In this study, we explored the effect of 1,25-dihydroxyvitamin D3 on systemic lupus erythematosus encephalopathy using MRL/lpr mice which have proven to be the most useful spontaneous model of NPSLE. Our results showed that 1,25-dihydroxyvitamin D3 was able to protect MRL/lpr mice from cerebral injury by presenting serum immunological markers and pathological changes. However, the pathogenesis of NPSLE is highly complex, with detailed mechanisms yet to be elucidated. In order to investigate the underlying mechanism of 1,25-dihydroxyvitamin D3 against NPSLE, the BCSFB integrity, the effects of PPARγ/NF-κB/TNF-α and TGF-β/Smads on BCSFB were detected. Our findings support the positive effect of 1,25-dihydroxyvitamin D3 on NPSLE in MRL/lpr mice, which may be related to the protection of BCSFB disruption through the activation of the anti-inflammatory PPARγ/NF-κB/TNF-α pathway and upregulation of BDNF and the inhibition of TGF-β/Smads signaling pathway expression.
However, definitive biomarkers of NPSLE are yet to be identified owing to its complex pathogenesis and polymorphic phenotype (20). And we analyzed the identification of pertinent, robust biomarkers of SLE. The MRL/lpr mice have a very similar overall disease pattern to human SLE including cutaneous manifestation (21). To expose the effect of 1,25-dihydroxyvitamin D3 on SLE, we firstly observed cutaneous manifestations in MRL/lpr mice. Skin lesions are one of the typical symptoms of SLE. The MRL/lpr mice began to have different degrees of skin lesions at the age of 8 weeks, especially in the skin of the head, back, mouth and nose. However, 1,25-dihydroxyvitamin D3 treatment significantly attenuated the degree of skin lesions. The immunopathogenic mechanisms underlying the development of end-organ disease in SLE have been made much progress (1). These involve complex immunological cascades, including humoral mediated immunity such as autoantibody and complement deposition. Autoantibodies to components of chromatin, which include dsDNA, are central in the pathogenesis of SLE (9). The activation of the complement system in SLE is characterized by the consumption of complement proteins. The degree of reduction in serum level of C3, which is composed in the complement pathway, is associated with the pathogenesis of SLE (22). Serum A-dsDNA and C3 activities showed markedly increased or decreased with time after onset, which may be associated with lupus activity. And it may cause subsequent leukocyte and cytokine driven serious multi-system damage including the nervous system; while 1,25-dihydroxyvitamin D3 treatment notably improved the activity of these serum markers. These findings demonstrate that 1,25-dihydroxyvitamin D3 has a protective effect against SLE and NPSLE, to some extent.
The symptoms of NPSLE in MRL/lpr mice are caused by autoimmune diseases in most cases (23). To further investigate the protective effect of 25-dihydroxyvitamin D3 on NPSLE, we observed neuropsychiatric manifestations in MRL/lpr mice. Neuropsychiatric manifestations are the most common diffuse clinical manifestation of the central nervous system in NPSLE. Neuropsychiatric impairment depends on the environment, genetic inheritance, and hormonal factors. Headaches, mood disorders, cognitive dysfunction, seizures, and cerebrovascular disease are the most frequent symptoms (24). Vascular abnormalities, self-autoantibodies, and inflammatory mediators are considered to be the primary contributory factors (25). In the present study, 1,25-dihydroxyvitamin D3 treatment improved notably the typical neurological symptoms of NPSLE, such as lack of curiosity, indifference to external stimuli, liking to be alone, and lack of activity. In addition, the HE and Nissl staining showed severe histological damages to brain tissue. Obviously, the nerve cells in the brain of MRL/lpr mice were extensively damaged, especially in the hippocampus, showing neuronal atrophy and nuclear chromatin condensation. Neuronal damage in the hippocampus may trigger NPSLE development (26). At the same time, previous studies showed that severe gene levels in the hippocampus were related to neuropsychiatric manifestations, including cognitive function (27). Similar damages of the immune cells were also observed in the choroid plexus of MRL/lpr mice. As shown in HE staining, lymphocytic infiltrated with hemorrhage in choroid plexus of MRL/lpr mice. It may be due to the influx of immune cells, self-antibodies, pro-inflammatory cytokines that occur after barrier disruption in the brain. The findings demonstrated that MRL/lpr lupus mice had brain damage. However, 1,25-dihydroxyvitamin D3 treatment significantly reduced the degree of brain damage of NPSLE. These findings explain that 1,25-dihydroxyvitamin D3 can ameliorate brain damage of NPSLE in the MRL/lpr mice by protecting neurons and decreasing inflammatory injury.
The pathogenesis of NPSLE remains poorly understood and there remain many unanswered questions regarding its mechanism. However, it is believed that important factors are the immune cell-mediated damage and pathological activity that may result in disruption of barrier in the brain (28). When its integrity is compromised, immune cells, self-autoantibodies, and proinflammatory cytokines can cross barrier by promoting an inflammatory environment with glial activation, neurodegeneration, and subsequent adverse behavioral consequences (29,30). There are three brain barriers: the BBB, the meningeal barrier, and the BCSFB. It has been shown that BBB disruption can trigger NPSLE (31). However, in our study, the primary site of leukocyte entry into the brain in NPSLE likely occurs at the choroid plexus. And considerable intrathecal lymphocyte infiltration likely occurs through the BCSFB, accompanied by epithelial hyper-permeability to antibodies. Thus, we report that the pathogenesis of NPSLE is believed to include the entry of circulating neuropathic antibodies to the brain via a pathologically permeable BCSFB, pathogenesis aligned with the previous report of Sivan Gelb (32). Based on our findings, we focus on the BCSFB dysfunction as a causative factor in NPSLE. The choroid plexus is an epithelial bilayer surrounded by a highly vascularized capillary plexus (33). The epithelium synthesizes cerebrospinal fluid (CSF) but also separates brain ventricles from the blood to form BCSFB (34). The CSF space is separated from the vascular system via BCSFB, whereas BBB, responsible for maintaining the brain homeostasis, is located between the vascular system and the brain parenchyma (35). The apical tight junctions (TJs) of the choroid plexus (CP) epithelial cells play a fundamental role in the regulation of BCSFB permeability and integrity (33). The TJs are composed of proteins related to the inner and outer leaflets of the cell membrane, whereas occludin and claudins are the main transmembrane molecules mediating epithelial contact (36). Epithelial TJ of the BCSFB contains the protein claudin-2 (37). Thus, the expressions of occludin and claudin-2 purportedly reflect the integrity of BCSFB. In this study, we found that 1,25-dihydroxyvitamin D3 protected the BCSFB permeability and integrity in NPSLE of MRL/lpr mice. And the cerebral injury on BCSFB got worse with time. It was found that reduced expressions of occludin and claudin-2 following 1,25-dihydroxyvitamin D3 treatment is time-dependent.
PPARγ has been implicated in the pathogenesis of numerous diseases including diabetes, stroke, inflammatory or immune diseases. It is expressed in multiple cells, involving immune cells, and neurons (38). PPARγ not only exerts effects on the regulation of cellular differentiation to control lipid metabolism and glucose homeostasis (39). PPARγ ligands also play a crucial role in attenuating degenerative processes in central nervous systems as well as in peripheral systems (40). And it is responsible for the control of neurogenesis, anti-inflammatory mechanisms, and neuronal death (38,39,40). Previous studies have shown the gratifying anti-inflammatory effect of PPARγ, mainly via negatively regulating several transcription factors such as NF-κB and its downstream pro-inflammatory cytokine TNF-α (41). Therefore, regulating PPARγ and PPARγ-related pathways has great potential in treating NPSLE. In this study, exploration of the potential mechanisms demonstrated that 1,25-dihydroxyvitamin D3 significantly activated PPARγ and subsequently suppressed NF-κB and TNF-α activation. And in the serum of MRL/lpr mice, the levels of NF-κB and TNF-α markedly reduced in the VitD3-treated groups, suggesting that the result again, to some extent. Furthermore, the expression of the PPARγ/NF-κB/TNF-α axis changed over time, as has been shown in the BCSFB. And there is a study has shown that the activation of the PPAR-γ/TNF-α/NF-κB axis decreases brain inflammation and protects neuronal by maintaining BBB function in the brain (15). Thus, it seems that the elevated expression of NF-κB along with upregulation of TNF-α, regulated by the PPARγ may induce alteration of BCSFB. And 1,25-dihydroxyvitamin D3 treatment protected BCSFB of NPSLE in the MRL/lpr mice by modulating the PPARγ/NF-κB/TNF-α axis.
BDNF, which has prominent functions of promoting neuroprotection and neurodegeneration, is one of the most widely extensively studied and distributed neurotrophins in the brain (42). Immune cell neurotrophin production could be neuroprotective against autoimmunity-driven CNS damage (43). However, there are many questions that whether BDNF is associated with damage severity in NPSLE. In this study, BDNF reduced with the damage severity in NPSLE, but 1,25-dihydroxyvitamin D3 treatment upregulated BDNF, which exerts a neuroprotective effect against brain injury of NPSLE.
To further investigate the underlying mechanism of 1,25-dihydroxyvitamin D3 on brain injury of NPSLE, the key TGF-β/Smads signaling pathway was detected. TGF-β1 is implicated in a large number of interactions and plays a variety of roles according to the cellular environment (44). Firstly, TGF-β is one of the most important cytokines to activate and promote the transformation, exacerbating the process of inflammation (45). And TGF-β is also associated with the immune dysregulation disorders such as Multiple Sclerosis (MS), in which a forceful increase in TGF-β expression and circulating TGF-β was observed during an MS attack in patients (46). Furthermore, TGF-β has been expressed in neural progenitor cells, differentiated neurons, and mature neural cells (19). In fact, TGF-β induces cell cycle exist the hippocampal neurons in mice (47) and is associated with the loss of adult neurogenesis by preventing the proliferation of progenitor cells (48). Previous studies of Alzheimer’s and Parkinson’s disease declared that TGF-β ligands were elevated in cerebrospinal fluid (49,50). At the same time, inflammatory response, neuronal damage, and autoimmune activation are the key factors in the pathogenesis of NPSLE (3). Smads are intracellular signal transduction molecules of the TGF-β superfamily. TβR-I and TβR-II-mediated TGF-β/Smads signal family members, and Smads were activated by TGF-β receptor phosphorylates downstream receptor. TGF-β causes phosphorylation of Smad2 and Smad3 and subsequently migrates to the nucleus, leading to the expression of target genes increased obviously (51). There was evidence to illustrate that the TGF-β/Smads signal pathway is related to NPSLE and 1,25-dihydroxyvitamin D3 treatment significantly down-regulated the expression of TGF-β1、TβR-I、Smad2/3 and phosphorylation of Smad2/3 in brain tissue, suggesting that the anti-inflammatory effect of 1,25-dihydroxyvitamin D3 may be due to the suppression of the TGF-β/Smads signaling pathway. However, the effect of the TGF-β/Smads on the BCSFB and its function in the brain remain unclear in MRL/lpr. Thus, in order to detect the effect of time points on the expression level of the TGF-β/Smads signaling pathway, the MRL/lpr mice were executed for 4-time points. In the present study, the expression of the TGF-β/Smads signaling pathway increased over time, as has been shown in the BCSFB. Based on these results, we inferred that the elevated expression of the TGF-β/Smads signaling pathway may induce alteration of BCSFB. And 1,25-dihydroxyvitamin D3 treatment protected BCSFB of NPSLE in the MRL/lpr mice by modulating TGF-β/Smads signaling pathway
Overall, this study demonstrates that the 1,25-dihydroxyvitamin D3 can ameliorate systemic lupus erythematosus encephalopathy in MRL/lpr mice, which may be related to the protection of BCSFB disruption through the activation of the anti-inflammatory PPARγ/NF-κB/TNF-α pathway as well as upregulation of BDNF and the inhibition of TGF-β/Smads signaling pathway. Moreover, it has few side effects or toxicity, suggesting that 1,25-dihydroxyvitamin D3 may be developed as a potential natural medicine for the treatment of systemic lupus erythematosus encephalopathy. Because of the limits of the experiments, this thesis is not included the inhibitor group but preliminary proves that the relationship between BCSFB and PPARγ/NF-κB/TNF-α pathway as well as TGF-β/Smads pathway, providing the foundation for successive study.