Relative Protein Levels Research Articles

Effects of Low Molecular Weight Peptides from Red Shrimp (Solenocera crassicornis) Head on Immune Response in Immunosuppressed Mice.

This study aimed to investigate the immunoenhancement effects of low molecular weight peptides (SCHPs-F1) from red shrimp (Solenocera crassicornis) head against cyclophosphamide (CTX)-induced immunosuppressed mice. ICR mice were intraperitoneally injected with 80 mg/kg CTX for 5 consecutive days to establish the immunosuppressive model and then intragastrically administered with SCHPs-F1 (100 mg/kg, 200 mg/kg, and 400 mg/kg) to investigate its improving effect on immunosuppressed mice and explore its potential mechanism using Western blot. SCHPs-F1 could effectively improve the spleen and thymus index, promoting serum cytokines and immunoglobulins production and upregulating the proliferative activity of splenic lymphocytes and peritoneal macrophages of the CTX-treated mice. Moreover, SCHPs-F1 could significantly promote the expression levels of related proteins in the NF-κB and MAPK pathways in the spleen tissues. Overall, the results suggested that SCHPs-F1 could effectively ameliorate the immune deficiency caused by CTX and had the potential to explore as an immunomodulator in functional foods or dietary supplements.

#2524 RESOLVIN D1 ATTENUATES SEPSIS INDUCED ACUTE KIDNEY INJURY TARGETING MITOCHONDRIA AND NF-κB SIGNALING PATHWAY

Abstract Background and Aims Acute kidney injury is a highly common and multifactorial renal disease resulting in significant morbidity and mortality, especially sepsis-induced acute kidney injury. There is no effective therapy available to treat or prevent sepsis-induced acute kidney injury. One of the specialized pro-resolving mediators, Resolvin D1 exhibits special anti-inflammatory effects in several inflammatory disease models, but there is little evidence about the effect and mechanism of Resolvin D1 in sepsis-induced acute kidney injury. To explore the renal protective role of RvD1 in SI-AKI and to further explore the inherent pathophysiological mechanism, especially the role of mitochondrial function and NF-κB signaling pathway, in vitro and in vivo experiments were conducted. Method We conducted experiments to explore the effect and mechanism of Resolvin D1 in sepsis-induced acute kidney injury. In vitro, human proximal tubular epithelial cells were used to test the apoptosis ratio, cell viability and reactive oxygen species level. In vivo, C57BL/6 mice were injected with lipopolysaccharide to establish a sepsis-induced acute kidney injury model. Renal function and structure, apoptosis ratio of kidney cells, mitochondrial structure and function and related protein and gene levels were assessed. Results In vitro, the resolvin D1-treated group showed higher cell viability and lower reactive oxygen species levels and apoptosis ratios than the LPS group. In vivo, Resolvin D1 can not only improve renal function and mitochondrial function but also reduce the apoptosis ratio, while mediating mitochondrial dynamics and inhibiting NF-κB pathway. Conclusion Resolvin D1 has a good renoprotective effect by maintaining mitochondrial dynamics and inhibiting the NF-κB pathway. This provides an important basis for us to further develop the clinical therapeutic value of RvD1 in acute renal injury in sepsis.

TL1A promotes the postoperative cognitive dysfunction in mice through NLRP3-mediated A1 differentiation of astrocytes.

We investigated the mechanism, whereby tumor necrosis factor-like ligand 1A (TL1A) mediates the A1 differentiation of astrocytes in postoperative cognitive dysfunction (POCD). The cognitive and behavioral abilities of mice were assessed by Morris water maze and open field tests, while the levels of key A1 and A2 astrocyte factors were detected by RT-qPCR. Immunohistochemical (IHC) staining was used to examine the expression of GFAP, western blot was used to assay the levels of related proteins, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory cytokines. The results showed that TL1A could promote the progression of cognitive dysfunction in mice. Astrocytes differentiated into A1 phenotype, while unobvious changes were noted in astrocyte A2 biomarkers. Knockout of NLRP3 or intervention with NLRP3 inhibitor could inhibit the effect of TL1A, improving the cognitive dysfunction and suppressing the A1 differentiation. Our results demonstrate that TL1A plays an important role in POCD in mice, which promotes the A1 differentiation of astrocytes through NLRP3, thereby exacerbating the progression of cognitive dysfunction.

Cholesterol‐related APOB Thr98Ile correlates with brain cortical APOB mRNA levels

AbstractBackgroundApolipoprotein B (APOB) is well‐known for its role in cholesterol metabolism. We recently showed that APOB protein levels are elevated in the CSF of patients suffering from sporadic Alzheimer’s disease (AD) compared to control individuals (Picard et al, Alzheimers Dement. https://doi.org/10.1002/alz.12442). Since rare coding APOB variants were discovered in association with early‐onset familial Alzheimer’s disease (Wingo et al, JAMA Neurol. 2019), we hypothesized that common APOB variants could influence molecular mechanisms leading to sporadic AD.MethodPublicly available genomic and transcriptomic data were screened for possible interactions between APOB single nucleotide polymorphisms (SNPs) and brain APOB mRNA levels. Potential regulatory SNPs were contrasted with blood cholesterol levels obtained from the Global Lipids Genetics Consortium (GLGC), in order to highlight their systemic effects. Brain tissue cholesterol levels were measured in frontal cortices of autopsy‐confirmed controls and AD subjects from the Douglas‐Bell Canada Brain Bank (DBCBB) using high performance liquid chromatography (HPLC). DNA extracted from those samples was used to perform APOB locus genotyping with the help of the Illumina Global Screening Array (GSA) chip.ResultThe APOB Thr98Ile variant commonly associated with familial hypercholesterolemia was found to correlate with a significant reduction of brain cortical APOB mRNA levels in three different cohorts. In GLGC cohort, this SNP strongly associates with elevated LDL‐cholesterol levels measured from blood samples of 188 578 individuals (p = 9.48E‐183). Our preliminary analysis shows that APOB Thr98Ile also correlates with elevated cholesterol levels (r2 = 0.36; p < 0.005) measured in brain cortical samples. Using public databases with available genotypic and AD‐related neuropathological data, inconsistent associations were found between APOB Thr98Ile variant and sporadic AD pathophysiological changes in the brain. Ongoing studies in our laboratory are examining the impact of APOB Thr98Ile variant on APOB mRNA and protein levels in relation to classical AD pathological markers in a large group of AD and control subjects.ConclusionAlthough APOB Thr98Ile is significantly associated with gene regulation and cholesterol levels measured in blood and brain samples, its effect on AD neuropathology remains to be clearly defined and is the subject of current investigations.

Effect of Nucleolin on Lymphoma Proliferation by Regulating Thymidine Kinase 1

To investigate the mechanism of nucleolin (NCL) involved in lymphoma proliferation by regulating thymidine kinase 1 (TK1). Twenty-three patients with diffuse large B-cell lymphoma (DLBCL) were selected and divided into initial treatment group (14 cases) and relapsed/refractory group (9 cases). Serum TK1 and C23 protein in peripheral blood mononuclear cells were detected. Cell models of CA46-NCL-KD (CA46-NCL-knockdown) and CA46-NCL-KNC (CA46-NCL-knockdown negative control) were established by lentivirus vector mediated transfection in Burkitt lymphoma cell line CA46. The half maximal inhibitory concentration (IC50) of CA46-NCL-KD, CA46-NCL-KNC, and CA46 to adriamycin were detected by cell proliferation assay (MTS). The expression of NCL mRNA and protein in CA46-NCL-KD and CA46-NCL-KNC cells were dectected by Q-PCR and Western blot, respectively. The cell cycle of CA46-NCL-KD, CA46-NCL-KNC, and CA46 cells were detected by flow cytometry. The expression of TK1 protein in CA46-NCL-KD and CA46-NCL-KNC cells was detected by an enhanced chemiluminescence (ECL) dot blot assay. The level of serum TK1 in the initial treatment group was 0.43(0-30-1.01) pmol/L, which was lower than 10.56(2.19-14.99) pmol/L in the relapsed/refractory group (P<0-01), and the relative expression level of NCL protein in peripheral blood was also significantly lower. The IC50 of CA46-C23-KD cells to adriamycin was (0.147±0.02) μg/ml, which was significantly lower than (0.301±0.04) μg/ml of CA46-C23-KNC cells and (0.338±0.05) μg/ml of CA46 cells (P<0.05). Compared with CA46-NCL-KNC cells, the expression of NCL mRNA and protein, TK1 protein decreased in CA46-NCL-KD cells, and the proportion of S phase and G2/M phase also decreased, while G0/G1 phase increased in cell cycle. The increased expression of NCL in DLBCL and CA46 cells indicates low sensitivity to drug. NCL may participate in regulation of lymphoma proliferation by affecting TK1 expression, thereby affecting the drug sensitivity.

Evaluation of small extracellular vesicles as biomarkers of efficacy with anti-PD-1 mAb therapy in patients with recurrent/metastatic HNSCC.

6042 Background: The development of biomarkers that reliably predict response to anti-PD-1 mAb therapy (IO) is a critical need. Small (30-150 nm) extracellular vesicles (sEV), aka exosomes, are “molecular mimics” of their parent cells. In cancer, plasma sEV are mixtures of tumor-derived exosomes (TEX) and vesicles produced by non-malignant cells. We conducted a retrospective study of R/M HNSCC patients treated with IO to evaluate sEV as predictive biomarkers. Methods: We evaluated sEV in plasma of R/M HNSCC patients (n = 24) obtained just prior to initiation of IO. Precleared ultrafiltered plasma was used to isolate total plasma sEV by size exclusion chromatography. Total plasma sEV were separated into two subsets, T cell-derived CD3(+) sEV and TEX-enriched CD3(-) sEV by immunocapture with anti-CD3 mAb. On-bead flow cytometry was used to estimate relative levels of proteins carried on the sEV surface. Specifically, the CD3(-) fraction was evaluated for stimulatory (stim; CD40, CD40L, OX40, OX40L, CD80) and suppressive (supp; TGFb, CTLA4, FasL, PD-L1, PD-1) proteins. Differences between responders (CR/PR/SD) and non-responders (PD) were assessed using Wilcoxon-Mann-Whitney test. Multivariate Cox proportional hazards regression was used to evaluate the relationship between the pre-treatment sEV characteristics and outcome. Results: Mean age was 62, 46% had oropharynx (82% HPV+), 17% oral cavity and 29% had larynx/hypopharynx primary. Treatment indication for IO was 46% platinum failure, remainder first line. Best response: 17% PR, 29% SD, and 54% PD. Median PFS and OS were 5.3 and 16.2 months, respectively. Total sEV protein level was not associated with efficacy. Total CD3(+) sEV level was low overall and did not correlate with lymphocyte count or neutrophil/lymphocyte ratio. High CD3(+) sEV level was associated with better OS [HR: 0.16 (95% CI 0.04-0.59), P = 0.007] and PFS [HR: 0.16 (95% CI 0.04-0.58), P = 0.005] but not response. While total TEX and levels of individual stim and supp proteins carried by TEX were not predictive of response, an increased supp/stim ratio was (P = 0.02). Importantly, high TEX supp score and stim score were both associated with better OS [HR: 0.19 (95% CI 0.05-0.77), P = 0.02, and HR: 0.26 (95% CI 0.07-0.95), P = 0.04, resp.]. High PD-L1, but not PD-1 on TEX, was associated with better OS [HR: 0.15 (95% CI 0.04-0.56), P = 0.005]; and PFS: HR: 0.33 (95% CI 0.11-0.94), P = 0.04]. Conclusions: Evaluation of two sEV subsets, T cell-derived-CD3(+) sEV and TEX-enriched CD3(-) sEV, indicated their potential utility as predictive biomarkers with IO. High T cell-derived sEV, TEX supp/stim ratio, and TEX PD-L1 expression levels were independently associated with significantly increased efficacy with IO. Our study uniquely analyzed the predictive value of plasma sEv subsets in IO treated R/M HNSCC patients, and evaluation in a larger cohort is warranted.

Ebracteolatain A Inhibits Lung Cancer Growth via p21 and p27 Mediated Cell-Cycle Arrest

Objective: Lung cancer is a refractory tumor with a poor five-year survival rate. Ebracteolatain A (EA), a key acetyl-phloroglucinol derived from the traditional Chinese herb Langdu, has been reported to have anti-tumor activity in human breast cancer. However, the therapeutic efficacy and underlying mechanism of lung cancer remain to be elucidated. In the present study, we intended to evaluate whether EA exhibits anti-lung cancer activity and elucidate the underlying mechanisms. Methods: ATPlite and clonogenic survival assays were used to assess the inhibiting effect of EA on two lung cancer cell lines in vitro. FACS was conducted to determine the cell-cycle profiling of lung cancer cells upon treatment with EA. Western blot was performed to detect the level of related proteins in EA-treated lung cancer cells. RNA silencing and other experiments were used to identify the molecular mechanism of EA-triggered cell-cycle arrest. Furthermore, a xenograft tumor model was constructed to evaluate the therapeutic effect and potential mechanism of EA in vivo in mice. Results: EA significantly inhibited the growth of lung cancer cells both in vitro and in vivo. Mechanistically, EA-induced G1 phase cell-cycle arrest is mediated by the accumulation of p21 and p27. Conclusion: Our findings revealed the anti-lung cancer effect of EA and the mechanisms, providing a solid scientific basis for EA as an attractive choice for lung cancer therapy.

Role of Fibrinogen in Type-2 Diabetes Mellitus with Diabetic Neuropathy and its Preliminary Mechanism.

Diabetic peripheral neuropathy (DN) is the most common complication of type 2 diabetes mellitus (T2DM). This study aimed to explore the role of fibrinogen (FIB) in T2DM neuropathy and its preliminary mechanism. Ten male Sprague-Dawley rats were divided into a normal control group (NC group) and a T2DM neuropathy model group (DN group). The DN group was given a high-energy diet and streptozotocin, while the NC group was given a normal diet and a citric acid buffer. The expression levels of related proteins were analysed. Electrophysiology: Compared with the NC group, the conduction latency of the somatosensory-evoked potential and nerve conduction velocity was prolonged in the DN group, while the motor nerve action potential was decreased. As seen under a light microscope, the peripheral nerve fibres in the DN group were swollen, and the nerve fibres in the posterior funiculus of the spinal cord were loose or missing. Moreover, as seen under an electron microscope, the peripheral nerve demyelination of the DN group was severe, with microvascular blood coagulation, luminal stenosis, and collapse. Compared with the NC group, in the DN group, the expression of FIB was positively correlated with the expression of both ionised calcium-binding adaptor molecule-1 and glial fibrillary acidic protein. Compared with the NC group, in the DN group, the expression of platelet/endothelial cell adhesion molecule-1 and B-cell lymphoma 2 was negatively correlated. The increased concentration of FIB may be the cause of neuropathy, and its mechanism may be related to its promotion of inflammatory response, blood coagulation, and vascular stenosis.

RNF220 promotes gastric cancer growth and stemness via modulating the USP22/wnt/β-catenin pathway

Gastric cancer (GC) is a prevalent malignancy that seriously threatens the health and life of patients. Although Ring finger 220 (RNF220) has been demonstrated to participate in the development of various cancers, its role and mechanism in GC remain undiscovered. The expression of RNF220 was determined by The Cancer Genome Atlas (TCGA) database and Western blot. Additionally, the overall survival (OS) and post-progression survival (PPS) were analyzed based on the levels of RNF220 in the TCGA database. The role and mechanism of RNF220 in growth and stemness were investigated using cell counting kit-8, colony formation, sphere-formation, co-immunoprecipitation, and Western blot experiments. Furthermore, the role of RNF220 was investigated in a xenografted mouse model. The expression of RNF220 was found to be upregulated in GC, which predicted unfavorable OS and PPS in patients with GC. Knockdown of RNF220 reduced cell viability, colony numbers, numbers of spheres formation, and the relative protein levels of Nanog, Sox2, and Oct4 in both AGS and MKN-45 cells. Moreover, overexpression of RNF220 increased cell viability and the numbers of spheres formation in MKN-45 cells. Mechanistically, RNF220 bound to USP22, and interference of RNF220 downregulated the Wnt/β-catenin axis via USP22, which was confirmed by the overexpression of USP22 in both cell lines. Furthermore, silencing of RNF220 significantly decreased tumor volume and weight, the level of Ki-67, and the relative protein levels of USP22, β-catenin, c-myc, Nanog, Sox2, and Oct4. Taken together, downregulation of RNF220 suppressed GC cell growth and stemness by downregulating the USP22/Wnt/β-catenin axis.

Arctigenin hinders the invasion and metastasis of cervical cancer cells via the FAK/paxillin pathway

ContextCervical cancer is the most common gynecological pernicious tumor with high morbidity and mortality worldwide, especially in developing countries. Arctigenin (ARG), a nature-derived component, has exhibited anti-tumor activity in various tumors. ObjectiveTo explore the effect of ARG on cervical cancer. Materials and methodsThe effect and mechanism of ARG on cervical cancer cells were explored by cell counting kit-8 (CCK-8), flow cytometry, transwell and Western blot assays. Additionally, in vivo experiment was conducted in xenografted mice by immunohistochemistry (IHC), terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) and Western blot assays. ResultsARG treatment induced both concentration-dependent and time-dependent reductions in the cell viability of SiHa and HeLa cells with a IC50 value of 9.34 μM and 14.45 μM, respectively. ARG increased the apoptosis rate and the protein levels of cleaved-caspase 3 and E-cadherin, but decreased the invaded cell numbers and the protein levels of Vimentin and N-cadherin in vitro. Mechanically, ARG inhibited the expression of focal adhesion kinase (FAK)/paxillin pathway, which was confirmed by the overexpression of FAK in SiHa cells. The inhibitory role of overexpression of FAK in proliferation and invasion, as well as its promoted role in apoptosis were reversed with ARG treatment. Meanwhile, ARG suppressed growth and metastasis, and enhanced apoptosis in vivo. Consistently, ARG administration reduced the relative protein level of p-FAK/FAK and p-paxillin/paxillin in tumor tissues of xenografted mice. ConclusionARG inhibited proliferation, invasion and metastasis, but enhanced apoptosis of cervical cancer via the FAK/paxillin axis.

LncRNA NKILA Exacerbates Alzheimer's Disease Progression by Regulating the FOXA1-Mediated Transcription of TNFAIP1.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases in the world, which seriously affects AD patients' life quality. Recently, long non-coding RNAs (lncRNAs) have been reported to play a key role in AD pathogenesis, however, the specific mechanism remains unclear. Herein, we aimed to investigate the role of lncRNA NKILA in AD.The learning and memory performance of rats from streptozotocin (STZ)-treated or other treated groups were tested by Morris water maze test. Relative levels of genes and proteins were measured using RT-qPCR and Western blotting. Mitochondrial membrane potential was tested by JC-1 staining. Levels of ROS, SOD, MDA, GSH-Px, and LDH were measured using corresponding commercial kits. Apoptosis was evaluated by TUNEL staining or Flow cytometry assay. RNA Immunoprecipitation (RIP), RNA pulldown, Chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays were utilized to test the interaction between indicated molecules.STZ treatment caused learning and memory impairment in rats and oxidative stress damage in SH-SY5Y cells. LncRNA NKILA was found to be elevated in the hippocampal tissues of rats and SH-SY5Y cells after STZ exposure. Knockdown of lncRNA NKILA alleviated STZ-induced neuronal damage. Furthermore, lncRNA NKILA could bind to ELAVL1, which regulate the stability of FOXA1 mRNA. Moreover, TNFAIP1 transcription process was controlled by FOXA1, which targeted the promoter of TNFAIP1. In vivo results demonstrated that lncRNA NKILA accelerated STZ-induced neuronal damage and oxidative stress by FOXA1/TNFAIP1 axis.Our findings indicated that knockdown of lncRNA NKILA inhibited the neuronal damage and oxidative stress induced by STZ through the FOXA1/TNFAIP1 axis, thereby alleviating the development of AD, revealing a promising therapeutic axis for AD treatment.

Berberine inhibits the progression of renal cell carcinoma cells by regulating reactive oxygen species generation and inducing DNA damage.

Berberine is a natural isoquinoline alkaloid that has been shown to have antitumor properties in a growing number of studies. However, its role in renal cell carcinoma remains unclear. This study investigates berberine's effect and mechanism in renal cell carcinoma. The methyl-tetrazolium, colony formation, and lactate dehydrogenase assay were used to detect proliferation and cytotoxicity, respectively. Flow cytometry, caspase-Glo 3/7 assay, and adenosine triphosphate assay were used to detect apoptosis and the adenosine triphosphate levels. Wound healing and transwell assay were used to examine the migration ability of renal cell carcinoma cells. Besides, the level of reactive oxygen species (ROS) was explored using a DCFH-DA-based kit. Additionally, western blot and Immunofluorescence assay was taken to determine the levels of relative proteins. In vitro, our findings indicated that the proliferation and migration of renal cell carcinoma cells treated with berberine in various concentrations were inhibited, while the level of ROS and apoptosis rate were increased. Furthermore, The results of western blot showed that the expression of Bax, Bad, Bak, Cyto c, Clv-Caspase 3, Clv-Caspase 9, E-cadherin, TIMP-1and γH2AX were up-regulated, while Bcl-2, N-cadherin, Vimentin, Snail, Rad51 and PCNA were down-regulated after treating with berberine with various concentration. The result of this study revealed that berberine inhibits renal cell carcinoma progression via regulating ROS generation and inducing DNA break.

DDX43 mRNA expression and protein levels in relation to clinicopathological profile of breast cancer.

Breast cancer (BC) is the most often diagnosed cancer in women globally. Cancer cells appear to rely heavily on RNA helicases. DDX43 is one of DEAD- box RNA helicase family members. But, the relationship between clinicopathological, prognostic significance in different BC subtypes and DDX43 expression remains unclear. Therefore, the purpose of this study was to assess the clinicopathological significance of DDX43 protein and mRNA expression in different BC subtypes. A total of 80 females newly diagnosed with BC and 20 control females that were age-matched were recruited for this study. DDX43 protein levels were measured by ELISA technique. We used a real-time polymerase chain reaction quantification (real-time PCR) to measure the levels of DDX43 mRNA expression. Levels of DDX43 protein and mRNA expression within BC patients had been compared to those of control subjects and correlated with clinicopathological data. The mean normalized serum levels of DDX43 protein were slightly higher in control than in both benign and malignant groups, but this result was non-significant. The mean normalized level of DDX43 mRNA expression was higher in the control than in both benign and malignant cases, although the results were not statistically significant and marginally significant, respectively. Moreover, the mean normalized level of DDX43 mRNA expression was significantly higher in benign than in malignant cases. In malignant cases, low DDX43 protein expression was linked to higher nuclear grade and invasive duct carcinoma (IDC), whereas high mRNA expression was linked to the aggressive types of breast cancer such as TNBC, higher tumor and nuclear grades. This study explored the potential of using blood DDX43 mRNA expression or protein levels, or both in clinical settings as a marker of disease progression in human breast cancer. DDX43 mRNA expression proposes a less invasive method for discriminating benign from malignant BC.

Wuzang Wenyang Huayu Tang promoting learning-memory ability in vascular dementia rats via brain-gut-microbiome axis

IntroductionLots of research has revealed that vascular dementia (VD), which is closely related to the metabolic disorders of intestinal microbiota, can be treated through traditional Chinese drug by regulating the brain-gut-microbiome axis. However, the specific interaction between VD and intestinal microflora, as well as the mechanism of Wuzang Wenyang Huayu Tang (WWHT) in treating VD by regulating the brain-gut-microbiome axi remains unknown on the whole and needs to be further studied. ObjectivesTo explore the effect of WWHT on the learning and memory ability of rats with VD based on brain-gut-microbiome axis. MethodsIn this study, taking the specific pathogen-free (SPF) SD rats as the subjects, a vascular dementia rat model was created by using the two-vessel occlusion model (2-VO). The subjects were divided into six groups, namely control group, sham group, VD model group, the piracetam group, the WWHT group, and the WWHT combined with piracetam (WCP) group. After 22 days of the gavage administration method, the Morris water maze experiment was adopted to determine the learning and memory abilities of each group through the water maze, observe the effects of hematoxylin and eosin (HE) staining and transmission electron microscopy in the hippocampal area of each group, and analyze the relevant proteins in the whole brain by Western Blot (WB). In addition, the feces of each group were collected and analyzed by fecal metabolomics combined with 16S rRNA gene sequencing to explore the interaction between VD and intestinal microbiota. ResultsThrough Morris water maze experiment, it was found that the learning and memory ability of VD rats in WWHT, piracetam and WCP groups was improved. The morphology of hippocampal neuron cells were improved in the WWHT group and the number of hippocampal neuron cells were increased in the VD rats by HE. By observing the ultrastructure of hippocampal cells, it can be found that the VD model group showed cell membrane edema and large organelle lysis. The ultrastructure of the hippocampus of all the administered groups was improved. In terms of the effect on the expression levels of related proteins in the whole brain of rats, compared with the VD model group, the expression levels of Caspase-3 protein decreased in all groups after drug administration. The protein expression levels of APG5L/ATG5, Beclin-1 and LC3A/B were upregulated, indicating that the drug administration group could regulate cellular autophagy in the whole brain to further protect brain tissue. Fecal metabolite profiles revealed 27 different metabolites related to VD. These metabolites are mainly involved in related metabolic pathways, such as vitamin B metabolism, purine metabolism, pyrimidine metabolism, and histidine metabolism. Further analysis of the gut microbiota by 16S rRNA gene sequencing data showed that WWHT-treated VD rats were different in gut microbiota composition. DiscussionThrough the effect of brain-gut-microbiome axis, WWHT regulates the metabolism of vitamin B6 and tryptophan in VD rats, which can improve the intestinal microbiota of VD rats and regulate autophagy in the brain to improve cell morphology in the hippocampus, thus increasing the number of hippocampal cells to protect hippocampal neuron cells2510100, and finally improving the learning and memory ability of VD rats.

MiR-21-5p promotes sorafenib resistance and hepatocellular carcinoma progression by regulating SIRT7 ubiquitination through USP24

ObjectiveTo validate the mechanism by which miR-21-5p mediates autophagy in drug-resistant cells in hepatocellular carcinoma (HCC), aggravating sorafenib resistance and progression of HCC. MethodsHCC cells were treated with sorafenib to establish sorafenib-resistant cells, and nude mice were subcutaneously injected with hepatoma cells to establish animal models. RT–qPCR was used to determine the level of miR-21-5p, and Western blotting was used to determine the level of related proteins. Cell apoptosis, cell migration, the level of LC3 were accessed. Immunohistochemical staining was used for detection of Ki-67 and LC3. A dual-luciferase reporter assay certified that miR-21-5p targets USP42, and a co-immunoprecipitation assay validated the mutual effect between USP24 and SIRT7. ResultsmiR-21-5p and USP42 were highly expressed in HCC tissue and cells. Inhibition of miR-21-5p or knockdown of USP42 inhibited cell proliferation and cell migration, upregulated the level of E-cadherin, and downregulated the level of vimentin, fibronectin and N-cadherin. Overexpression of miR-21-5p reversed the knockdown of USP42. Inhibition of miR-21-5p downregulated the ubiquitination level of SIRT7, downregulated the levels of LC3II/I ratio and Beclin1, and upregulated the expression of p62. The tumor size in the miR-21-5p inhibitor group was smaller, and Ki-67 and LC3 in tumor tissue were reduced, while the overexpression of USP42 reversed the effect of the miR-21-5p inhibitor. ConclusionmiR-21-5p promotes deterioration and sorafenib resistance in hepatocellular carcinoma by upregulating autophagy levels. Knockdown of miR-21-5p inhibits the development of sorafenib-resistant tumors by USP24-mediated SIRT7 ubiquitination.

Butyrate inhibits iILC2-mediated lung inflammation via lung-gut axis in chronic obstructive pulmonary disease (COPD)

BackgroundThe study investigated the effects and underlying mechanisms of intestinal flora metabolite butyrate on inflammatory ILC2 cells (iILC2s)-mediated lung inflammation in chronic obstructive pulmonary disease (COPD).MethodsMouse models of COPD and acute exacerbation of COPD (AECOPD) were established. Flow cytometry was used to detect natural ILC2 cells (nILC2s) and iILC2s in lung and colon tissues. The 16s rRNA and GC-MS were used to detect microbial flora and short chain fatty acids (SCFAs) in feces. ELISA was used to detect IL-13 and IL-4. Western blot and qRT-PCR were used to detect the relative protein and mRNA levels, respectively. In vitro experiments were performed with sorted ILC2s from colon tissues of control mice. Mice with AECOPD were treated with butyrate.ResultsThe nILC2s and iILC2s in lung and colon tissues of AECOPD mice were significantly higher than control groups. The abundance of the flora Clostridiaceae was significantly reduced, and the content of SCFAs, including acetate and butyrate, was significantly reduced. The in vitro experiments showed that butyrate inhibited iILC2 cell phenotype and cytokine secretion. Butyrate treatment reduced the proportion of iILC2 cells in the colon and lung tissues of mice with AECOPD.ConclusionsThe nILC2s and iILC2s in the colon tissues are involved in the course of COPD. Decreased Clostridiaceae and butyrate in AECOPD mice caused the accumulation of iILC2 cells in the intestines and lungs. Supplementation of butyrate can reduce iILC2 in the intestine and lung tissues. Our data may provide new ideas for prevention and treatment of COPD.

ZeXieYin Formula alleviates TMAO-induced cognitive impairment by restoring synaptic plasticity damage

Ethnopharmacological relevanceTreating cognitive impairment is a challenging and necessary research topic. ZeXieYin Formula (ZXYF), is a traditional herbal formula documented in the book of HuangDiNeiJing. Our previous studies demonstrated the ameliorative effects of ZXYF on atherosclerosis by reducing the plasma trimethylamine oxide (TMAO) level. TMAO is a metabolite of gut microorganisms, our recent research found that the increasing level of TMAO may have adverse effects on cognitive functions. Aim of the studyOur study mainly focused on the therapeutic effects of ZXYF on TMAO-induced cognitive impairment in mice and explored its underlying mechanism. Materials and methodsAfter the TMAO-induced cognitive impairment mice models were established, we applied behavioral tests to estimate the learning and memory ability of the ZXYF intervention mice. Liquid chromatography-mass spectrometry (LC-MS) was used to quantify the TMAO levels in plasma and the brain. The effects of ZXYF on the hippocampal synaptic structure and the neurons were observed by transmission electron microscopy (TEM) and Nissl staining. In addition, western-blotting (WB) and immunohistochemical (IHC) staining were used to detect the level of related proteins in the synaptic structure and further verify the changes in synaptic plasticity and the mTOR pathway after ZXYF administration. ResultsBehavioral tests showed that the learning and memory ability of mice impaired after a period of TMAO intervention and ZXYF could alleviate these changes. A series of results showed that ZXYF partly restored the damage of hippocampal synapse and neurons in TMAO-induced mice, at the same time, the expression of synapse-related proteins and mTOR pathway-related proteins were significantly regulated compared with the damage caused by TMAO. ConclusionZXYF could alleviate TMAO-induced cognitive impairment by improving synaptic function, reducing neuronal damage, regulating synapse-associated proteins, and regulating the mTOR signaling pathway.

Effects of melatonin on phosphorylation of memory-related proteins in the hippocampus and the perirhinal cortex in male mice.

We recently demonstrated that a single post-training administration of either melatonin, an MT1/MT2 melatonin receptor agonist ramelteon, or a brain melatonin metabolite N1-acetyl-5-methoxyquinuramine (AMK) enhanced object recognition memory. The present study aims to investigate the effects of melatonin, ramelteon, and AMK on relative phosphorylation levels of memory-related proteins in order to explore candidate signaling pathways associated with the receptor-mediated and nonreceptor-mediated memory-enhancing effects of melatonin. We first confirmed that post-training administration of either melatonin, ramelteon, or AMK at 1 mg/kg promoted long-term memory formation, using the novel object recognition task. Next, the effects of the same doses of these drugs on relative phosphorylation levels of the extracellular signal-regulated kinase (ERK) and calcium/calmodulin-dependent kinases (CaMKs) in the hippocampus and the perirhinal cortex (PRC) were examined by western blot analysis. In the hippocampus, treatment with ramelteon or AMK significantly increased and decreased phosphorylation levels of ERK and cAMP-response element binding protein (CREB) and those of CaMKIIα and β, respectively. In the PRC, phosphorylation levels of ERK and those of CaMKIIβ were significantly increased by both ramelteon and AMK and by ramelteon, respectively. Neither ramelteon nor AMK altered the phosphorylation levels of CaMKIV in either hippocampus or PRC. These results suggest that melatonin may be involved in promoting the formation of long-term object recognition memory in a similar, if not identical, manner by modulating the phosphorylation levels of memory-related proteins such as ERK, CaMKIIs, and CREB in both receptor-mediated and nonreceptor-mediated signaling pathways.

Red Ginseng Improves D-galactose-Induced Premature Ovarian Failure in Mice Based on Network Pharmacology.

In this study, we evaluated the ameliorative effect and molecular mechanism of red ginseng (Panax ginseng C.A. Meyer) extract (RGE) on D-galactose (D-gal)-induced premature ovarian failure (POF) using network pharmacology analysis. Ginsenosides are important active ingredients in ginseng, which also contains some sugar and amino acid derivatives. We aimed to determine the key proteins through which RGE regulates POF. In this work, we retrieved and screened for active ingredients in ginseng and the corresponding POF disease targets in multiple databases. A PPI network of genes was constructed in the STRING database and core targets were screened using topological analysis. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were conducted in R software. Finally, molecular docking was conducted to validate the results. Female ICR mice were used to establish a POF mouse model for in vivo experiments. Serum levels of relevant estrogens were determined using ELISA and expression levels of relevant proteins in ovarian tissues were detected using immunofluorescence and western blot analysis. Network pharmacology analysis predicted that PI3K, Akt, Bax, Bcl-2, p16, and other proteins were highly correlated with POF and RGE. The results clearly showed that RGE could increase estradiol (E2) and lower follicle-stimulating hormone (FSH) levels in D-gal-fed mice. RGE restored the expression levels of related proteins by reducing Nrf2-mediated oxidative stress, PI3K/Akt-mediated apoptosis, and senescence signaling pathways. Overall, RGE has the potential to prevent and treat POF and is likely to be a promising natural protector of the ovaries.

Taohong siwu decoction ameliorates cognitive dysfunction through SIRT6/ER stress pathway in Alzheimer's disease

Ethnopharmacological relevanceA growing number of people suffer from Alzheimer's disease (AD), but there is currently no effective treatment yet. Taohong Siwu Decoction (TSD) has been proved to take strong neuropharmacological activity on dementia, but the effect and mechanism of TSD against AD are still elusive. Aim of studyTo investigate whether TSD could be effective in ameliorating cognitive deficits through SIRT6/ER stress pathway. Materials and methodsHerein, the APP/PS1 mice, an AD model, and HT-22 cell lines were utilized. Different dosages of TSD (4.25, 8.50 and 17.00 g/kg/d) were administered to the mice for 10 weeks by gavage. Following the behavioral tests, oxidative stress levels were measured using malondialdehyde (MDA) and superoxide dismutase (SOD) kits. Nissl staining and Western blot analyses were used to detect the neuronal function. Then, immunofluorescence and Western blot analysis were applied to evaluate silent information regulator 6 (SIRT6) and ER Stress related protein levels in APP/PS1 mice and HT-22 cells. ResultsBehavioral tests revealed that APP/PS1 mice administered with TSD orally took more time in the target quadrant, crossed more times in the target quadrant, had a higher recognition coefficient, and spent more time in the central region. In addition, TSD could ameliorate oxidative stress and inhibit neuronal apoptosis in APP/PS1 mice. Furthermore, TSD could up-regulate the SIRT6 protein expression and inhibit ER sensing proteins expressions, such as p-PERK and ATF6, in APP/PS1 mice and Aβ1-42-treated HT22 cells. ConclusionAccording to the abovementioned findings, TSD could alleviate cognitive dysfunction in AD by modulating the SIRT6/ER stress pathway.