Ketogenic Diets Alter the Gut Microbiome, Resulting in Decreased Susceptibility to and Cognitive Impairment in Rats with Pilocarpine-Induced Status Epilepticus.

A ketogenic diet (KD) is a high-fat, low-carbohydrate, and low-protein diet that exerts antiepileptic effects by attenuating spontaneous recurrent seizures, ameliorating learning and memory impairments, and modulating the gut microbiota composition. However, the role of the gut microbiome in the antiepileptic effects of a KD on temporal lobe epilepsy (TLE) induced by lithium-pilocarpine in adult rats is still unknown. Our study provides evidence demonstrating that a KD effectively mitigates seizure behavior and reduces acute-phase epileptic brain activity and that KD treatment alleviates hippocampal neuronal damage and improves cognitive impairment induced by TLE. We also observed that the beneficial effects of a KD are compromised when the gut microbiota is disrupted through antibiotic administration. Analysis of gut microbiota components via 16S rRNA gene sequencing in fecal samples collected from TLE rats fed either a KD or a normal diet. The Chao1 and ACE indices showed decreased species variety in KD-fed rats compared to TLE rats fed a normal diet. A KD increased the levels of Actinobacteriota, Verrucomicrobiota and Proteobacteria and decreased the level of Bacteroidetes. Interestingly, the abundances of Actinobacteriota and Verrucomicrobiota were positively correlated with learning and memory ability, and the abundance of Proteobacteria was positively correlated with seizure susceptibility. In conclusion, our study revealed the significant antiepileptic and neuroprotective effects of a KD on pilocarpine-induced epilepsy in rats, primarily mediated through the modulation of the gut microbiota. However, whether the gut microbiota mediates the antiseizure effects of a KD still needs to be better elucidated.

Establishment of high performance liquid chromatographic fingerprint and determination of 4 kinds of phenolic acid bioactive substances of fruitless Lycium barbarum leaves from Ningxia at different harvesting periods.

"Fruitless Lycium barbarum leaf (FLBL) are the leaves of a new variety of Lycium barbarum in Ningxia, which exhibit higher content of various nutrients, trace elements, and bioactive substances compared to Lycium barbarum fruits and leaves. However, the health and medicinal value as well as the by-products derived from FLBL have not received sufficient attention, and the contents of main components vary at different harvesting periods. Therefore, for the first time this study aimed to establish high-performance liquid chromatography (HPLC) fingerprints and determine the contents of four phenolic acid bioactive substances during different harvesting periods in order to provide an experimental basis for cultivation, collection, and research on FLBL. The results revealed 17 common peaks among 10 batches samples with a similarity ranging from 0.71 to 0.976. The linear relationships R2 for catechin, epicatechin-catechin, chlorogenic acid, and rutin were determined as 0.9999 each; meanwhile, the average recovery rate ranged from 93.92 % to 120.11 %, with an RSD between 0.91 % and 2.82 %. The precision, repeatability stability (24 h), and recovery rate met the requirements outlined in "Chinese Pharmacopoeia". Catechin, epicatechin, and rutin exhibited higher levels from June to August, while chlorogenic acid showed increased levels from July to September. The findings serve as a foundation for quality control measures such as identifying optimal harvest periods or facilitating development and production processes related to Ningxia FLBL."

Activation of RhoA pathway participated in the changes of emotion, cognitive function and hippocampal synaptic plasticity in juvenile chronic stress rats.

Neuropsychiatric diseases are related to early life stress (ELS), patients often have abnormal learning, memory and emotion. But the regulatory mechanism is unclear. Hippocampal synaptic plasticity (HSP) changes are important mechanism. RhoA pathway is known to regulate HSP by modulating of dendritic spines (DS), whether it's involved in HSP changes in ELS hasn't been reported. So we investigated whether and how RhoA participates in HSP regulation in ELS. The ELS model was established by separation-rearing in juvenile. Results of IntelliCage detection etc. showed simple learning and memory wasn't affected, but spatial, punitive learning and memories reduced, the desire to explore novel things reduced, the anxiety-like emotion increased. We further found hippocampus was activated, the hippocampal neurons dendritic complexities reduced, the proportion of mature DS decreased. The full-length transcriptome sequencing techniques was used to screen for differentially expressed genes involved in regulating HSP changes, we found RhoA gene was up-regulated. We detected RhoA protein, RhoA phosphorylation and downstream molecules expression changes, results shown RhoA and p-RhoA, p-ROCK2 expression increased, p-LIMK, p-cofilin expression and F-actin/G-actin ratio decreased. Our study revealed HSP changes in ELS maybe regulate by activation RhoA through ROCK2/LIMK/cofilin pathway regulated F-actin/G-actin balance and DS plasticity, affecting emotion and cognition.

Elevated Expression of ADAP2 is Associated With Aggressive Behavior of Human Clear-Cell Renal Cell Carcinoma and Poor Patient Survival.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common and lethal cancer of the adult kidney. ADAP2 is a GTPase-activating protein was upregulated in clear cell renal cell carcinoma. The role of ADAP2 in ccRCC progression is unknown. METHODS: ADAP2 expression in ccRCC cell lines and tissues was examined via real-time PCR, Western blot and IHC. MTS, colony formation and transwell assay to explore the role of ADAP2 in ccRCC. ADAP2 in growth and metastasis of ccRCC were evaluated in vivo through ccRCC xenograft tumor growth, lung metastatic mice model. The prognostic role of ADAP2 was evaluated by survival analysis. RESULTS: ADAP2 mRNA was expressed at significantly higher levels in 23 pairs of ccRCC tissues than in normal kidney tissues (P < 0.01). Immunohistochemical analysis of 298 ccRCC tissues revealed elevated ADAP2 expression as an independent unfavorable prognostic factor for the overall survival (P = 0.0042) and progression-free survival (P = 0.0232) of patients. The KaplanMeier survival curve showed that patients with a higher expression of ADAP2 showed a significantly lower overall survival rate and disease-free survival rate. Moreover, high expression of ADAP2 at the mRNA level was associated with a worse prognosis for overall survival (P = 0.0083) in The Cancer Genome Atlas (TCGA) cohort. In vivo and in vitro functional study showed that overexpression of ADAP2 promotes ccRCC cell proliferation and metastasis ability, whereas knockdown of ADAP2 inhibited cell proliferation, colony formation, migration and invasion. CONCLUSION: ADAP2 is a novel prognostic marker and could promotes tumor progression in ccRCC.

A preliminary study on fetal cardiac morphology and systolic function of normal and anemic pregnant women by fetal heart quantification technology.

Background: Maternal anemia is a common nutritional problem during pregnancy. Fetal heart quantification (fetal HQ) technology is used to quantitatively evaluate the size, shape, and contractile function of the fetal heart, which can reflect the development of the fetus in the uterus. Methods: We used fetal HQ technology to evaluate the basal-apical length (BAL), the transverse width (TW) and the area (A) of the four chamber view at end-diastole in 77 normal fetuses and 40 fetuses of women with anemia. We analyzed the changes of fetal heart size and measured the global sphericity index (GSI), the fraction area change (FAC), and the global longitudinal strain (GLS). The sphericity index (SI) and the fractional shortening (FS) of 24 segments were analyzed to identify any changes of fetal heart morphology and systolic function. The normal range of Z value was set at -2 to 2. Results: Fetal BAL, TW, A, and gestational age (GA) were positively linearly correlated, while GSI, GLS, and FAC had no significant correlation with GA. There was no significant difference in fetal BAL, TW, A, GLS, and FAC between the two groups (P>0.05). There was no significant difference in the FS of the 24 segments of the left and right ventricles between the two groups (P>0.05). There was no significant difference in the SI of the 1-24 segments of the right ventricle between the two groups (P>0.05). The difference in fetal GSI between the two groups was statistically significant (P<0.05). There was no significant difference in SI between the 1-22 segments of the left ventricle between the two groups (P>0.05), but there was a statistically significant difference between the 23-24 segments (P<0.05). Conclusions: The fetal HQ analysis technology can quickly and simply quantitatively assess the size, shape, and contractility of the fetal heart. Anemia in pregnant women has no significant effect on fetal heart size and systolic function; it only affects the morphology of the heart, showing that the heart tends to be spherical as a whole and some segments of the apical segment of the left ventricle are abnormal.

Ursolic Acid Protects Neurons in Temporal Lobe Epilepsy and Cognitive Impairment by Repressing Inflammation and Oxidation.

Temporal lobe epilepsy (TLE) is characterized as an impaired ability of learning and memory with periodic and unpredictable seizures. Status epilepticus (SE) is one of the main causes of TLE. Neuroinflammation and oxidative stress are directly involved in epileptogenesis and neurodegeneration, promoting chronic epilepsy and cognitive deficit. Previous studies have shown that ursolic acid (UA) represses inflammation and oxidative stress, contributing to neuroprotection. Herein, we demonstrated that UA treatment alleviated seizure behavior and cognitive impairment induced by epilepsy. Moreover, UA treatment rescued hippocampal neuronal damage, aberrant neurogenesis, and ectopic migration, which are commonly accompanied by epilepsy occurrence. Our study also demonstrated that UA treatment remarkably suppressed the SE-induced neuroinflammation, evidenced by activated microglial cells and decreased inflammation factors, including TNF-α and IL-1ß. Likewise, the expression levels of oxidative stress damage markers and oxidative phosphorylation (OXPHOS) enzyme complexes of mitochondria were also remarkably downregulated following the UA treatment, suggesting that UA suppressed the damage caused by the high oxidative stress and the defect mitochondrial function induced by SE. Furthermore, UA treatment attenuated GABAergic interneuron loss. In summary, our study clarified the notable anti-seizure and neuroprotective properties of UA in pilocarpine-induced epileptic rats, which is mainly achieved by abilities of anti-inflammation and anti-oxidation. Our study indicates the potential advantage of UA application in ameliorating epileptic sequelae.

Predicting signaling pathways regulating demyelination in a rat model of lithium-pilocarpine-induced acute epilepsy: A proteomics study.

Demyelination is observed in animal models of intractable epilepsy (IE). Epileptogenesis damages the myelin sheath and dysregulates oligodendrocyte precursor cell (OPC) development. However, the molecular pathways regulating demyelination in epilepsy are unclear. Here, we predicted the molecular mechanisms regulating demyelination in a rat model of lithium-pilocarpine hydrochloride-induced epilepsy. We identified DGKA/Mboat2/Inpp5j and NOS/Keratin 28 as the main target molecules that regulate demyelination via glycerolipid and glycerophospholipid metabolism, phosphatidylinositol signaling, and estrogen signaling in demyelinated forebrain slice cultures (FSCs). In seizure-like FCSs, the actin cytoskeleton was regulated by Cnp and MBP via Pak4/Tmsb4x (also known as Tß4) and Kif5c/Kntc1. Tß4 possibly prevented OPC differentiation and maturation and inhibited MBP phosphorylation via the p38MAPK/ERK1/JNK1 pathway. The MAPK signaling pathway was more likely activated in seizure-like FCSs than in demyelinated FCSs. pMBP expression was decreased in the hippocampus of lithium-pilocarpine hydrochloride-induced acute epilepsy rats. The expression of remyelination-related factors was suppressed in the hippocampus and corpus callosum in lithium-pilocarpine hydrochloride-induced epilepsy rats. These findings suggest that the actin cytoskeleton, Tß4, and MAPK signaling pathways regulate the decrease in pMBP in the hippocampus in a rat model of epilepsy. Our results indicate that regulating the actin cytoskeleton, Tß4, and MAPK signaling pathways may facilitate the prevention of demyelination in IE.

GPER1 Modulates Synaptic Plasticity During the Development of Temporal Lobe Epilepsy in Rats.

G-protein coupled estrogen receptor 1 (GPER1) is a novel type of estrogen receptor. Several studies have shown that it has an anti-inflammatory action,which plays an important role in remyelination and cognitive ability adjustment. However, whether it is involved in the development of temporal lobe epilepsy (TLE) is still unknown. The present study established a TLE model by intraperitoneal injection of lithium chloride (3 mmol/kg) and pilocarpine (50 mg/kg) in rats to study the effect of GPER1 in the synaptic plasticity during the development of temporal lobe epilepsy. A microinjection cannula was implanted into the lateral ventricle region of rats via a stereotaxic instrument. G-1 is the specific GPER1 agonist and G15 is the specific GPER1 antagonist. The G1 or G15 and Dimethyl sulfoxide were injected into the rat brains in the intervention groups and control group, respectively. After G1 intervention, the learning and memory abilities and hippocampal neuron damage in epileptic rats were significantly improved, while G15 weakened the neuroprotective effect of GPER1. Meanwhile, G1 controlled the abnormal formation of hippocampal mossy fiber sprouting caused by seizures, and participated in the regulation of synaptic plasticity by reducing the expression of Synapsin I and increasing the expression of gephyrin. Inhibitory synapse gephyrin may play a significant role in synaptic plasticity.

Discovery and characterization of the novel conotoxin Lv1d from Conus lividus that presents analgesic activity.

Cone snails are predatory gastropod mollusks that are distributed in all tropical marine environments and contain small peptides (conotoxins) in their venom to capture prey. However, the biochemical and molecular aspects of conotoxins remain poorly understood. In this article, a novel α4/7-conotoxin, Lv1d, was obtained from the venom duct cDNA library of the worm-hunting Conus lividus collected from the South China Sea. The cDNA of Lv1c encodes a 65 residue conopeptide precursor, which consists of a 21 residue signal peptide, a 27 residue Pro region, and 17 residues of mature peptide. The mature peptide Lv1d was chemically synthesized according to the sequence GCCSDPPCRHKHQDLCG. It was found that 10 µM Lv1d can completely inhibit frog sciatic nerve-gastrocnemius muscle contractility within 60 min. Moreover, 100 µg/kg Lv1d showed good analgesic effects in mouse hot plate model and formalin test. Patch clamp experiments showed that 5 µM Lv1d can inhibit the cholinergic microexcitatory postsynaptic currents (mEPSCs) requency and amplitude of projection neurons in Drosophila. In conclusion, the synthesis of Lv1d and its biological and physiological data might contribute to the development of this peptide as a novel potential drug for therapeutic applications. This finding also expands the knowledge of the targeting mechanism of the α4/7-subfamily conotoxins.

Silencing of Long Noncoding RNA LINC00346 Inhibits the Tumorigenesis of Colorectal Cancer Through Targeting MicroRNA-148b.

PURPOSE: This study was aimed to explore the regulatory effect of long noncoding RNA LINC00346 (LINC00346) on colorectal cancer (CRC) and the potential molecular mechanisms. METHODS: The expression of LINC00346 and microRNA-148b (miR-148b) in CRC tissues and cells was detected by qRT-PCR. LINC00346 was overexpressed and silenced in HT29 and HCT116 cells by the transfection of pcDNA-LINC00346 and si-LINC00346, respectively. The cell proliferation, migration, invasion, and apoptosis were analyzed by cell counting kit-8 (CCK-8), wound-healing, transwell, and flow cytometry assay, respectively. The targeting relationship between LINC00346 and miR-148b was predicted by TargetScan and determined by dual-luciferase reporter assay. A tumor xenograft model was established in mice to evaluate the tumor growth in vivo. RESULTS: The expression of LINC00346 was up-regulated in CRC tissues and cells. The expression of LINC00346 was positively associated with the TNM stage, lymphoma metastasis and histological grade. Overexpression of LINC00346 promoted the proliferation, migration and invasion and inhibited the apoptosis of HT29 and HCT116 cells. MiR-148b was a target of LINC00346. Silencing of miR-148b reversed the anti-tumor effect of si-LINC00346 on CRC cells. Furthermore, silencing of LINC00346 inhibited the tumor growth in mice through up-regulating miR-148b. CONCLUSION: Silencing of LINC00346 inhibited the proliferation, migration and invasion, and promoted the apoptosis of CRC cells through targeting miR-148b.

[Modification of calcium sulfate bone cement by gentamicin and oxygen-carboxymethylated chitosan].

Objective: To extend its application in the field of bone repair by adding oxygen-carboxymethylated chitosan (O-CMC) and gentamicin for modification of the calcium sulfate cement (CSC). Methods: The O-CMC/CSC was prepared by adding O-CMC with different concentrations (0.1wt%, 0.3wt%, 0.5wt%, 0.7wt%, and 1.0wt%) in the CSC liquid phase. The effect of O-CMC on the CSC was evaluated by testing the injectability, compressive strength, degradation rate, pH value, cytotoxicity and osteogenesis. After the optimal concentration of O-CMC was determined, gentamicin with different concentrations (0.5wt%, 1.5wt%, and 2.5wt%) was added in the O-CMC/CSC, and then the compressive strength and antibacterial properties were investigated. Results: After adding O-CMC in the CSC liquid phase, the injection time of O-CMC/CSC was increased to more than 5 minutes; it significantly prolonged with increased concentration of O-CMC ( P<0.05). The compressive strength of the modified bone cement was in the range of 11-18 MPa and it was the highest when the concentration of O-CMC was 0.5wt% ( P<0.05). The degradation rate of O-CMC/CSC was not influenced obviously by O-CMC ( P>0.05). The pH value was in the range of 7.2-7.4 and Ca 2+ concentration was in the range of 6-8 mmol/L. In vitro mineralization experiment indicated that the induced mineralization ability of O-CMC/CSC was much higher than that of pure CSC. The 0.5wt% O-CMC/CSC had the best performance; the compressive strength of the composite bone cement was above 5 MPa after gentamicin was added, which had antibacterial effect. Conclusion: O-CMC is able to effectively improve the injection, compressive strength, and osteogenic activity of CSC; in addition, antibacterial properties is obtained in the CSC after adding gentamicin.

Neurochemical features of endomorphin-2-containing neurons in the submucosal plexus of the rat colon.

AIM: To investigate the distribution and neurochemical phenotype of endomorphin-2 (EM-2)-containing neurons in the submucosal plexus of the rat colon. METHODS: The mid-colons between the right and left flexures were removed from rats, and transferred into Kreb's solution. For whole-mount preparations, the mucosal, outer longitudinal muscle and inner circular muscle layers of the tissues were separated from the submucosal layer attached to the submucosal plexus. The whole-mount preparations from each rat mid-colon were mounted onto seven gelatin-coated glass slides, and processed for immunofluorescence histochemical double-staining of EM-2 with calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT), nitric oxide synthetase (NOS), neuron-specific enolase (NSE), substance P (SP) and vasoactive intestinal peptide (VIP). After staining, all the fluorescence-labeled sections were observed with a confocal laser scanning microscope. To estimate the extent of the co-localization of EM-2 with CGRP, ChAT, NOS, NSE, SP and VIP, ganglia, which have a clear boundary and neuronal cell outline, were randomly selected from each specimen for this analysis. RESULTS: In the submucosal plexus of the mid-colon, many EM-2-immunoreactive (IR) and NSE-IR neuronal cell bodies were found in the submucosal plexus of the rat mid-colon. Approximately 6 ± 4.2 EM-2-IR neurons aggregated within each ganglion and a few EM-2-IR neurons were also found outside the ganglia. The EM-2-IR neurons were also immunopositive for ChAT, SP, VIP or NOS. EM-2-IR nerve fibers coursed near ChAT-IR neurons, and some of these fibers were even distributed around ChAT-IR neuronal cell bodies. Some EM-2-IR neuronal cell bodies were surrounded by SP-IR nerve fibers, but many long processes connecting adjacent ganglia were negative for EM-2 immunostaining. Long VIP-IR processes with many branches coursed through the ganglia and surrounded the EM-2-IR neurons. The percentages of the EM-2-IR neurons that were also positive for ChAT, SP, VIP or NOS were approximately 91% ± 2.6%, 36% ± 2.4%, 44% ± 2.5% and 44% ± 4.7%, respectively, but EM-2 did not co-localize with CGRP. CONCLUSION: EM-2-IR neurons are present in the submucosal plexus of the rat colon and express distinct neurochemical markers.

[Changes and significance of P-selectin and PAC-1 in coronary heart disease before and after stenting].

OBJECTIVE: To explore the platelet activation status before and after coronary artery stent implantation at different timepoints. METHODS: The contents of CD62P (soluble P-selectin) and PAC-1 (platelet glycoprotein GPIIb/IIIa fibrinogen receptor) in patients after coronary stent implantation were detected by flow cytometry (FCM) before, immediately after surgery, postoperative 2, 4, 6 and 24 h in peripheral blood. RESULTS: In 65 cases, the levels of CD62P[40.63(25.14-51.01)%] and PAC-1[36.59(20.37-48.41)%] immediately after surgery were significantly higher than their preoperative values [14.79 (9.85-26.66) %, 13.99(11.42-30.133)%] (P < 0.05). And the postoperative levels of 2 h [8.64(4.48-15.67)%, 12.64(6.45-18.83)%], 4 h [7.91(4.56-12.94)%, 12.81(6.89-18.66)%], 6 h [6.53(4.12-9.27)%, 9.43(5.27-15.65)%] and 24 h [6.28(4.36-9.63)%, 10.38(4.63-18.11)%] decreased significantly versus their preoperative values (P < 0.05). CONCLUSION: Stenting of coronary endothelial damage within 2h may enhance platelet activation and boost the risk of thrombosis.

Tranexamic acid reduces postoperative bleeding in off-pump coronary artery bypass grafting.

OBJECTIVE: Tranexamic acid (TA) reduces blood loss in coronary artery surgery with cardiopulmonary bypass. The present prospective study was designed to investigate its hemostatic effect in off-pump coronary artery bypass (OPCAB). METHOD: Seventy-six patients undergoing elective OPCAB were randomized into two groups, received TA (0.75 g loading dose before surgery and 250 mg/h during surgery, gross dose: 1.5 g, n=36) and saline solution (control, n=40), respectively. Perioperative blood samples were collected. Hematochemical parameters including platelet adhesion rate, D-dimer and fibrinopeptide-A (FPA) were analysis. Volume of blood loss, blood transfusion and other clinical data were recorded throughout the perioperative period. RESULTS: Cumulative blood loss was significantly reduced in the TA group as compared to the controls postoperatively (6 hrs (median [25th-75th]): TA: 200.0 [140.0-230.0] ml, CONTROL: 225.0 [200.0-347.5.0] ml, p=0.009; 24 hrs: TA: 440.0 [270.0-605.0] ml, CONTROL: 655.0 [500.0-920.0] ml, p<0.001). Number of patients received blood transfusion in each group was similar. Levels of D-dimer rose significantly after surgery, and were significantly lower in the TA group than that in controls. Platelet adhesion rate and FPA levels remained at baseline levels after the operation in two groups. Early clinical outcomes were similar between groups. CONCLUSION: The results indicated that tranexamic acid limits fibrinolysis and reduces blood loss after off-pump coronary artery bypass surgery.

[Research on cellular immune function of patients with lung cancer before and after operation].

BACKGROUND: To explore the perioperative changes of T subsets and NK cell and analyze the related factors in patients with lung cancer. METHODS: The T subsets and NK cell from peripheral blood of 60 patients with lung cancer, 15 patients with lung benign tumor and 15 healthy people were detected by immunofluorescence. These indexes of the patients with lung cancer were detected also at postoperative 2nd, 7th, 14th and 28th days. RESULTS: 1.There were significant differences in the indexes between the lung cancer group and the groups of lung benign tumor and normal people except for CD8+ (P < 0.05). 2.At postoperative 2nd day CD3+, CD4+, CD4+/CD8+ and NK cell of the patients with lung cancer were decreased and CD8+ was increased significantly than those before operation (P < 0.05). During postoperative 1 to 2 weeks, all indexes had recovered basically to the preoperative level. At postoperative 28th day, CD3+, CD4+ , CD4+/CD8+ and NK cell were increased and CD8+ was decreased than those before operation (P < 0.05). 3. There was significant difference in the indexes among preoperative stage IIIA, IIIB and IB, and between preoperative N2 diseases and N0 group (P < 0.05). There was significant difference between the groups of radical and palliative operation and the group of thoracic exploration at postoperative 28th day (P < 0.05). There was significant difference in T subsets between the groups of blood transfusion and non-transfusion at postoperative 14th day (P < 0.05). CONCLUSIONS: The cellular immune function of the patients with lung cancer was lower than that of the patients with lung benign tumor and normal people. The perioperative immunity of patients with lung cancer decreases after operation and increases later. TNM stage and lymph node metastasis are relative to preoperative but not postoperative immunity. There is no significant correlation between cellular immune function and pathological type of the tumor. Radical and palliative operations can both significantly increase the patients' cellular immune function. Therefore the palliative operation is better than thoracic exploration. Blood transfusion can depress the immune function of the patients, so it is better to avoid perioperative blood transfusion.