Blueberry Phenolics Reduce Gastrointestinal Infection of Patients with Cerebral Venous Thrombosis by Improving Depressant-Induced Autoimmune Disorder via miR-155-Mediated Brain-Derived Neurotrophic Factor

Cerebral venous thrombosis (CVT) often causes human depression, whereas depression-induced low immunity makes the patients susceptible to gastrointestinal infection. Blueberry possesses antidepressant properties which may improve autoimmunity and reduce gastrointestinal infection. Brain-derived neurotrophic factor (BDNF) performs antidepressant function and can be regulated by miR-155, which may be affected by blueberry. To explore the possible molecular mechanism, blueberry compounds were analyzed by high-performance liquid chromatography. Activity of compounds was tested by using HT22 cells. The present study tested 124 patients with CVT-induced mild-to-moderate depressive symptoms (Center for Epidemiologic Studies—Depression Scale [CES-D] ≥16) and gastrointestinal infection. Patients were randomly assigned to blueberry extract group (BG, received 10 mg blueberry extract daily) and placebo group (PG, received 10 mg placebo daily). After 3 months, depression, gastrointestinal infection and lipid profiles were investigated. Serum miR-155 and BDNF were measured using real-time quantitative polymerase chain reaction and or Western Blot. Blueberry treatment improved depressive symptoms and lipid profiles, and also reduced gastrointestinal infection in the BG group (P < 0.05) but those of the PG group (P = 1). These changes were paralleled by increase in serum levels of BDNF and miR-155 (P < 0.05). HPLC analysis showed that blueberry extracts were the main phenolic acids with 0.18, 0.85, 0.26, 0.72, 0.66, 0.4,1, and 1.92 mg/g of gentisic acid, chlorogenic acid, [2]-epicatechin, p-coumaric acid, benzoic acid, p-anisic acid, and quercetin in blueberry extracts, respectively. Phenolics in blueberry are possible causal agents in improving antidepressant activity and reducing gastrointestinal infection. Administration of blueberry increased BDNF expression and miR-155. Blueberry cannot affect BDNF level when miR-155 is overexpressed or inhibited. Phenolics from blueberry reduced gastrointestinal infection of patients with CVT by improving antidepressant activity via upregulation of miR-155-mediated BDNF.


INTRODUCTION
Cerebral venous thrombosis (CVT) is an arterial disease, and it often results in cerebral infarction and even potential morbidity. Thrombosis may result in mental retardation (Lohiya et al., 2005), which is often associated with long-term depression (Niere et al., 2012). Depression is linked with some autoimmune diseases (Benros, 2016), which can increase incidence of bacterial infection .Epidemiological studies strongly suggest a link between depression and thrombosis (Stein et al., 2017). Mental depression may cause gastrointestinal disturbances (Ledochowski et al., 2000), whereas pathogens may increase in numbers and infect or invade tissue. The association of depressant symptoms with gastrointestinal infection remains unclear. Chronic psychological stress is assumed to be related to the diseases in which the immune activity fails to exert protecting functions. Chronic stress may also result in bacterial infection (Kiank et al., 2006).
On the other hand, gastrointestinal infection is often associated with thrombotic events (Altamirano et al., 2010). As reported in literature, infection can cause hemostatic abnormalities (Choi et al., 2004;Levi et al., 2012), which can lead to thrombotic complications or intravascular coagulation (Cummins et al., 2001;Asakura et al., 2006). Intestinal inflammation may activate coagulation (Schoots et al., 2004). Thrombin is associated with blood coagulation and its dysregulation may result in hemostatic abnormalities, which range from subtle subclinical to life-threatening coagulopathies (Danckwardt et al., 2013). Proinflammatory cytokines and immune cells also exert differential effects on the coagulation and fibrinolysis (Daubie et al., 2006;Pang et al., 2016). In some cases, some infections may cause hemorrhagic fever or thrombotic microangiopathy.
Phenolic-rich food was proven effective in antidepressant treatment (Chang et al., 2016;Sawamoto et al., 2016). Benefits of phenolics in human health have been well-known. Abundance of flavonoid-derived products in the gastrointestinal tract has been reported to contribute to health benefits of phenolics (Goto et al., 2012;Wang et al., 2015).
Phenolics and their metabolites have been found to transverse blood-brain-barrier and exhibit neuropharmacological properties (Shen et al., 2015;Lan et al., 2016). For instance, blueberry has been shown to increase brain-derived neurotrophic factor (BDNF) (Williams et al., 2008;Rendeiro et al., 2012). Considerable evidence suggests that BDNF plays a key role in antidepressant treatment (Wang et al., 2017). On the other hand, an earlier report has shown that miR-155 is a target of inflammatory mediators and associated with psychiatric and neurodevelopmental disorders (Thounaojam et al., 2014). Furthermore, miR-155 has been found to be a novel regulator of BDNF (Varendi et al., 2014). Thus, miR-155 may affect depressant situation of patients via BDNF.
Above information suggest that CVT often causes human depression, whereas depression-induced low immunity makes the patients susceptible to gastrointestinal infection. BDNF performs antidepressant function and can be regulated by miR-155. Blueberry may improve depression-induced autoimmunity disorder and reduce gastrointestinal infection by affecting BDNF level via miR-155. To explore the possible molecular mechanism, the effects of blueberry juice on the depression and gastrointestinal infection investigated here. Meanwhile, the main bioactive compounds of blueberry juice were measured here.

Materials
Antibodies anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-BDNF and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody were purchased from Abcam. ECL reagent and Hyperfilm ECL were purchased from Amersham Biosciences (Amersham, UK

Blueberry Extracts
Blueberry powder was prepared as follows: fresh blueberries were purchased from local supermarkets. Then, 100 g blueberry fruits were blended by using a blender (DS-1, Shanghai. Precision Instruments Co., Ltd, Shanghai, China). The fresh juice was filtered by using a Whatman #2 (Whatman Inc.; Florham, NJ, USA) under vacuum. Clear juice was freeze-dried and powdered by using a miller. A dried powder sample (5 g) was extracted with 50 ml 80% methanol at 30 • C for 1 day in a THZ-Q shaking incubator (HZQ-F, Harbin Donglian Electronic Technology Development Co., LTD., Harbin, China) at 100 r/min. The extracts were collected via centrifugation at 10,000 rpm for half an hour and filtered via a Millipore filter (0.22 µm) at room temperature.

HPLC Analysis of Phenolic Contents in Blueberry Extracts
Phenolic contents in blueberry extracts were identified by using corresponding standards. Standard cures were made as follows: standard concentration was chosen based on final testing of phenolic contents in blueberry juice within the linear range. Regression equation was calculated by using peak area as Y (vertical axis), and the concentration as X (horizontal axis). Each compound was calculated correspond to the standard curve after detecting HPLC peak area and substituting corresponding standard curve equation.

Exclusion Criteria
The following exclusion criteria were used: (1) current psychiatric symptoms that necessitated treatment as determined by clinical interview; (2) presence of a major medical problem; (3) undergoing medication that can affect gastrointestinal infection, CVT, and depression symptoms; (4) current involvement in weight loss or psychotherapy; and (5) pregnancy.

Patients Grouping
Before experiments, all procedures were approved by the ethical committee of the First Hospital of Jilin University. Informed consent was obtained in writing from all patients. A total of 124 patients were selected after excluding the patients did not pass selection criteria. Final selected patients were randomly and evenly assigned into two groups: blueberry extract group (BG, n = 62, received 10 mg of blueberry extract daily) and placebo group (PG, n = 62, received placebo daily, Figure 1). Follow-up period was 6 weeks.

Measurement of Depression Symptoms
Depression symptoms were measured by a total score ≥ 16 on the 20-item CES-D. Effects of blueberry on depressant symptoms were measured between BG and PG groups. Severity of depression symptoms of CVT patients were compared by using CES-D scores.

Lipid Profile Measurement
Serum lipid profile (triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C)), which is closely associated with CVT progression, was measured. Serum concentration of TG was measured by immunometric assay (Immulite 2000 Thyroglobulin; Diagnostic Products, Los Angeles, CA, USA). Serum concentration of TC was measured by using an automated clinical chemistry analyzer kit (Biosino Biotech, Beijing, China). HDL-C was measured by using an automated chemistry analyzer (Hitachi, Tokyo, Japan). Serum concentration of LDL-C was measured by using a LDL-cholesterol kit (Sekisui Medical Co., Ltd., Tokyo, Japan). Serum malondialdehyde (MDA) was analyzed by using a MDA kit in Nanjing Jiancheng Institute of Biotechnology (Nanjing, China).

CVT Measurement
Venous thrombi were detected with computed tomography (CT) venography. Brain CT was carried out in a CT 64-slice multidetector row CT scan (Toshiba Medical Systems Corporation, Otawara, Tochigi, Japan). CT images were obtained with 2.5 mm thickness via the posterior fossa with 5 mm section thickness via supratentorial hemispheres.

Measurement of Gastrointestinal Infection
Gastrointestinal infection was first observed by endoscopy. After determination, stool specimens were received by our laboratory and were submitted with orders by providers of stool culture. Genome from the specimen aliquots was extracted by using Genomic DNA purification kit (Promega, Madison, WI, USA) according to manufacture's instruction. Bacterial species were identified by using rRNA gene bidirectional sequencing.

Western Blot
Protein was extracted by using Protein Extraction Kit (Millipore, Bedford, MD, USA) according to manufacturer's protocol. Protein concentration was determined by using a BCA protein concentration determination Kit (Beyotime, Beijing, China). Samples were incubated for 10 min at 95 • C in boiling buffer (50 mM Tris, pH 8.8, 1% sodium dodecyl sulfate (SDS); 5% 2-mercaptoethanol, 10% glycerol, and 0.002% bromophenol blue). Protein samples were run on12% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to nitrocellulose membranes. Membranes were then blocked with 5% nonfat milk (w/v) and incubated with rabbit anti-human anti-BDNF (1:1,000) or anti-GAPDH (1:3,000); and a goat anti-rabbit secondary antibody. Bands were analyzed with software Image J. 1.42q (National Institutes of Health, United States). GAPDH was used to normalize BDNF protein levels.

Statistical Analysis
For immunoblot data, statistical comparisons were carried out using to one-way ANOVA with the diet group as the main factor. Correlation coefficients were calculated using Pearson product-moment correlation coefficient. All the data were expressed as mean ±standard deviation (S.D.) and analyzed using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA).

Blueberry Juice Is Rich in Phenolics
For HPLC analysis, the maximum absorption of phenolic compounds is at 280 nm for gallic acid, [1]-catechin, [2]epicatechin, benzoic acid and p-anisic acid; 320 nm for p-coumaric acid, gentisic acid, chlorogenic acid, sinapic acid and 3,4,5-trimethoxycinnamic acid; and 370 nm for myricetin and quercetin (Figure 2A). HPLC analysis showed that blueberry extracts are the main phenolic acids ( Figure 2B, gentisic acid, chlorogenic acid, [2]-epicatechin, p-coumaric acid, benzoic acid, p-anisic acid, and quercetin) compared with standard (Figure 2A). Table 1 showed 7 kinds of phenolic compounds detected by HPLC via standards. Detection response values of standard solution had a good linear relationship between phenolic concentration and HPLC peak area, and the limit of detection are low, suggesting that this method is very sensitive. There are 0.18, 0.85, 0.26, 0.72, 0.66, 0.41, and 1.92 mg/g of gentisic acid, chlorogenic acid, [2]-epicatechin, p-coumaric acid, benzoic acid, panisic acid, and quercetin in blueberry extracts, respectively ( Table 1). Table 2 showed baseline clinical characteristics among all CVT patients with gastrointestinal infection. No statistically significant difference was observed in body mass index (BMI), daily food intake, baseline depressive symptom severity, and demographic characteristics between BG and PG groups (P > 0.05).

Blueberry Improves Depressive Symptoms of CVT Patients
As shown in Figure 3, all outcomes improved significantly between baseline and subsequent follow-ups in the BG group (P < 0.05) but the PG group (P > 0.05). Improvements from baseline to 3 months included the following: CES-D from mean (S.D.) of 36.58 (6.54) to 18.76 (9.28) (P < 0.001) in the BG group. Comparatively, the PG group showed no improvement from baseline to 3 months: CES-D from mean (S.D.) of 37.29 (7.13) to 36.54 (7.98) (P > 0.05). All patients did not use antidepressants or psychotherapy during these 3 months. After 3-month therapy, CES-D scores for depressant symptoms were reduced 48.7% when compared with before therapy in the BG group. Comparatively, there was no change for the CES-D scores for depressant symptoms in the PG group.

CT Venography Analysis of CVT Patients
CT venography is a rapid, simple, and accurate technique for detecting CVT. The technique provides detailed information for CVT measurement. All patients were diagnosed with CVT as indicated by arrows in Figure 4. One disadvantage of CT venography is difficulty in reconstructing maximum intensity projection image from image data, which require the subtraction of bone adjacent to the venous sinus.

Blueberry Reduces the Cases of Gastrointestinal Infection
Before therapy, no statistically significant difference was noted between BG and PG groups (P > 0.05). Gastrointestinal symptoms were mainly caused by infection with Clostridium difficile, Campylobacter spp., Aeromonas spp., Plesiomonas shigelloides, and Shigella spp., and mixture infection. After 3 months of blueberry therapy, the BG group presented lower cases of gastrointestinal infection than the PG group (P < 0.05, Table 3). After 1-month therapy, gastrointestinal infection rates were reduced by 33.9% in the BG group while the rates were only reduced by 8.1% in the PG group. After 2-month therapy, gastrointestinal infection rates were reduced by 74.2% in the BG group while the rates were reduced by 16.1% in the PG group. After 3-month therapy, gastrointestinal infection rates were reduced by 83.9% in the BG group while the rates were reduced by 21.0% in the PG group.

Blueberry Fails to Inhibit Bacterial Pathogens
Blueberry may inhibit gastrointestinal infection via inhibition of pathogens. To explore this possibility, the effects of blueberry on the growth of pathogens isolated from CVT patients were measured here. However, blueberry failed to inhibit the growth of bacterial pathogens even after using 10 mg/ml blueberry juice was used. Thus, blueberry extract cannot inhibit pathogens Clostridium difficile, Campylobacter spp., Aeromonas spp., Plesiomonas shigelloides, and Shigella spp., and mixture infection directly even high concentration was used (data were not shown). However, gastrointestinal infection can be inhibited by blueberry via improvement of autoimmune activity.

Blueberry Improves Lipid Profiles of CVT Patients
Long-term blueberry consumption improved lipid profile in CVT patients from the BG group when compared with PG group. In the BG group, serum levels of TG, TC, LDL-C, and MDA decreased, whereas serum level of HDL-C changed after 1 month (Table 4). However, no statistically significant  difference between the two groups (P > 0.05, Table 4). Serum lipid profile improved further in the BG group after 2 months, and statistically significant differences were observed between the two groups (P < 0.05, Table 4). Furthermore, the lipid profile remarkably improved in the BG group after 3 months, and the two groups featured statistically significant differences (P < 0.05, Table 4). These findings demonstrate that blueberry improved lipid profile in CVT patients. Therefore, blueberry treatment changed the serum levels of lipid profiles in CVT patients. Blueberry dietary supplementation not only performs antidepressant-like function but also improves lipid profile of CVT patients. Present findings demonstrate that blueberry treatment exerts antidepressant effect and regulates lipid profile simultaneously.

Blueberry Increases BDNF Level via miR-155
Real-time quantitative polymerase chain reaction (qRT-PCR) analysis showed t no statistically significant difference for serum miR-155 levels between BG and PG groups before therapy (P > 0.05). After 3 months of blueberry therapy, miR-155 level was higher in the BG than PG group (P < 0.05, Figure 5A). Relative level of miR-155 was lowest level when it was silenced whereas the level was highest when it was overexpressed ( Figure 5B). Meanwhile, no statistically significant difference in BDNF mRNA levels between BG and PG groups before the therapy (P > 0.05, Figures 5C,E). After 3-month blueberry therapy, the BG group exhibited higher BDNF mRNA levels  than the PG group (P < 0.05, Figure 5C). Cellular level test showed that miR-155 overexpression or inhibition can increase or reduce mRNA levels of BDNF ( Figure 5D). A significant positive correlation was observed between miR-155 or BDNF levels and blueberry treatment (P < 0.05, Figure 5D). Blueberry treatment cannot affect the mRNA levels of BDNF when miR-155 was overexpressed or inhibited (P > 0.05, Figure 5D). Western Blot analysis showed no statistically significant difference in BDNF levels between BG and PG groups before the therapy (P > 0.05, Figure 5E). After 3 months of blueberry therapy, BDNF level was higher in BG than the PG group (P < 0.05, Figures 5E).Additionally, miR-155 overexpression or inhibition increased or reduced protein levels of BDNF (P > 0.05, Figures 5F). Blueberry treatment cannot affect t protein levels of BDNF when miR-155 was overexpressed or inhibited. All these results suggest that blueberry increases BDNF level via miR-155.

DISCUSSION
Blueberry has been reported to be highly effective in controlling inflammatory conditions . However, the effects of blueberry on gastrointestinal infection and related molecular mechanisms remain unknown. Consumption of blueberries has been reported to improve spatial learning performance in an animal model, and this improvement is associated with the activation of BDNF pathway in the hippocampus (Rendeiro et al., 2012). Blueberry is rich in phenolics and may be useful for antidepressant treatment. Phenolic-rich food has been reported to increase the levels of BDNF, which mediates neuroplasticity, contributing critically in antidepressant treatment (Bouckaert et al., 2016). However, whether phenolics are the causal agents in inducing such behavioral responses still requires proof. We hypothesized that blueberry extracts can decrease depressive symptoms by regulating BDNF, and reducing gastrointestinal infection.
To make sure the functions of blueberry extracts, the phenolic contents should be clear. Folin-Ciocalteau method is often used to phenolic quantitation but it suffers the interference of reducing molecules other than phenolic compounds. Therefore, the phenolic quantification in blueberry extracts were performed by using HPLC analysis. HPLC analysis showed that blueberry extracts are the main phenolic acids with 0. 18, 0.85, 0.26, 0.72, 0.66, 0.41, and 1.92 mg/g of gentisic acid, chlorogenic acid, [2]epicatechin, p-coumaric acid, benzoic acid, p-anisic acid, and quercetin in blueberry extracts, respectively ( Table 1).
Blueberry polyphenols have been found to improve symptoms of neurological diseases (Dodd, 2012). Blueberries can maintain good mood of children and adults and reduce depression (Khalid et al., 2017).On the other hand, chronic depression can result in bacterial translocation or leaky gut (Maes et al., 2012). Therefore, depression may be associated with gastrointestinal infection. Antidepressant properties of blueberry may be beneficial for treatment of gastrointestinal infection. In this study, we observed that blueberry treatment can increase serum level of miR-155, resulting in increased BDNF level. BDNF can improve patients' spiritual well-being and autoimmunity, and can increase the antidepressant properties, reducing the rate of gastrointestinal infection (Figure 6).     Nachamkin and Yang, 1992;Locht and Krogfelt, 2002 These findings were supported by the increase in antidepressant activity induced by the phenolic-rich plants, as reported in previous reports. An interesting research discussed antioxidant properties of natural products, which can prevent brain damage from depressant disease. Enriched phenolic fraction of C. pachystachya exerts antidepressant function via its antioxidant activities (Ortmann et al., 2017). Antidepressant-like effect of phenolics in chronically stressed animal models was previously demonstrated by another study. Unpredictable chronic stress in an animal model also involves synthesis of pro-inflammatory cytokines, kynurenine pathway (KP), 5-hydroxytryptamine metabolism and caspase activities. Phenolics can reduce the levels or metabolism of these molecules and their activities (Filho et al., 2016). On the other hand, phenolics can prevent age-associated memory loss by scavenging free radicals and modulating BDNF levels (Souza et al., 2015). These results also suggest that the effect of phenolics in blueberry may be the main mechanism for its antidepressant function. The present data indicate that daily dose of 10 g of blueberry for 3 months can improve depressant symptoms.
According to previous reports, phenolics can inhibit pathogens, such as Candidatropicalis, Trichophyton rubrum, Staphylococcus species, Escherichia coli, Clostridium species, Proponbacteriumacnes, Aeromonas species, Proteus species, Klebsiella species, Salmonella species and Campylobacter species (Table 5). However, the blueberry cannot inhibit the growth of the pathogens isolated from the CVT patients with gastrointestinal infection. Therefore, blueberry inhibits gastrointestinal infection by improving autoimmune activity of CVT patients via miR-155-mediated regulation of BDNF level. On the other hand, blueberry improves lipid profiles and can inhibit CVT development. Low level of HDL-C has been found to be associated with recurrence of CVT (Ma et al., 2015).
The main limitation of the present work is that there are many components in blueberry juice. Which component playing an important role in the therapy of CVT patients with gastrointestinal infection remains unclear. Therefore, further work is still needed to be performed to confirm the results in the future.

CONCLUSIONS
A tight link exists between CVT and depression, whereas activation of depression results in gastrointestinal infection. miR-155 play a crucial role in mediating these effects by regulating BDNF levels. Several mechanisms contribute to enhanced risk of depression and accelerate gastrointestinal infection. Thus, blueberry treatment is possibly important in improving CVT, preventing depression symptoms, and reducing cases of gastrointestinal infection. Clinical evidence of efficacy and safety of blueberry is still needed in the future.

ETHICS STATEMENT
Before the experiments, all procedures were approved by the ethical committee of the First Hospital of Jilin University. Informed consent and assent were obtained in writing from all patients.

AUTHOR CONTRIBUTIONS
NX and HM: conceived and designed the experiments; TL and YF: performed the experiments; YQ and DZ: analyzed the data; HM: contributed reagents, materials, analysis tools, and wrote the paper. All authors reviewed and approved this manuscript.