consumption/(sucrose solution consumption + tap water consumption) * 100%.

| Forced swim test
The Forced swim test (FST) is one of the most commonly used animal models for assessing antidepressant-like behavior (Slattery & Cryan, 2012). According to literature, the FST setup consisted of a cylinder (40 cm height Â28 cm internal diameter). In the pretest, the rats were individually placed in the cylinder with water at 25 C filled to 30 cm. The rats were removed after 15 min, dried, and returned to their home cages. Then on the test day, they were immersed in a swimming tank for 6 min one by one. Immediately after the testing, rats were removed from the water, gently dried with paper towels, The following groups were used: CUMS and Control. The data (mean ± SEM) were analyzed by Student's t-test for two-group comparisons, n = 8. *p < .05, **p < .001 versus Control group and placed inside a cage warmed by a heating pad. The duration of climbing and immobility was videotaped and analyzed. The immobility time was defined as floating time in the water without struggling and only performing the movements to keep the head above water during the final 4 min. And Climbing time was defined as upward-directed movement time of the forepaws.

| Sacrifice and sample preparation
After completion of behavioral tests, all rats were sacrificed by decapitation. Hippocampus were dissected and flash-frozen in liquid nitrogen immediately after decapitation. The remaining rats were transcardially perfused with 0.9% saline, then with 4% paraformaldehyde in phosphate buffer. And then, the brains were rapidly collected and fixed in 4% paraformaldehyde for 1 week. Finally, the brains were embedded in paraffin. Serial 5 μm sections were cut in the coronal plane.

| Immunostaining and morphometric analysis
According to the previous study (Rho & Swanson, 1989), the Paraffin- (v/v) glycerin and 2.5% (w/v) triethylenediamine. The confocal images were obtained, and digital images were captured using a Fluoview laser scanning confocal microscopes (Olympus) equipped with the FV1000 (Ver.1.7a) software.

| Western blot analysis
The hippocampus and prefrontal cortex were homogenized in the ice- Beyotime Biotechnology, China) and exposed to X-ray film (BIORAD).
For analysis, the levels of target proteins were normalized against those of GAPDH (Martinet et al., 2004).

| Immunoprecipitation
For detecting the interaction of NEK7 and NLRP3 by coimmunoprecipitation (Co-IP) (Ponzielli et al., 2008). According to the manufacturer's instructions (P2012, Beyotime Biotechnology, China), the centrifuged lysates were incubated with a 1:200 dilution of anti-NEK7 or anti-NLRP3 antibody overnight, at 4 C. Then, 40 μl Protein A/G Agarose (Beyotime, China) was added and incubated for an additional 4 h in a shaker. The immune complexes were boiled in the sample buffer after washing with PBS five times. The samples were then immunoblotted with anti-NLRP3 or anti-NEK7, respectively.

| Cell culture and treatment
Mouse microglial (BV2 cells) purchased from China Center for Type Culture Collection (Wuhan, China), were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and incubated in a humidified atmosphere of 5% CO 2 and 95% air at 37 C. The cells (passages from 10 to 15) were pretreated with Ori (respectively, 5, 10, 20, or 40 μmol/L) for 12 h followed by incubation with LPS (1 μg/mL) for 24 h.

| Cell viability analysis
Cell viability was measured using MTT (C0009, Beyotime Biotechnology, China) assay (Cai et al., 2021). The microglia cells were plated in cell culture plates at a density of 1.5 Â 10 3 cells/well and cultured overnight. Various concentrations of Ori (respectively, 5, 10, 20, or 40 μmol/L) were added to each well and incubated for 12 h.
According to the manufacturer's instructions, the MTT solution (5 mg/mL) was added to each well and incubated in a humidified atmosphere of 5% CO 2 and 95% air at 37 C for 4 h. Thereafter, the formazan solution was added and incubated at 37 C for 3 h. After the formazan crystals had dissolved, absorbance was read at 570 nm.

| Limulus amebocyte lysate (LAL) test
The effect of Ori on LPS activity was measured by using the LAL test (Rat Lipopolysaccharides ELISA Kit, CSB-E14247r, CUSABIO, Wuhan, China) (Lamprou et al., 2022). Briefly, a series of concentrations of the Ori (5, 10, 20, and 40 μmol/L) were incubated with LPS (1 μg/mL) for 30 min at 37 C. The absorbance was measured at 450 nm after the addition of 100 μl of the chromogenic substrate.

| Statistical analysis
All data were analyzed with GraphPad Prism 6 software (San Diego, CA, USA) and are presented as the mean ± SD. The normality of the data was tested using the D'Agostino and Pearson omnibus normality test. In addition, the data from behavioral tests at one point in time and Western blot analysis were analyzed by Student's t-test for twogroup comparisons or one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test for the various CUMS groups.
p-Values less than 0.05 were considered statistically significant.

| 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 3 weeks. To determine the potential anti-depressive effect of Ori, behavioral analyses were performed. Firstly, no significant differences were detected between CUMS and CUMS + Veh in all tests. As compared to Control, CUMS rats showed significantly increased immobility time (t = 11.71, p < .001. Figure 2a) and decreased climbing time in the FST (t = 9.216, p < .001. Figure 2b). In addition, in comparison with control group, CUMS also showed fewer percentage of sucrose preference (t = 13.71, p < 0.001. Figure 2c) and lighter body weight (t = 8.56, p < .001. Figure 2d) at fourth weekend. 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 < .05; t = 9.114, p < .001) (Figure 2a). And only 10 mg/kg of Ori significantly increased climbing time (t = 3.749, p < .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 < .05; t = 4.051, p < .001. The following groups were used: Control, CUMS, CUMS+Vehicle, CUMS+Ori (5 mg/kg), CUMS+Ori (10 mg/kg), and CUMS+Ori (20 mg/kg). The data (mean ± SEM) were analyzed by one-way analysis of variance (ANOVA), n = 8. *p < .05, **p < .001 versus Control group; #p < .05, ##p < .001 versus CUMS + Veh group increased sucrose preference in the SPT at fourth weekend (respectively, p < .05; p < .001. Figure 3c). The extents of improvement in anti-depressive by FLX at 10 mg/kg were significantly weaker compared with those caused by Ori at 10 mg/kg in all tests (immobility time, p < .05; climbing time, p < .05; sucrose preference level, p < .05 Figure 3a-c). 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 (10 mg/kg) alone in all tests, especially 10 mg/kg dose of Ori (immobility time, p < .001; climbing time, p < .001; sucrose preference level, p < .001 Figure 3a-c). Interestingly, further observation revealed the anti-depressive effect of Ori (10 mg/kg) plus FLX (10 mg/kg) was even apparently better than that of FLX at maximum effective dose (18 mg/kg) (immobility time, p < .05; climbing time, p < .05; sucrose preference level, p < .05. Figure 3a-c). However, no significant differences were detected among all groups in body weight gain measurement (Figure 3d). Altogether, these results suggest that the anti-depressive effects of Ori at 10 mg/kg are stronger than those of FLX at 10 mg/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 (Colasanti et al., 2016;Pan, Chen, Zhang, & Kong, 2014). 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, there was no significant difference in Iba-1(a marker of microglia) expression levels F I G U R E 3 Comparison of the anti-depressive actions between Ori and FLX and the total effects of these two drugs. (a) Immobility time in the FST. (b) Climbing time in the FST. (c) Consumption of sucrose water in the SPT. (d) Body weight gain. The following groups were used: CUMS+Vehicle, CUMS+Ori (5 mg/kg), CUMS+Ori (10 mg/kg), CUMS+FLX (10 mg/kg), CUMS+Ori (10 mg/kg) + FLX (10 mg/kg), CUMS+Ori (5 mg/kg) + FLX (10 mg/kg), and CUMS+FLX (18 mg/kg). The data (mean ± SEM) were analyzed by one-way analysis of variance (ANOVA), n = 8. #p < .05, ##p < .001 versus CUMS + Veh group; &p < .05, &&p < .001 versus FLX 10 mg/kg group; +p < .05 versus FLX 18 mg/kg between CUMS and CUMS + Veh in prefrontal cortex. In addition, CUMS and CUMS + Veh exhibited significantly increased expression levels of Iba-1 in the prefrontal cortex compared with Control group (respectively, t = 5.994, p < .001; t = 6.114, p < .001), suggesting the activation of microglia in the prefrontal cortex of CUMS rats. Ori at 10 mg/kg significantly decreased expression levels of Iba-1(t = 3.334, p < .05). However, FLX at 10 mg/kg could not significantly decrease expression levels of Iba-1 (Figure 4a,b). Similarly, there was no significant change for Iba-1 expression levels between CUMS and CUMS + Veh in hippocampus. Both CUMS and CUMS + Veh exhibited significantly increased expression levels of Iba-1 in the hippocampus compared with Control group (respectively, t = 15.41, p < .001; t = 17.36, p < .001), while prophylactic application of FLX at 10 mg/kg and Ori at 10 mg/kg remarkably inhibited the increase of Iba-1 compared with CUMS + Veh group (respectively, t = 4.901, p < .05; t = 6.206, p < .001) (Figure 4c,d). The 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 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) (Figure 7a). According to immunoprecipitation and subsequent immunoblotting analyses, Ori treatment dose-dependently blocked the interaction between NLRP3 and NEK7 (Figure 7b). Coimmunoprecipitation of NEK7 and NLRP3 was then detected (Figure 7c, 5 mg/kg group, t = 21.86, p < .001; 10 mg/kg group, t = 32.2, p < .001. Figure 7e, 5 mg/kg group, t = 22.39, p < .001; 10 mg/kg group, t = 55.6, p < .001). As shown in Figure 7d and f, furthermore, western blotting analysis showed no alteration in expressions of NEK7 or NLRP3 with Ori treatment, suggesting that Ori could not decrease endogenous levels of NEK7 or NLRP3.
F I G U R E 6 Detection of related protein levels of autophagy-related proteins in hippocampus. (a) Densitometry analyses of the bands. (b-e) Western blot analysis of Beclin-1 (b), p62 (c), Atg5 (d), and LC3B (e) protein expression. The following groups were used: Control, Control+Ori (10 mg/kg), CUMS, CUMS+FLX (10 mg/kg), and CUMS+Ori (10 mg/kg). The data (mean ± SEM) were analyzed by one-way analysis of variance (ANOVA), n = 3-4. The experiment was repeated three times. *p < .05 versus Control group; #p < .05 versus CUMS + Veh group 3.7 | 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 (Figure 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 μmol/L). As shown in Figure 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 μmol/L (t = 8.362, p < .001). Ori was not cytotoxic at concentrations below 20 μmol/L, but it decreased cell viability to 93.62% and 92.27% at 40 μmol/L in DMSO group and LPS group, respectively (t = 3.889, p < .05; t = 3.015, p < .05. vs. DMSO group) ( Figure 8c). Due to Ori at 40 μmol/L has certain toxicity to cells, the final concentration of ≦ 20 μmol/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 < .001; t = 8.586, p < .001). In addition, treatment with Ori 10 μmol/L decreased the expression levels of IL-1β and IL-18 compared with DMSO group (respectively, t = 7.4, p < .05; t = 3.932, F I G U R E 7 Detection of the interaction between NLRP3 and NEK7 in hippocampus. (a) Immunofluorescent double staining. (b) Coimmunoprecipitation of NEK7 and NLRP3. (c-f) Western blot analysis of NEK7 and NLRP3. The data (mean ± SEM) were analyzed by Student's t-test for two-group comparisons, n = 3-4. The experiment was repeated three times. **p < .001 versus Control group p < .05). Furthermore, treatment with Ori 20 μmol/L has stronger inhibition of IL-1β and IL-18 expressions compared with DMSO group (respectively, t = 11.77, p < .001; t = 5.782, p < .001) (Figure 8d,e).
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 LPSactivated BV2 microglia. As shown in Figure 9a, LPS treatment altered the expression of autophagy-related proteins including Beclin-1, p62, Atg5, and LC3B, while Ori treatment reversed these changes.

| DISCUSSION
The link between excessive inflammatory response and autophagy impairment is considered a hallmark of various neurodegenerative F I G U R E 8 Detection of cytokine productions in LPSactivated BV2 cells. (a) Experimental paradigms illustrating the drug application. (b) LPS activity. (c) Cell Viability. *p < .05, **p < .001 versus DMSO group. (d and e) ELISA analysis of IL-1β (d) and IL-18 (e) expression. The following groups were used: Control, DMSO, CUMS+Ori (5 mg/kg), CUMS+Ori (10 mg/kg), and CUMS+Ori (20 mg/kg). The data (mean ± SEM) were analyzed by Student's t-test for two-group comparisons, n = 3-4. The experiment was repeated three times. **p < .001 versus Control group; #p < .05 versus DMSO group diseases including Parkinson's disease (PD) and Alzheimer's disease (AD) (El Sayed & Ghoneum, 2020;Yao et al., 2019). However, the mechanistic interaction between neuroinflammation, autophagy, and depression is still largely unknown. Moreover, several studies showed that Ori has not only anti-inflammatory effect, but it has the function of regulating autophagy (Xu et al., 2019;Yao et al., 2017). Thus, this study aimed to determine whether autophagy and neuroinflammation mediate the antidepressant effects of Ori.
Although Ori has recently been recommended as a potential drug for the treatment of depression (Liu & Du, 2020), its mechanism of action and dosage have not been thoroughly studied. In the present study, we found that Ori treatment is able to inhibit chronic cytokinemediated inflammatory responses characterized by activation of NLRP3 inflammasome and consequent productions (IL-1β, IL-18, and caspase-1) in the brain. In addition, it ameliorates microglia activity and most importantly autophagy impairment induced by CUMS, which may underlie its anti-depressive effects. We tend to attribute this effect partially to the function of blocking the interaction between NLRP3 and NEK7 via Ori directly binding with NLRP3. Furthermore, it is also confirmed that Ori is a potent adjuvant to increase the antidepressive effects of FLX. In this study, both prophylactic and therapeutic treatments with Ori effectively improved depressive-like behaviors induced by CUMS, with the effects of the former being more pronounced than the latter. However, our results showed that Ori could not improve depressive-like behaviors at 20 mg/kg. A previous study demonstrated that Ori did not attenuate the memory impairment in AD mice at 20 mg/kg , which also confirmed our finding through other models. Therefore, it seems that Ori has the dose ceiling effect in anti-depressive treatment. In the prophylactic paradigm, interestingly, the effects of Ori still on depressive-like behaviors of rats after withdrawal 1 week, although we applied Ori for the first 3 weeks throughout CUMS treatment.
This demonstrated that Ori affected depression with a slow onset and offset property. Therefore, Ori may be an ideal candidate for preventing the occurrence of depression when used at the early stages of chronic stress. FLX, as the first specific serotonin reuptake inhibitor, has been an essential medicine for major depression in clinics (Zhou et al., 2020). Furthermore, it is also frequently employed in animal experiments to explore the efficacy of other new drugs as a reference (Ma, Wang, Xu, Wang, & Wang, 2018;Szewczyk et al., 2019). In this study, our results showed that the anti-depressive effect of Ori at 10 mg/kg was slightly stronger than that of FLX at 10 mg/kg in all behavioral tests. In addition, the combined application of Ori with FLX dose-dependently enhanced the anti-depressive of FLX alone, with the most prominent effect of Ori observed at 10 mg/kg, which is even better than maximum effective dose of FLX alone.
The NLRP3 is an intracellular signaling molecule that binds to ASC upon activation, and then interacts with pro-caspase-1 to create a complex referred to as the inflammasome, which leads to the F I G U R E 9 Detection of related protein levels of autophagy-related proteins in LPS-activated BV2 cells. (a) Densitometry analyses of the bands. (B-E) Western blot analysis of Beclin-1 (b), p62 (c), Atg5 (d), and LC3B (e) protein expression. The following groups were used: Control, DMSO, CUMS+Ori (5 mg/kg), CUMS+Ori (10 mg/kg), and CUMS+Ori (20 mg/kg). The data (mean ± SEM) were analyzed by Student's t-test for two-group comparisons, n = 3-4. The experiment was repeated three times. *p < .05, **p < .001 versus Control group; #p < .05 versus DMSO group activation of IL-1β, IL-18, and caspase-1. In recent years, more and more evidence indicated that activation of NLRP3 inflammasomes is involved in altered prefrontal cortex and hippocampal function and consequent mood disorders of neuropathic states, which can be ameliorated by their pharmacological antagonisms (Liu, Li, Su, Wang, & Jiang, 2019;Pan et al., 2014). NEK7 is an important component of the NLRP3 inflammasome in macrophages. However, it is not known that whether NEK7 is also expressed in brain cells. In this study, our findings showed that it was highly expressed in microglia but not in astrocytes and neuron-like cells. Furthermore, Wu et al. (Wu et al., 2020) indicated that IL-18 expression was mainly found in microglia at a later phase of post-stroke depression. Therefore, we believe that NEK7 binding NLRP3 activated IL-1β, IL-18, and caspase-1 maturation in microglia at a later phase of depression. In this study, prophylactic application of Ori significantly reduced neuroinflammation in the hippocampus, as manifested by suppression of microglial activation, reversal of cytokine levels, and blocking the interaction between NEK7 and NLRP3.
Previous studies on the role of autophagy in depression yielded inconclusive results (Jia & Le, 2015;Song et al., 2017;Tan et al., 2018). However, some recent animal experiments have shown that CUMS reduced the expression of autophagy-related proteins (Shu et al., 2019;Zhao et al., 2017). Therefore, one of our aims was to investigate the mechanistic relationship among neuroinflammation, depression, and autophagy function after Ori treatment. In this study, our results indicated that CUMS decreased the expression of autophagy-related proteins. And CUMS-induced depressive-like behavior and neuroinflammation are associated with autophagy impairment via NLRP3 inflammasome activation. Furthermore, Ori, as well as fluoxetine treatment, significantly reversed the expression of these proteins, suggesting that Ori plays a key role in improving autophagy impairment in microglia cells under stress conditions. The mechanism regulating autophagy might be involved in tissue or cell types.
Currently, there are many inhibitors of NLRP3 inflammasome, but the mechanism of action is different. BAY11-7082, as NF-κB inhibitor, indirectly inhibited NLRP3 inflammasome activation to alleviate neuroinflammation (Jiang, Li, He, Zhou, & Zhu, 2017). In addition, MCC950, another small-molecule inhibitor, has been used to treat NLRP3-associated autoinflammatory and autoimmune diseases (Coll et al., 2015). And several observations demonstrated that MCC950 completely abrogated neuroinflammation (Chivero et al., 2021;Fu et al., 2020;Huang et al., 2021). It directly interacts with the Walker B motif within the NLRP3 NACHT domain, thereby inhibiting NLRP3 activation and inflammasome formation (Coll et al., 2019). According to the latest research, we found that Oridonin forms a covalent bond with the cysteine279 of NLRP3 in NACHT domain to block the interaction between NLRP3 and NEK7, thereby inhibiting NLRP3 inflammasome assembly and activation (He et al., 2018). Thus, MCC950 and Ori may have a similar pharmacological mechanism. Ori is a commonly used traditional Chinese medicine for the treatment of inflammatory diseases and a high-lipophilic small molecule diterpene compound that passes through the blood-brain barrier through passive diffusion. Therefore, we believed that blocking the interaction between NLRP3 and NEK7 by Ori-mediated is common cause for the anti-depressive effect.
In conclusion, our study demonstrated that chronic stress stimuli strongly induce pro-inflammatory cytokines including IL-1β, IL-18, and caspase-1 along with depressive-like behaviors. Further NLRP3 inflammasome activation was associated with autophagy impairment under CUMS-induced stress conditions. Ori acts as an antidepressant by attenuating neuroinflammation and autophagy impairment via blocking the interaction between NLRP3 and NEK7. Our findings suggest that treatment with Ori could be a valuable therapeutic strategy to treat neuroinflammation associated with autophagy impairment and depressive-like behaviors. In addition, it is noteworthy that Ori is a potent adjuvant to increase the anti-depressive effects of FLX.
However, there was a limitation in our study. We did not conduct in vivo toxicological study, because it was previously reported that oridonin has effects on liver function.

AUTHOR CONTRIBUTIONS
Liang Liang and Gaohua Wang performed design and wrote the manuscript, Liang Liang, Hui Wang, Ying Hu, Hetao Bian, and Ling Xiao performed research and analyzed data. The authors read and approved the final manuscript.