Propofol inhibits inflammatory response by regulating the miR-494/ Nrdp1 pathway in ICH mice model

Background: Propofol is an anesthetic agent with neuro-protective effect in neuronal injury. However, the mechanism of propofol in M1 macrophage polarization following ICH has not been well studied. Ubiquitination mediated M1/M2 macrophage polarization plays important roles in pathogenesis of immune disease. The experiment analyzed anti-inflammatory effects of propofol in macrophages following ICH. Methods: In the experiment, macrophages were administrated with erythrocyte lysates, and then miR-494, Nrdp1 and M1 related markers were analyzed. In addition, brain inflammatory response, brain edema, and neurological functions of ICH mice were also assessed. Results: We found that propofol decreased miR-494 levels while increased Nrdp1 levels in macrophages after ICH. We also demonstrated that miR-494 inhibited Nrdp1 expression by directly binding its 3′-untranslated region. MiR-494 attenuated Nrdp1 levels and downstream proinflammatory factors production. Upregulation of Nrdp1 in macrophages significantly decreased M1 macrophage polarization. Conclusion: Taken together, these results suggest that propofol can attenuate the neuroinflammatory response of macrophages after ICH through regulation of the miR-494/Nrdp1 pathway. were transduced with miR-494 or controls, and further treated by propofol. The results promote hematoma resolution in experimental intracerebral

Propofol is an intravenous hypnotic agent utilized in anesthesia and intensive care (7).
Propofol acts by potentiating the γ-aminobutyric acid type A (GABA-A) receptor-mediated inhibition in the central nervous system (8). In addition, propofol has been identified to play neuroprotective role by anti-inflammatory properties in brain injury (9)(10)(11). However, whether propofol contributes to M1/M2 macrophage polarization following ICH and the specific molecular mechanism has not been studied. miRNAs are small noncoding (NC) RNA molecules (~ 20-22 nucleotides) and contribute to mRNA post-transcriptional regulation (12)(13)(14). In recent years, miRNAs have acted as potential biomarkers in inflammatory diseases (15). In addition, miRNAs are involved in the development and regulation of both innate and adaptive immunity and contribute to regulate M1/M2 macrophage polarization (16)(17)(18). Recent studies have suggested that altered expression of miR-494 contributed to several inflammation-mediated diseases (19). Notably, targeting miR-494 has been found to be potential biomarker and therapeutic target to the inflammatory response.
Ubiquitination is a common post-translational modification of protein and regulates many cell processes, such as growth, cycle, and apoptosis (20). Ubiquitination has also been identified in many aspects of immune responses (21). Nrdp1, an E3 ubiquitin ligase, has previously been demonstrated to inhibit M1 activation of macrophages (22).
In this experiment, we made a hypothesis that the miR-494 signaling pathway contributes to the protective roles of propofol in ICH-induced neuroinflammation. Erythrocyte lysatestreated macrophages and ICH mice have been used as an in vitro and in vivo model to study the mechanisms underlying neuronal injury. The results demonstrated that propofol could attenuate pro-inflammatory mediator production by downregulating miR-494 levels, which targeted Nrdp1 signaling pathways. BMDMs (bone marrow derived macrophages) were isolated from the marrow of the femurs and tibias of C57BL/6 mice. The legs of the animals were sprayed with 70% EtOH, and the skin and muscle tissue were removed from the bones. The bones were sprayed with 70% EtOH, transferred to a sterile-flow hood and cut at both ends. The marrow was flushed out into a sterile falcon tube in Dulbecco's modified Eagle's medium supplemented with heatinactivated fetal bovine serum (FBS; 50 ml; 10%) and penicillin-streptomycin (5 ml; 1%; Gibco). The cell suspension was triturated using a sterile Pasteur pipette, filtered through a nylon mesh filter into a sterile tube and centrifuged (400 × g, 5 min). The supernatant was removed, and the pellet was resuspended in red blood cell lysis buffer (Sigma-Aldrich, Gillingham). The suspension was centrifuged (400 × g, 5 min), the supernatant was discarded and the cells were washed using DMEM and centrifuged once more (400 × g, 5 min). The pellet was resuspended in 20 ml of DMEM supplemented with L929-conditioned media (20%). Cells were seeded in sterile cell culture flasks (T175 cm 2 flasks). On day 2, non-adherent cells were removed from the flask, the media was replaced and the remaining adherent cells were maintained in culture for a further 6 days.
Neuronal cultures cerebral cortices and hippocampus of fetus mice were dissected out and the meninges were carefully removed. Cells (1 × 10 6 cells/mL) were maintained in poly-d-lysine (Sigma, St. Louis, MO) coated plates in Dulbecco's modified eagle medium (DMEM) medium (Life Technologies) with 10% fetal bovine serum (FBS) (Life Technologies). After 4-6 h of culture, the cultures were replenished with Neurobasal medium (Life Technologies) containing 100 U/mL penicillin, 100 µg/mL streptomycin, 2% B27, and 0.5 mM glutamine (Life Technologies) at 37 • C with 5% CO 2 . The medium was changed every three days.

Preparation of erythrocyte lysates
Whole blood collected from 30 to 50 mice was pooled and leuko reduced using a Neonatal High-Efficiency Leukocyte Reduction Filter (Purecell Neo; Pall Corporation). Blood was centrifuged at 400 × g for 15 minutes, and the volume reduced to a final hemoglobin level ranging from 17.0 to 17.5 g/dL, as determined by a modified Drabkin hemoglobin assay at a 1:251 dilution of stored RBCs to Drabkin reagent (Ricca Chemical Company). Washed stored RBCs were prepared with 3 washes using 10 volumes of phosphate-buffered saline (PBS) and centrifugation at 400 g. After the final wash, the washed stored RBCs were resuspended in PBS to a final hemoglobin concentration of 17.0 to 17.5 g/dL for transfusion. Supernatant was obtained using a 400gspin of stored RBCs and 400 µL of this solution were transfused undiluted. RBC ghosts were obtained by hypotonic lysis of twice the volume of stored RBCs (ie, for 400 µL of ghosts, 800 µL of stored RBCs were hemolyzed) with PBS to distilled water (1:15), followed by multiple washes with the same buffer and centrifugation at 30 000 g until a white pellet was obtained. The white pellet of RBC ghosts was resuspended in PBS. Stroma-free RBC lysate was prepared by freeze-thaw of washed stored RBCs followed by centrifugation at 16 000 × g to pellet and remove the stroma.

Cell treatment
Macrophages (1 × 10 5 ) were stimulated with propofol (0 ~ 100 µM) plus 10 µl PBS or erythrocyte lysates for 24 hs. After then, the supernatants were removed and further analyzed for cytokine production with ELISA.

Real-time PCR
The ipsilateral hemisphere was homogenized using RNAiso Plus (Takara) and ceramic beads for 1 min in a speedmill plus according to the instructions of the manufacturer (Alytik Jena). RNA was isolated according to the instructions of the manufacturer and reverse transcripted to obtain cDNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara). Real-time PCR was performed using cDNA samples with SYBR@Premix ExTaq™II (Takara, Tli RNaseH Plus) by the One-step Plus analyzer (ABI). We normalized the results for each individual gene using the housekeeping gene beta-actin. The 2 − ΔΔCT method was used to calculate relative gene expression levels.

Western blotting analysis
Proteins from cultured macrophages were resolved using SDS-PAGE and transferred onto polyvinylidene fluoride membranes using electroblotting. The membranes were incubated with primary antibodies, all diluted to 1:1000 (Cell Signaling Technology), at 4˚C overnight. GAPDH (1:200; Santa Cruz Biotechnology, Dallas, TX) was used as the loading control. The membranes were incubated with HRP-conjugated goat anti-rabbit secondary Abs (1:2500; Sigma-Aldrich, St. Louis, MO) at 25˚C for 1 h. Bound Abs were visualized using a chemiluminescence detection system. Protein levels were calculated as the ratio of the target protein value to the GAPDH value.

Enzyme-linked immunosorbent assay
The supernatants or brain tissue extracts were harvested, and TNF-α, IL-1β and IL-6 productions were determined by ELISA. The specimens were assayed using respective Enzyme Linked Immunosorbent Assay (ELISA) kits (Minneapolis, MN, USA) according to the instruction manuals.

Cytotoxicity assay
The cytotoxic activity of macrophages was measured by a 6 h lactate dehydrogenase release assay using CytoTox96 Non-radioactive Cytotoxicity Assay kit (Promega, Charbonnie'res-les-Bains, France) on 5 × 10 3 neuron/well. Neuron was then added to the Vector construction and luciferase reporter assays Luciferase reporter constructs were used, and luciferase assays were performed as described previously. Briefly, the mouse Nrdp1 3′-UTR sequence was amplified by PCR from mouse genomic DNA, and ligated into the pMIR-REPORT luciferase vector multiple cloning site (Ambion, Austin, TX) to yield pMIR-Nrdp1 3′-UTR (NRDP1 3′-UTR). Another pMIR-REPORT luciferase construct containing the Nrdp1 mRNA 3′-UTR with a mutation by site-directed mutagenesis was generated as a negative control and named Mut-Nrdp1 3′-UTR. Macrophage were plated in 6-well plates and allowed to reach 60%-80% confluence overnight. Cells were then co-transfected with a reporter construct (pMIR-null REPORT plasmid, pMIR-Nrdp1 3′-UTR, pMIR-Nrdp1 3′-UTR-Mut). After 24 h, cells were harvested, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's recommendations. Luciferase activity was normalized to control TK Renilla construct expression (pRL-TK, Promega).

ICH model
After anesthetizing mice with 1-3% isoflurane inhalation and ventilating them with Sham-operated mice received the same treatment, including needle insertion, but collagenase was not injected. The mortality rate in untreated animals is 4.6%.

Evaluation of neurological scores
A standardized battery of behavioral tests was used to quantify neurological function at 3ds post-ICH. The neurological scores were determined by Neurological Severity Scores, a composite of motor, sensory, reflex, and balance tests. Neurological function was graded on a scale of 1-18; a score of 1 point is awarded for the inability to perform the test or for the lack of a tested reflex. The higher the score, the more severe the injury (normal score 2-3; maximal deficit score 18). Tests were conducted by an observer blinded to the treatment group.
Brain water content measurement Brain water content was measured in mouse cerebral tissues after ICH. Briefly, mice were randomly sampled from each group and anesthetized by intraperitoneal injection with chloral hydrate (n = 5). Next, the cerebral tissues were removed, and the surface water on the cerebral tissues was blotted with filter paper. Brain samples were immediately weighed on an electric analytic balance to obtain the wet weight and then dried at 100 °C for 24 h to obtain the dry weight. Brain water content was calculated using the following formula: brain water content (%) =(wet weight -dry weight) / wet weight × 100%.

Statistical Analysis
All experiments were independently performed three times. The differences between groups were determined with the one-way analysis of variance (ANOVA) using SPSS 13.0 software. P values of less than 0.05 were considered to be statistically significant.

Propofol attenuates erythrocyte lysates-induced M1 polarization
To explore the effects of propofol on erythrocyte lysates-induced macrophage polarization, macrophages were pretreated with various concentrations of propofol and stimulated with erythrocyte lysates for 24 hs. The results showed that stimulation of erythrocyte lysates increased the production of TNF-α and IL-1β respectively. However, these pro-inflammatory factors were significantly inhibited by propofol in a concentrationdependent manner (Fig. 1). The data demonstrated that propofol attenuated erythrocyte lysates-induced M1 polarization.

Propofol suppresses erythrocyte lysates-induced miR-494 levels
Recent studies indicated that miR-494 contributes to the regulation of inflammation and innate immune responses. Thus, we investigated miR-494 levels on erythrocyte lysates stimulation by using a real-time PCR assay. The treatment of macrophages with erythrocyte lysates showed increased miR-494 levels. To test the hypothesis that propofol downregulates inflammation by targeting miR-494, macrophages were pretreated with various concentrations of propofol and stimulated with erythrocyte lysates. The results demonstrated that miR-494 levels decreased after erythrocyte lysates treatment compared with control groups (Fig. 2). The data suggests that propofol suppresses erythrocyte lysates-induced miR-494 levels.

MiR-494 is required for propofol inhibition of inflammation
To further assess the role of miR-494 in the anti-inflammation of propofol, miR-494 inhibitor was used to inhibit miR-494 levels in macrophages. Macrophages were transduced with miR-494 inhibitor or controls, and the inhibition was assessed by realtime PCR. The miR-494 levels were significantly decreased in the cells treated with the miR-494 inhibitor compared with controls (Fig. 3A). Next, we detected the proinflammatory factors levels of macrophages after propofol treatment, and found that these cytokines decreased in miR-494 knockdown group compared with controls (Fig. 3B). The data indicate that miR-494 is critical to the anti-inflammation of propofol.
Nrdp1 was a direct target of miR-494 in macrophage The target prediction program TargetScan (www.targetscan.org) suggests 3'-UTR of Nrdp1 mRNA includes a putative miR-494 target sequence (Fig. 4A). To identify Nrdp1 was a direct target of miR-494 in macrophages, we analyzed this relationship by a Dual-Luciferase reporter system. Our data found that co-expression with miR-494 mimics significantly attenuated the activity of a firefly luciferase reporter containing wild-type Nrdp1 3' -UTR, while miR-494 mimics could not attenuate the activity of a firefly luciferase reporter containing a mutated Nrdp1 3 '-UTR (Fig. 4B). The phenomenon suggested that miR-494 likely attenuated Nrdp1 expression by directly binding target sites in the Nrdp1 3'-UTR.

Propofol upregulates Nrdp1 by suppressing miR-494
In addition, we investigated whether propofol inhibition of miR-494 would increase Nrdp1 levels. The Nrdp1 levels of macrophage were evaluated after different treatments. We detected Nrdp1 levels of macrophages after propofol treatment, and found that Nrdp1 levels increased compared with controls (Fig. 5A). In addition, macrophages were transduced with miR-494 or controls, and further treated by propofol. The results demonstrated that the increased levels of Nrdp1 were not significant after miR-494 transduction. However, levels of Nrdp1 were much more robust after miR-494 inhibitors transduction (Fig. 5B). The results suggest that propofol upregulates Nrdp1 by suppressing miR-494.

Downregulation of Nrdp1 attenuates the anti-inflammatory effects of propofol
To explore the role of Nrdp1 in propofol mediated anti-inflammatory effect, we detected cytokine levels in macrophage in which Nrdp1 was downregulated. Macrophages were transduced with Nrdp1 siRNA or control siRNA, and the inhibition efficiency was assessed by Western blot. Nrdp1 levels were significantly decreased in Nrdp1 siRNA group compared with control group (Fig. 6A). TNF-α and IL-6 production of macrophages transduced with Nrdp1 siRNA after erythrocyte lysates treatment was significantly increased compared with macrophages transduced with control siRNA (Fig. 6B). Therefore, Nrdp1 plays an important role in the anti-inflammatory activity of propofol.
Propofol attenuated macrophage accumulation of the perihematomal region Macrophage accumulation was involved in the brain inflammatory damage. To detect the effect of propofol in macrophage accumulation, we detected the Iba-1-positive macrophages in the perihematomal brain tissue at 3 days after ICH. Our data indicated that the number of macrophage in the perihematomal brain tissues increased at 3 days in ICH mice compared with sham controls. However, the number of macrophage in ICH mice after propofol treatment decreased compared with that in control ICH mice (Fig. 7A).
These results suggested that propofol attenuated macrophage accumulation of the perihematomal region in ICH mice.
Propofol inhibits inflammatory injury in vivo. To explore the role of propofol to neurological function, i.c.v. adminstration of propofol or PBS were administered 10 min after ICH. BBB integrity, Brain water content and neurological injury of mice were observed 3 days after ICH. We found that propofol significantly inhibited BBB injury, water content and neurological damage (Fig. 7B-D). These data suggested that propofol could inhibit inflammatory injury in vivo.

Discussion
Spontaneous intracerebral hemorrhage (ICH) is a subtype of stroke, accounting for 15 to 20% of all stroke types. While the high mortality and morbidity makes ICH a challenging problem, there are no effective therapies for ICH patients (23)(24)(25). Much study demonstrates that inflammatory responses are involved in the progress and progression of brain injury following ICH, including macrophage activation and neutrophil infiltration (26)(27)(28). Various factors, such as thrombin and glutamate, could activate macrophages and elicit inflammatory response, and subsequently generate neuroinflammation following ICH (29)(30)(31). Therefore, the strategies based on inhibition of macrophage activation might represent promising way for ICH.
Macrophage produces nutrition factors and nerve toxicity factors, and shows the dual role of proinflammatory and anti-inflammatory, characterized by M1 and M2 polarization (32)(33)(34). M1 macrophage secretes high levels of oxide metabolites and proinflammatory factor, such as IL-6 and TNF-α. Its role is to eliminate pathogenic microorganisms and cancer cells, but also lead to the normal cell and tissue injury (35). M2 macrophage secretes high level of IL-10 and TGF-β. Its role is immune suppression, tissue repair and functional remodeling (36). Much evidence demonstrated that miRNA level varied in different macrophage polarization. It was also identified that miRNAs contributed to macrophage polarization (37). For example, miR-155 and miR-146a contribute to M1 polarization and their expressions are promoted by IFN-γ or LPS stimulation (38)(39). Recent research demonstrated that miR-21 modulates the polarization of macrophages and increases the effects of M2 macrophages on promoting the chemoresistance of ovarian cancer (40).
Propofol is a widely used intravenous anesthetic agent with potential neuroprotective effect in neuronal damage, including ischemic stroke and traumatic brain injury (41)(42).
However, the effects and molecular mechanisms of propofol in M1 macrophage polarization after ICH have not been identified.
Firstly, we utilized erythrocyte lysates-treated macrophage model to explore the effects of propofol on erythrocyte lysates-induced macrophage polarization. We found that that propofol attenuated erythrocyte lysates-induced M1 polarization. Secondly, we investigated miR-494 levels on erythrocyte lysates stimulation by using a real-time PCR assay. The data suggested that propofol suppressed erythrocyte lysates-induced miR-494 levels and miR-494 is critical to the anti-inflammation of propofol. Thirdly, we identified that Nrdp1 was a direct target of miR-494 in macrophage and propofol upregulated Nrdp1 by suppressing miR-494. Downregulation of Nrdp1 attenuated the anti-inflammatory effects of propofol. Lastly, we proved that propofol attenuated macrophage accumulation of the perihematomal region and significantly inhibited BBB injury, water content and neurological damage following ICH in vivo.

Conclusion:
Our data suggests that propofol can attenuate the neuroinflammatory response of macrophages after ICH through the regulation of the miR-494/Nrdp1 pathway. In addition, propofol also represent anti-inflammatory therapy strategy in ICH.     The neurological deficit tests were performed by behavioral measurement, including composite of motor, sensory, reflex, and balance tests. Experiments