Progesterone induces neuroprotection associated with immune/inflammatory modulation in experimental traumatic brain injury

Abstract

An imbalance of immune/inflammatory reactions aggravates secondary brain injury after traumatic brain injury (TBI) and can deteriorate clinical prognosis. So far, not enough therapeutic avenues have been found to prevent such an imbalance in the clinical setting. Progesterone has been shown to regulate immune/inflammatory reactions in many diseases and conveys a potential protective role in TBI. This study was designed to investigate the neuroprotective effects of progesterone associated with immune/inflammatory modulation in experimental TBI. A TBI model in adult male C57BL/6J mice was created using a controlled contusion instrument. After injury, the mice received consecutive progesterone therapy (8 mg/kg per day, i.p.) until euthanized. Neurological deficits were assessed via Morris water maze test. Brain edema was measured via the dry–wet weight method. Immunohistochemical staining and flow cytometry were used to examine the numbers of immune/inflammatory cells, including IBA-1⁺ microglia, myeloperoxidase⁺ neutrophils, and regulatory T cells (Tregs). ELISA was used to detect the concentrations of IL-1β, TNF-α, IL-10, and TGF-β. Our data showed that progesterone therapy significantly improved neurological deficits and brain edema in experimental TBI, remarkably increased regulatory T cell numbers in the spleen, and dramatically reduced the activation and infiltration of inflammatory cells (microglia and neutrophils) in injured brain tissue. In addition, progesterone therapy decreased the expression of the pro-inflammatory cytokines IL-1β and TNF-α but increased the expression of the anti-inflammatory cytokine IL-10 after TBI. These findings suggest that progesterone administration could be used to regulate immune/inflammatory reactions and improve outcomes in TBI.

Introduction

Traumatic brain injury (TBI) is a leading cause of death, disability, and cognitive deficits in adults with trauma [1,2]. The secondary brain injury induced by hyperactive immune/inflammatory reactions is heavily responsible for poor clinical prognosis after TBI. Previously, the brain was considered to be an immune-privileged organ, as the blood-brain barrier (BBB) tightly controls the entry of larger particles, such as cells and proteins, into the central nervous system [3]. However, recent studies suggest that immune activity can occur in the brain, especially after BBB damage, such as that induced by TBI [4]. After such damage, peripheral immune cells, including macrophages, neutrophils, and T lymphocytes can invade the whole brain, upon which microglia cells are activated. As a consequence, secondary brain injury occurs, further aggravating brain trauma [5]. Although researchers have made great progress in understanding the pathophysiological mechanisms, developing new pharmaceutic targets, and optimizing the treatment strategies for TBI, we still face the desperate reality that there are not enough therapeutic avenues that can effectively improve TBI prognosis in the clinical setting.

Progesterone, as a fat-soluble steroid hormone, can easily penetrate the BBB and plays important roles in the reproductive and endocrine systems. It can promote the development of the embryonic nervous system along with its receptor, which is broadly expressed throughout brain tissue. After patients undergo physiological stress and trauma, progesterone maintains immune and inflammatory homeostasis through a variety of proteins and receptors [6]. In addition, it also exhibits a role in immunomodulation by reducing post-ischemic stroke systemic inflammation [7]. The neuroprotective effect of progesterone in the brain has been confirmed with previous research. Studies have reported that progesterone treatment can limit infarct size and reduce motor function defects [8]. Female rats with high levels of progesterone were reported to have a less severe post-traumatic stress response and faster recovery [9]. Progesterone can also reduce brain edema, as it increases the activity of sodium-potassium pumps in nerve cells [10]. Studies have also reported that progesterone can promote angiogenesis and nerve regeneration in brain tissue in traumatized areas [11,12], increase the expression of the anti-apoptotic protein Bcl-2 and brain-derived neurotrophic factor, and reduce the activity of caspase-3 [13]. Our previous study found that progesterone increases the number of endothelial progenitor cells in rats; promotes the expression of vascular makers, such as VWF, CD31, and CD34, around brain tissue in the injured area; and repairs cognitive defects after TBI [14].

Considering the neuroprotective effects of progesterone in central nervous system diseases, as well as our previous research findings, we repeated experiments and confirmed its neuroprotective role in an experimental TBI model. Neutrophils and microglia are important participants in secondary brain injury after TBI, and they can exert effects through various cytokines, such as IL-1β, TNF-α, IL-10, and TGF-β, etc. In addition, our previous studies found that regulatory T cells had immune/inflammatory regulatory and neuroprotective effects in a mouse model of TBI. Therefore, we hypothesized that progesterone can alleviate brain edema, regulate immune/inflammatory reactions, and improve neurological outcomes in mice subjected to TBI. Progesterone administration is potentially a highly effective therapeutic method for TBI.

Materials and methods

Animals and groups

Adult male C57BL/6 mice (20–22 g) were purchased from Beijing Huafukang Biotechnology. All mice were given free access to food and water in a temperature-controlled (25 ± 0.5 °C) vivarium under a 12-h light/dark cycle with dark hours between 19:00–07:00. They were randomly divided into a Sham group (subjected to surgery without controlled contusion injury), TBI+PROG group (received controlled contusion injury and treated with progesterone), and TBI Control group (received controlled contusion injury and treated with dimethyl sulfoxide, which is the solvent of progesterone). We also strictly abided by the guidelines of China’s small animal protection organization. These research protocols were approved by the Ethics Committee of Tianjin Medical University General Hospital and are in accordance with the guidelines of the National Institutes of Health.

Model preparation and progesterone treatment protocol

A mouse TBI model was made using a controlled cortical impactor (CCI, Custom Design & Fabrication, USA). The mice were anesthetized with 1.25% avertin (0.02 mL/g) (purchased from Nanjing Aibei Biotechnology Company) via intraperitoneal injection before surgery. The mice were then placed in a stereotaxic frame. To expose the dura, a 5.0-mm diameter hole was drilled in the right parietal bone (1.0 mm lateral to the midline and 1.0 mm anterior to the herringbone) after the surgical site was clipped and cleaned. To induce moderate TBI, the CCI device was set to a depth of 1.3 mm, a velocity of 4.5 m/s, and a dwell time of 200 ms [15]. After the injury was induced, the incision was sutured immediately with 5-0 silk sutures, and the mice were placed on a heating pad to recover from anesthesia. Progesterone was purchased from Sigma-Aldrich Commercial Company. Mice were injected intraperitoneally with progesterone (8 mg/kg) or equal volume of solvent (dimethyl sulfoxide, DMSO) at 1-h post-injury, and then the same dose of progesterone or DMSO was injected at the same time each day until they were euthanized [12].

Water maze test

A Morris water maze (MWM) was used to test for differences in cognitive function 7 to 11 days after TBI (n = 10/group) [16]. Briefly, the water maze (120-cm diameter, 40-cm high; DMS-2, Chinese Academy of Sciences, China) was filled with water (20–22 °C) and colored with milk. The platform (8-cm diameter) was located 0.5 cm below the water surface in the center of the southwest quadrant. Each mouse was trained to adapt to the maze for 1 min for 3 consecutive days prior to training. A computerized tracking system (Etho-vision 3.0; Noldus Information Technology, Wageningen, Netherlands) was used to record the latency time. Each mouse was trained with four daily trials from four random start positions (east, north, southeast, and northwest) for 5 consecutive days. Subsequently, a probe trial was performed from a novel start position (facing the tank wall) to evaluate memory retention when the platform was removed 12 days after TBI. The time each mouse stayed in the goal quadrant during a 30-s period was accurately recorded, and the percentage time was calculated via the formula:

(time in the goal quadrant)/ 30 × 100%.

Brain water content

To obtain the brains, all mice were anesthetized with avertin and immediately euthanized 3 days after TBI (n = 12/group). The injured cerebral hemisphere was isolated from the whole brain and its wet weight measured with an electronic analytical balance (Mettler Toledo Co., Switzerland). Then, the cerebral hemisphere was dried in an electrothermostatic blast oven at 65 °C for 72 h and weighed (to obtain dry weight). The brain water content was calculated via the formula:

brain water content (%) = (wet weight − dry weight)/wet weight × 100%.

Immunohistochemistry staining

The mice (n = 6/group) were anesthetized with 10% chloral hydrate (3 mL/kg) and perfused with saline, followed by paraformaldehyde (pH 7.4). Brain tissue was removed and fixed in 4% paraformaldehyde for 24 h. Then, brain sections of 6-μm thickness were made from the paraffin tissue blocks.

Brain tissue sections were dewaxed with ethanol and xylene. Sodium citrate solution was used for antigen retrieval, and 3% hydrogen peroxide was used for blocking endogenous peroxidase. After blocking with 3% BSA for 1 h at room temperature, the primary antibody anti-ionized calcium-binding adapter molecule-1 (IBA-1) (dilution 1:500, Abcam, Cambridge, UK) was added and incubated overnight at 4 °C. The binding of the antibodies was recognized using a secondary antibody (dilution 1:100, ZSGB-Bio Co., Beijing, China), and 3,3′-diaminobenzidine solution (ZSGB-Bio Co.) was used to detect HRP activity under light microscopy (Nikon, Japan).

The frozen sections were incubated with anti-myeloperoxidase (MPO) antibody (dilution 1:100, Abcam). After being washed three times with PBS for 5 min, the sections were incubated with FITC-IgG (dilution 1:500, Cell Signaling Technology, USA) at 37 °C for 1 h.

The brown-branched cells are IBA-positive cells. The green round cells are MPO-positive cells. The number of positive cells in the 5-mm area surrounding the injured lesion was counted in five random fields under a 100× objective by two independent observers who were blind to the research aims.

ELISA

To quantify the level of cytokines in the brain tissue 3 days after TBI (n = 6/group), brain homogenates were centrifuged at 300g for 15 min at 4 °C. The cytokines IL-10, IL-1β, TGF-β, and TNF-α in the supernatant were quantified using commercial ELISA kits (eBioscience, San Diego, USA). All procedures are performed according to the manufacturers’ protocols.

Flow cytometry

Mice were anesthetized and euthanized 3 days after TBI (n = 6/group). The spleens were surgically removed from the mice. After washing off the blood, each spleen was ground and homogenized. The tissue suspension was centrifuged at 300g for 15 min at 4 °C. The supernatant was discarded to obtain the mononuclear cell suspension. Tregs were labeled using a commercial Treg test kit (eBioscience). A FACSAria flow cytometer (BD, Bioscience, USA) was used to quantify cell populations.

Statistical analysis

Data processing and analysis were performed using SPSS 16.0 statistical software. Numerical values are presented as mean ± SD. The data from the MWM test were analyzed using repeated measured ANOVA and one-way ANOVA. The other data for statistical comparisons were analyzed using repeated measured ANOVA with the post hoc least significance difference (LSD) test. Statistical significance was defined as P < 0.05.

Results

Progesterone improves neurological function after TBI

To detect the effects of progesterone on neurological function, an MWM test was performed with all mice. During the 5-day spatial acquisition test, the latency time gradually shortened, which suggested spatial memory was developed in all mice (F_{(4, 25)} = 177.186, P = 0.000; Fig. 1a). However, the spatial memory abilities of the three groups were significantly different (F_{(2, 27)} = 25.430, P = 0.000; Fig. 1a). Our data show that the TBI Control group had a longer latency time than the Sham group 7 (P = 0.002; Fig. 1a), 8 (P = 0.000; Fig. 1a), 9 (P = 0.000; Fig. 1a), 10 (P = 0.000; Fig. 1a), and 11 (P = 0.000; Fig. 1a) days after TBI. Compared with the TBI Control group, the TBI+PROG group had a shorter latency time 8 (P = 0.027; Fig. 1a), 9 (P = 0.005; Fig. 1a), 10 (P = 0.000; Fig. 1a) and 11 (P = 0.000; Fig. 1a) days after TBI, which suggested the latter had better spatial memory recovery.

Fig. 1.

Neurological function detection using a Morris water maze test. (a) After the 5 days spatial acquisition to the Morris water maze (MWM), the changes in latency time suggested that spatial memory was developed in all mice. However, spatial memory varied significantly by group. TBI Control group had longer latency time than the Sham group 7, 8, 9, 10, and 11 days after TBI. Compared with the TBI Control group, the TBI+PROG group had a shorter latency time 8, 9, 10 and 11 days after TBI. (b) The MWM probe test 12 days after TBI showed that the TBI Control group spent a significantly lower percentage of time in the goal quadrant (reference memory) than the Sham group. Compared with the TBI Control group, the TBI+PROG group spent a significantly increased percentage of time in the goal quadrant. All data are expressed as means ± SD; n = 10, *P < 0.05 TBI + PROG group vs. TBI Control group; ^#P < 0.05 TBI + PROG group or TBI Control group vs. Sham group.

The MWM probe test conducted 12 days post-TBI showed that the TBI Control group spent a significantly lower percentage of time in the goal quadrant (reference memory) than the Sham group (P = 0.001; Fig. 1b), which demonstrated that TBI impaired reference memory. Compared with the TBI Control group, the TBI+PROG group spent a significantly increased percentage of time in the goal quadrant (P = 0.002; Fig. 1b), which suggested that progesterone treatment improved the impaired reference memory induced by TBI.

Progesterone reduced brain edema induced by TBI

To verify the effects of progesterone on brain edema, we applied the dry/wet weighing method to determine brain water content 1, 3, and 7 days after TBI. The data showed that TBI-induced brain edema 1 (P = 0.030; Fig. 2) and 3 (P = 0.000; Fig. 2) days post-injury. Compared with the TBI Control group, the water content of brain samples from the TBI+PROG group was significantly reduced 3 days post-injury (P = 0.003; Fig. 2).

Fig. 2.

Brain edema measured by water content. The data suggested that TBI significantly increased brain water content 1 and 3 days after TBI. Progesterone treatment significantly decreased brain edema 3 days after TBI compared to TBI Control group. All data are expressed as means ± SD; n = 12, *P < 0.05 TBI + PROG group vs. TBI Control group; ^#P < 0.05 TBI + PROG group or TBI Control group vs. Sham group.

Progesterone attenuated the activation of microglia and the infiltration of neutrophils

To validate the effects of progesterone, the numbers of microglia and neutrophils around the injured brain tissue were investigated with IHC and IFC staining. The ionized calcium-binding adapter molecule 1 (IBA-1) is known to be an activator of microglia. Our results revealed that microglial cells were noticeably activated 1, 3, and 7 days after brain trauma, as IBA-1⁺ microglial cells from the TBI groups were significantly increased compared with those from the Sham group, and numbers peaked 3 days after TBI (P = 0.000; Fig. 3d). After treatment with progesterone, the elevated expression of IBA-1 was significantly reduced in microglial cells from the TBI+PROG group 3 (P = 0.008; Fig. 3d) and 7 (P = 0.013; Fig. 3d) days post-TBI. Our data also showed that neutrophils stained with MPO antibody immediately infiltrated injured brain tissue 1 day after TBI, then gradually decreased in numbers (P = 0.000; Fig. 4d). Progesterone treatment significantly decreased the numbers of MPO⁺ neutrophils compared with those in the TBI Control group 3 (P = 0.000; Fig. 4d) and 7 (P = 0.045; Fig. 4d) days post-TBI, which suggested that it inhibited the infiltration of neutrophils after TBI.

Fig. 3.

Detection of activation of microglia in injured hemisphere by IHC staining 1, 3, and 7 days after TBI. (a) TBI Control group, (b) TBI + PROG group, (c) Sham group, (d) Statistical chart. The cortex around the injury is the area of interest and is shown via the yellow boxes. The brown-branched cells indicated by the black arrowheads are positive cells. (d) TBI induced the activation of microglial cells 1, 3, and 7 days after brain trauma compared with Sham group, and the highest peak appeared at 3 days. Compared with the TBI Control group, progesterone treatment group showed decreased IBA-1⁺ microglia cell numbers in injured brain tissue 3 and 7 days post-TBI. Scale bar = 50 μm. All data are expressed as means ± SD; n = 6, *P < 0.05 TBI + PROG group vs. TBI Control group; #P < 0.05 TBI + PROG group or TBI Control group vs. Sham group.

Fig. 4.

Detection of infiltration of neutrophils in injured hemisphere using immunofluorescence staining 1, 3, and 7 days after TBI. (a) TBI Control group, (b) TBI + PROG group, (c) Sham group, (d) Statistical chart. The green round cells indicated by the red arrowheads are MPO⁺ neutrophil cells. The data in (d) show that MPO⁺ neutrophils infiltrated injured brain tissue 1, 3, and 7 days after TBI. Progesterone treatment significantly decreased the numbers of MPO⁺ neutrophils compared with TBI Control group 3 and 7 days post-TBI. Scale bar = 50 μm. All data are expressed as means ± SD; n = 6, *P < 0.05 TBI + PROG group vs. TBI Control group; #P < 0.05 TBI + PROG group or TBI Control group vs. Sham group.

Progesterone increased anti-inflammatory and reduced pro-inflammatory cytokines of brain tissue after TBI

To investigate whether progesterone treatment regulates pro-inflammatory and anti-inflammatory cytokine levels in injured brain tissue, the concentrations of IL-1β, TNF-α, TGF-β, and IL-10 were measured using the ELISA test. Homogenates of injured brain tissue were tested 1, 3, and 7 days after TBI. We found that the stress of TBI up-regulated the expression of the above-mentioned cytokines. Interestingly, the cytokine expression of IL-1β (P₁ = 0.000, P₃ = 0.000; Fig. 5a), TNF-α (P₁ = 0.000, P₃ = 0.000; Fig. 5b), and TGF-β (P₁ = 0.028, P₃ = 0.008; Fig. 5d) were most increased 1 and 3 days post-TBI, but IL-10 was up-regulated at 3 and 7 days post-TBI (P₃ = 0.038, P₇ = 0.009; Fig. 5c). Compared with the TBI Control group, TBI + PROG group exhibited significantly inhibited expression of pro-inflammatory cytokines, including IL-1β (P₁ = 0.007, P₃ = 0.001; Fig. 5a) and TNF-α (P₁ = 0.002, P₃ = 0.000; Fig. 5b) 1 and 3 days after TBI. The data also show that TBI + PROG group significantly increased the expression of the anti-inflammatory cytokine IL-10 (P₃ = 0.012, P₇ = 0.028; Fig. 5c) 3 and 7 days after TBI, but not that of TGF-β, compared to the TBI Control group (Fig. 5d).

Fig. 5.

Detection of inflammatory cytokines in brain tissue using ELISA 1, 3, and 7 days after TBI. (a), (b), (c) and (d) The stress of TBI up-regulated expression of IL-1β, TNF-α, IL-10, and TGF-β cytokines. Compared with the mice of the Sham group, the expression of IL-1β, TNF-α, and TGF-β in the mice subjected with TBI were increased 1 and 3 days after TBI, but that of IL-10 increased 3 and 7 days. Compared with the TBI Control group, (a) and (b) the progesterone treatment group showed significantly inhibited expression of pro-inflammatory cytokines IL-1β and TNF-α 1 and 3 days post-TBI. (c) Progesterone treatment significantly increased the expression of anti-inflammatory cytokines IL-10 3 and 7 days after TBI. (d) There was no difference between the TBI + PROG and TBI Control group. All data are expressed as means ± SD; n = 6, *P < 0.05 TBI + PROG group vs. TBI Control group; ^#P < 0.05 TBI + PROG group or TBI Control group vs. Sham group.

Progesterone treatment increased peripheral regulatory T cells

To identify the cell origin of the discrepancies in the various inflammatory cells and cytokines induced by progesterone treatment, we detected the level of peripheral Tregs by flow cytometry. CD4 and CD25 double-positive cells were seen in the outer and upper quadrants, indicating the presence of Tregs. Compared with the Sham group, the stress of TBI decreased the percentage of peripheral Tregs among mononuclear cells 1, 3, and 7 days after TBI (P₁ = 0.030, P₃ = 0.007, P₇ = 0.001; Fig. 6). However, progesterone treatment significantly reversed the decline in the proportion of Tregs compared with the change in the TBI Control group 3 and 7 days post-TBI (P₃ = 0.015, P₇ = 0.018; Fig. 6).

Fig. 6.

Detection of changes in Treg numbers in spleen using FACS. (a) TBI Control group, (b) TBI + PROG group, (c) Sham group, (d) Statistical chart. Tregs were stained using CD4 and CD25 antibodies. After mononuclear cells were gated, the double-positive cells in outer and upper quadrant were defined as Tregs. (d) The stress of TBI decreased the percentage of peripheral Tregs among mononuclear cells 1, 3, and 7 days after TBI. Moreover, progesterone treatment significantly reversed the decline in the percentage of Tregs compared to the TBI Control group 3 and 7 days post-TBI. All data are expressed as means ± SD; n = 6, *P < 0.05 TBI + PROG group vs. TBI Control group; #P < 0.05 TBI + PROG group or TBI Control group vs. Sham group.

Discussion

After the primary damage induced by brain trauma, the imbalance in immune/inflammatory reactions aggravates secondary brain injury and deteriorates clinical prognosis [17]. Progesterone can regulate immune/inflammatory responses and reduce glutamate neurotoxicity in an animal model of ischemic stroke [7,8]. Recent researches have also shown that various dosage of progesterone administration methods(8 mg/kg, 16 mg/kg and 32 mg/kg)exhibit neuroprotective effects in animal experiments [18–20]. In this study, we utilized the lower dosage administration method(8 mg/kg)according to the previous research [18], and demonstrated that progesterone treatment of TBI significantly improved cognitive deficits and reduced brain edema after TBI. At the same time, it also attenuated the activation of microglia and the infiltration of neutrophils, up-regulated the expression of the anti-inflammatory cytokine IL-10, and downregulated the expression of pro-inflammatory cytokines IL-1β and TNF-α. Moreover, we showed that progesterone treatment significantly increased Treg numbers, which are reported to have the ability to regulate immune balance.

The cognitive deficits induced by TBI are an important cause of disability in patients [21]. Progesterone administration was shown to improve spatial memory and neurobehavioral outcomes in ovariectomized rats with TBI [22]. Here, we also demonstrated that progesterone can significantly reduce the cognitive deficits observed in the MWM test. Related research has indicated that the hormone can exert neuroprotective effects by activating the PI3K/Akt pathway in brain tissue [23]. Brain edema that occurs in the early stages of TBI exacerbates cognitive deficits. In this study, we found that progesterone can reduce edema 3 days after TBI. Recent research into the management of peritumoral brain edema confirmed that the mechanism of progesterone action on brain edema may be partially mediated by a decrease in the expression of aquaporin 4, which can regulate the intracellular and extracellular water balance [24].

The excessive infiltration of neutrophils and the activation of microglia play important roles in secondary injury after brain trauma. Both partially contribute to post-traumatic cognitive deficits and brain edema and lead to worse neurological outcomes [4]. Recent research demonstrated that progesterone therapy reduced the activation of microglia and induced an M1 to M2 switch in the microglia phenotype in a cuprizone-induced demyelination mouse model [25]. The influx of neutrophils may also have been prevented by progesterone in a murine model of preterm birth [26]. In the present study, our data showed that progesterone decreased the activation of IBA-1⁺ microglia and inhibited MPO⁺ neutrophil infiltration 3 and 7 days after brain trauma. There was no difference between the TBI + PROG group and TBI Control group 1 day after TBI, which may have been because progesterone did not reach a sufficient concentration. Although we have not delved into the molecular mechanisms, some studies have shown that this may be related to progesterone’s regulation of the expression of NLRC4 inflammasomes, which can regulate the infiltration of neutrophils and activation of microglia [27].

After TBI, peripheral cells (neutrophils, macrophages, T cells, etc.) and intrinsic cells (astrocytes, microglia, etc.) in brain tissue release large amounts of cytokines, including pro-inflammatory and anti-inflammatory cytokines. Pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6, etc.) increase the infiltration of monocytes, promote the secretion of endothelial cell adhesion factors, and induce BBB damage [28]. The upregulation of anti-inflammatory cytokines (IL-10 and TGF-β) by IL-4 administration can regulate post-traumatic neuroinflammation and inhibit cell apoptosis [29]. In this study, we found that the expression time of each immune/inflammatory cytokine was not entirely consistent after TBI. We also confirmed that progesterone treatment significantly decreased the expression of pro-inflammatory cytokines (IL-1β and TNF-α) 1 and 3 days after TBI and increased the expression of the anti-inflammatory cytokine IL-10, but not TGF-β, 3 and 7 days post-TBI. The data suggest that the neuroprotective role of progesterone can be partially attributed to its ability to regulate immune/inflammatory cytokine expression by decreasing the infiltration of neutrophils and activation of microglia.

A very important finding of our study is that the stress of TBI decreases the numbers of Tregs, and progesterone can reverse this phenomenon 3 and 7 days after TBI. Tregs are a subset of lymphocyte cells with immune/inflammatory-regulation ability [30]. Upregulation of Treg numbers through allogeneic transplantation inhibited microglia activation, reduced the infiltration of neutrophils and CD3⁺ T cells, and improved the neurological outcomes in an experimental stroke model [31]. Our previous study also demonstrated that an IL-2/anti-IL-2 complex regulates the immune/inflammatory responses in brain tissue by mobilizing Tregs after TBI [32]. Therefore, the increased Treg population induced by progesterone may be an important factor in the neuroprotection associated with immune/inflammatory modulation. Previous studies have confirmed that Tregs regulate neuroinflammatory responses through IL-10 in the early stage of subarachnoid hemorrhage [33]. In our study, we found that progesterone can increase the number of Tregs and the expression of IL-10. These suggested that progesterone may regulate immune/inflammatory responses through Tregs/IL-10 pathway.

In contrast to the findings of our experiments, a few previous clinical trials have suggested that TBI patients did not benefit from progesterone [34,35]. One reason for this may be attributed to differences in the experimental designs. The clinical trials paid too much attention to the mortality and disability of severe TBI, while ignoring the prognostic index of mild and moderate TBI, such as cognitive deficits. The other reason may be the unavoidable differences in the species studied. After all, the conditions of TBI patients are more complicated than those of C57/BL6 mice.

In conclusion, although our study had some limitations, such as the lack of a dosage gradient and details of the molecular mechanism, we have provided new evidence for the neuroprotective effects of progesterone. Progesterone administration improves cognitive deficits, reduces brain edema, decreases pro-inflammatory cytokine levels, and inhibits the infiltration and activation of immune/inflammatory cells, but it also increases Treg numbers and anti-inflammatory cytokine levels. Thus, it may be an effective therapeutic candidate for TBI patients in the future.

Acknowledgements

The authors are grateful to Fanglian Chen, Weiyun Cui, and Li Liu from the Tianjin Neurological Institute for their excellent technical support. We wish to thank the valuable help given by Dr. Guoqiang Chang and Liqun He in revising this manuscript.

Ziwei Zhou and Yadan Li designed and performed the experiments, analyzed data and wrote this paper. Ruilong Peng, Mingming Shi and Weiwei Gao performed experiments and analyzed data. Ping Lei and Jianning Zhang developed hypotheses, designed experiments, analyzed data, and wrote the paper. All authors approved the final version of the paper.

Conflicts of interest

There are no conflicts of interest.