fnins-12-00448 July 3, 2018 Time: 18:26 # 1 ORIGINAL RESEARCH published: 05 July 2018 doi: 10.3389/fnins.2018.00448 Total Salvianolic Acid Balances Brain Functional Network Topology in Rat Hippocampi Overexpressing miR-30e Qi Li1†, Liang Wang2,3†, Xin-Yi Li4†, Xiao Chen5, Bin Lu5, Long Cheng2, Chao-Gan Yan5* and Yong Xu2,6,7* 1 Drug Clinical Trial Institution, Taiyuan Center Hospital of Shanxi Medical University, Taiyuan, China, 2 Department of Psychiatry, First Hospital/First Clinical Medical College of Shanxi Medical University, Taiyuan, China, 3 Shanxi Province Mental Health Center/Taiyuan Psychiatric Hospital, Taiyuan, China, 4 Department of Neurology, Shanxi DaYi Hospital, Taiyuan, China, 5 Institute of Psychology, Chinese Academy of Sciences, Beijing, China, 6 MDT Center for Cognitive Impairment and Sleep Disorders, First Hospital of Shanxi Medical University, Taiyuan, China, 7 National Key Disciplines, Key Laboratory for Cellular Physiology of Ministry of Education, Department of Neurobiology, Shanxi Medical University, Taiyuan, China We investigated the therapeutic effects and underlying brain functional network topology mechanisms of total salvianolic acid (TSA) treatment for memory dysfunction by using miR-30e overexpression-induced memory deficit in rat hippocampi. Model rats were developed by lentivirus vectors carrying miR-30e into bilateral hippocampus CA1 region through stereo-surgery. Two weeks after surgery, TSA (20 or 10 mg/mL/kg) or saline were administrated for 14 consecutive days. Memory function was assessed Edited by: by behavioral tests (Y maze and Morris water maze [MWM]); resting-state functional Ashok Kumar, MRI (RS-fMRI); and molecular alterations of BCL-2, UBC9, and Caspase-3 in the University of Florida, United States hippocampus CA1 region, as detected by immunohistochemistry. Compared to Reviewed by: Luigia Trabace, controls, model rats exhibited significantly impaired working and long-term memory University of Foggia, Italy in the Y maze and MWM tests (p < 0.01). The brain functional network topology Harshini Sarojini, analyzed based on RS-fMRI data demonstrated that miR-30e disturbed the global University of Louisville, United States integration and segregation balance of the brain (p < 0.01), and reduced edge strength *Correspondence: Chao-Gan Yan between CA1 and the posterior cingulate, temporal lobe, and thalamus (p < 0.05, false [email protected] discovery rate corrected). At the molecular level, BCL-2 and UBC9 were downregulated, Yong Xu [email protected] while Caspase-3 was upregulated (p < 0.01). After TSA (20 mg/mL/kg) treatment, †These authors have contributed the biomarkers for behavioral performance, global integration and segregation, edge equally to this work. strength, and expression levels of BCL-2, UBC9, and Caspase3 returned to normal levels. The correlation analyses of these results showed that global brain functional Specialty section: This article was submitted to network topologic parameters can be intermediate biomarkers correlated with both Neuropharmacology, behavioral changes and molecular alterations. This indicated that the effects of TSA were a section of the journal Frontiers in Neuroscience achieved by inhibiting apoptosis of CA1 neurons to improve global functional network Received: 19 March 2018 topology. Accepted: 12 June 2018 Keywords: miR-30e, memory dysfunction, RS-fMRI, graph theory, hippocampus Published: 05 July 2018 Citation: Li Q, Wang L, Li X-Y, Chen X, Lu B, INTRODUCTION Cheng L, Yan C-G and Xu Y (2018) Total Salvianolic Acid Balances Brain MicroRNAs (miRNAs) are endogenous approximately 22nt RNAs that participate in many Functional Network Topology in Rat Hippocampi Overexpressing physiological activities or pathological process by targeting mRNAs for cleavage or translational miR-30e. Front. Neurosci. 12:448. repression (Bartel, 2004). Increasing evidence has demonstrated that dysregulation of doi: 10.3389/fnins.2018.00448 miRNAs affected mental process and were associated to various neuropsychiatric disorders Frontiers in Neuroscience| www.frontiersin.org 1 July 2018| Volume 12| Article 448 fnins-12-00448 July 3, 2018 Time: 18:26 # 2 Li et al. TSA Regulates Brain Network (Sharma and Lu, 2018; Sun et al., 2018). Among these miR-30e-induced memory deficit would hopefully be better miRNAs, recent investigations implied that miR-30e may have understood. complex effects. Previous researches indicated that miR-30e was an oncogenic miRNA in lung adenocarcinoma (Zhuang et al., 2017) or tumor suppressing miRNA in breast cancer MATERIALS AND METHODS (Liu et al., 2017), but in central nervous system miR-30e influenced many mental process and correlated to many Lentivirus Vector Construction psychiatric disorders, such as schizophrenia (Xu et al., 2010), To deliver the target gene safely and stably into the hippocampus, cognitive impairment (Labouesse et al., 2018) and vulnerability lentiviral vectors that were replication-deficient and did not leave to stress (Pearson-Leary et al., 2017). Our previous results the site of injection were used to recombine with miR-30e (Xu also indicated that miR-30e overexpression in rat hippocampus et al., 2016). Rat pre-miR-30e (Gene ID: 100314153) fragment could induce learning and memory impairment (Xu et al., was amplified by PCR using the following primers: forward 0 2015). Therefore, a miR-30e-induced memory deficit model 5 -CAACAGAAGGCTCGAGCTGTTGGAGAAGTGGGCATC 0 0 was chosen to explore the therapeutic strategy of memory -3 and reverse 5 -ATTCTGATCAGGATC CCTCCA AAC 0 dysfunctions. GAAGAGAGACAGTC-3 , which carried restriction sites for Total salvianolic acid (TSA) is an injectable solution of BamHI and XhoI, respectively. After PCR cycling and sequence the major water-soluble ingredients extracted from Salvia identification, the miR-30e fragment was cut with BamHI and miltiorrhiza, including 3, 4-dihydroxy-phenyl lactic acid, XhoI, and then cloned into the PLVX-IRES-ZsGreen1 Expression salvianolic acid A, salvianolic acid B, and other salvianolic acids, System (Shanghai SBO Medical Biotechnology Co., Ltd., which can ameliorate the microcirculation disturbance induced Shanghai, China). Subsequently, the miR-30e overexpression by ischemia-reperfusion in rats (Wang et al., 2010). Further lentiviral vectors and lentiviral packaging plasmid were mixed investigation suggests that TSA can bring about anti-oxidation and co-transferred into 293T cells. The medium was collected and neuron protection via inactivation of NADPH oxidase 48 h post-transfection (Figure 1). All lentiviral vectors expressed through a signaling pathway implicating AMP-activated protein green fluorescent protein (GFP) that helped to measure efficiency kinase (AMPK)/Akt/protein kinase C (PKC) (Tang et al., 2014). of transfection. Additionally, the other lentiviral vectors without Besides the microcirculation protection effects of TSA, a recent carrying miR-30e were also packaged and collected, which were study reported that TSA showed a remarkable improvement used in sham group and aimed to exclude the influence of in learning and memory impairment in APPswe/PS1dE9 mice lentivirus itself on cognition. (Shen et al., 2017). However, the mechanisms that underline the memory protection effects of TSA are still unclear. Animals and Groups To evaluate curative effects of therapeutic strategies, many Forty male adult Sprague–Dawley rats were obtained from the investigations mainly acquired data by behavioral test and Animal center of Military Medical Science Academy, Beijing, molecular biomarkers (Sun et al., 2015; Wan et al., 2015). China (SCXK-2014-0013). Rats were group-housed in poly However, there is always a gap existing between behavioral propylene cages (475 mm × 345 mm × 200 mm) with ad libitum changes and molecular alterations. To address this concern, access to food and water. Cages were kept in a temperature magnetic resonance imaging (MRI), especially resting-state (23◦C) and 12 h/12 h light/dark-cycle controlled room. Rats were functional MRI (RS-fMRI), has been applied as a valuable allowed to adapt to the new environment for 7 days before the translational tool to investigate alterations in brain structure start of experiments. and function in both patients and animal models of disease Forty rats were randomly arranged into five groups (n = 8 (McIntosh et al., 2017). RS-fMRI has recently been highlighted each): Rats without surgery C saline treatment (control), as a new technique for functional connectivity changes in empty lentiviral vectors C saline treatment (sham), miR-30e subjects with different subtypes of mild cognitive impairment lentiviral vectors C saline treatment (model), miR-30e lentiviral (MCI) and different stages of cognitive impairment, which vectors C TSA 10 mL/kg, 1 mg/mL (TSA low), and miR- avoids variability in task fMRI (Joo et al., 2016; Li et al., 2017; 30e lentiviral vectors C TSA 10 mL/kg, 2 mg/mL (TSA high) Pan et al., 2017). Similar findings were obtained in animal (Table 1). models, as resting-state functional connectivity data supports the Model rats were constructed by lentivirus vectors carrying detection of cognition in the rodent brain (Nasrallah et al., 2016). mir-30e into the CA1 region of the hippocampus and continually These findings were mainly acquired by independent component expressed to produce memory deficit. After anaesthetization analysis or seed-based approaches to delineate functionally by 10% choral hydrate (2 mg/kg), rats were placed on a connected brain regions or networks of regions. Recently, stereotaxic device