Expression Of Related Proteins Research Articles

Protective Effects of Dioscin and Diosgenin on Plateau Hyperuricemia by Attenuating Renal Inflammation via EPHX2

Plateau hyperuricemia is a common disease in the plateau area, and the incidence is much higher than that in the plain area. Dioscin (DIO) and its active metabolite Diosgenin (DG) exert therapeutic effects on hyperuricemia through oxidative stress and inflammation. In this study, DIO and its active metabolite DG were taken as the research objects to explore their therapeutic effects on high-altitude hyperuricemia in rats. To evaluate the therapeutic effect of DIO on the rat model of high-altitude hyperuricemia, the evaluation indexes include blood biochemical indexes, renal histopathology, oil red O staining of the kidney, rat kidney index, and rat renal inflammatory factors. Transcriptomics was used to analyze the control group, model group, and drug-administered group to preliminarily explore the protective mechanism of DIO in rats with high-altitude hyperuricemia. An HK-2 high-altitude hyperuricemia cell injury model was established to verify the therapeutic mechanism of DIO in rats with high-altitude hyperuricemia. Western blot was used to detect the expression of related proteins in renal tissues and cell models. The results showed that DIO and its active metabolite DG regulate renal lipid metabolism through the EPHX2 gene, attenuate renal inflammatory reaction, and then promote the excretion of uric acid and reduce its reabsorption, which ultimately achieves the effect of treating plateau hyperuricemia.

Skin as outermost immune organ of vertebrates that elicits robust early immune responses after immunization with glycoprotein of spring viraemia of carp virus.

As the outermost immune organ in vertebrates, the skin serves as the primary interface with the external environment and plays a crucial role in initiating the early immune response. The skin contains a variety of immune cells that induce mucosal and systemic immune responses, rendering it a prime target for vaccination strategies. Insight into the mechanisms through which vaccination triggers early immune responses is paramount for advancing animal and human health, yet our current understanding remains limited. Given its significance in vertebrate evolution, teleost fish emerges as an excellent model for investigating the early immune response of skin. In this study, we demonstrate that significant quantities of vaccine can be absorbed by the skin and transported to the body through dermis and muscle metabolism by immerses immune zebrafish with glycoprotein of spring viraemia of carp virus. Immersion immunization can elicit robust and enduring immune protection, with the skin triggering a potent immune response early in the immunization process. Analysis of the skin transcriptome revealed the involvement of numerous immune-related genes in the immersion immune response, with indications that HSP70 and MAPK signals might play pivotal roles in the immune process induced by glycoprotein. Co-immunoprecipitation and cell co-localization studies confirmed the interaction between glycoprotein and HSP70. Subsequent research demonstrated that overexpression or inhibition of HSP70 could respectively enhance or impede the expression of JNK and related proteins. However, the survival rate and immune response of HSP70 inhibited zebrafish with glycoprotein treatment were significantly reduced. These findings propose that the interaction between glycoprotein and HSP70 may activate JNK, thereby modulating mucosal and systemic immune responses induced by glycoprotein. This investigation offers novel insights and a foundational understanding of early skin immune reactions.

Aerobic exercise training attenuates ischemia-reperfusion injury in mice by decreasing the methylation level of METTL3-associated m6A RNA in cardiomyocytes.

Ischemic heart disease (IHD) presents a significant global health challenge, with myocardial ischemia-reperfusion injury (MIRI) being a major pathophysiological contributor and lacking effective interventions. While aerobic exercise training (AET) enhances cardiovascular health, its protective mechanism in MIRI remains elusive. This study aims to elucidate the protective effect of AET in MIRI and its underlying mechanism. A mouse model of AET and MIRI was established to evaluate basic indices, cardiac ultrasound, and myocardial injury markers. Dot Blot, qRT-PCR, and Western blot were employed to assess m6A RNA methylation levels and related protein expression in myocardial tissue. In vitro, primary cardiomyocyte culture was utilized to mimic MIRI, evaluating cell viability, mitochondrial membrane potential, etc. Finally, myocardial tissues of MIRI mice were immunoprecipitated for m6A RNA methylation and sequenced to analyze related signaling pathways. AET significantly improved cardiac function and mitigated myocardial injury and fibrosis. Moreover, AET protected myocardium from MIRI by reducing m6A RNA methylation levels and modulating METTL3 expression. In vitro experiments demonstrated that the decrease in m6A RNA methylation levels and METTL3 expression conferred resistance to hypoxia/reoxygenation-induced injury. Furthermore, sequencing results indicated elevated myocardial tissue m6A RNA methylation levels during MIRI, activation of the Nrf2-related signaling pathway, and AET-mediated regulation of the Nrf2/HO-1 signaling pathway, thereby attenuating MIRI through modulation of METTL3-related m6A methylation. AET attenuates MIRI by reducing the level of METTL3-related m6A RNA methylation in cardiomyocytes and activating the Nrf2/HO-1 antioxidant signaling pathway. This finding provides a novel insight and strategy for the prevention and treatment of IHD, holding significant clinical implications.

The Roles of Cytoplasmic Polyadenylation Element Binding Protein 1 in Tumorigenesis.

CPEB1 is an alternative polyadenylation binding protein that promotes or suppresses the expression of related mRNAs and proteins by binding to a highly conserved Cytoplasmic Polyadenylation Element (CPE) in the mRNAs 3'UTR. It is found to express abnormally in multiple tumors and affect tumorigenesis through many pathways. This review summarizes the functions and mechanisms of CPEB1 in a variety of cancers and suggests new directions for future related treatments. A total of 95 articles were eligible for inclusion based on the year, quality of the research, and the strength of association with CPEB1. In this review, current research about how CPEB1 affects the initiation and progression of glioblastoma, breast cancer, hepatocellular carcinoma, gastric cancer, colorectal cancer, non-small cell lung cancer, prostate cancer, and melanoma are dissected, and the biomedical functions and mechanisms are summarized. CPEB1 mostly presents as a tumor suppressor for breast cancer, endometrial carcinoma, hepatocellular carcinoma, non-small cell lung cancer, prostate cancer, and melanoma. However, for glioblastoma, gastric cancer, and colorectal cancer, CPEB1 exhibts two opposing properties of tumorigenesis, either promoting or inhibiting it. CPEB1 is likely to serve as a target and dynamic detection index or prognostic indicator for its function of apoptosis, activity, proliferation, migration, invasion, stemness, drug resistance, and even ferroptosis in various cancers.

Lactic acid bacteria reduce polystyrene micro- and nanoplastics-induced toxicity through their bio-binding capacity and gut environment repair ability

Microplastics and nanoplastics (MNPs) are emerging environmental contaminants that have received significant attention in recent years. Currently, there are more studies on the toxic effects of MNPs exposure on animals (especially aquatic organisms and mammals), but data on the reduction of toxic effects caused by MNPs exposure are still very limited. Lactic acid bacteria (LAB), recognized as safe food-grade microorganisms, possess the capability to bioconjugate harmful substances. In this experiment, we chose lactic acid bacteria (LAB) with different binding capacities to MNPs in vitro to intervene in MNPs-exposed mice to investigate the reducing effect on the toxicity caused by MNPs exposure. Our study showed that LAB with a high intercalation capacity with MNPs in vitro were more effective in alleviating the toxicity caused by MNPs exposure. Notably, Lactobacillus plantarum DT22, despite its low inter-adsorption with MNPs, played a pivotal role in upregulating the relative expression of tight junction proteins and modulating the intestinal microbiota. Thus, LAB strains' mitigation of MNPs toxicity extends beyond bio-binding; their capacity to repair the damaged gut environment is also crucial. LAB strains are proposed as a dietary intervention to reduce MNPs-induced toxicity.

Effect of Curcumin-Mediated miR-449 on Diabetic Nephropathy

This study assessed the mechanism for curcumin-mediated miR-449 regulating PACS-2 in diabetic nephropathy under nursing intervention. A diabetic nephropathy model was constructed, in which rats were fasted for 12 hours before experiments and injected with Streptozotocin (STZ) at a ratio of 45–65 mg/kg (30–60 mg STZ). When the rats aged 8 weeks, they were given intraperitoneal injection of curcumin and tissue histological morphology was detected to ensure successful model construction. HE staining observed the morphological changes in renal tissue, while immunohistochemistry detected the expression of PACS-2 and PACS-2 related proteins. Nursing intervention could reduce serum creatinine, fasting blood glucose, urea nitrogen and proteinuria levels. After nursing intervention, the levels of IL-6, hs-CRP and TNF-α in patients were significantly decreased (p < 0.05), and miR-499 expression increased after nursing intervention. After curcumin treatment, the red staining of entire kidney cytoplasm in the control and experimental groups became lighter. Moreover, the expression of miR-449 in the kidney tissue was significantly increased after curcumin treatment. Since PACS-2 plays an important role in kidney injury, curcumin largely normalized kidney injury as shown by serum BUN levels after curcumin treatment in the rat model. Turmeric, a traditional Chinese medicine, has a bitter taste and warm nature. It might facilitate blood circulation and remove blood stasis, and relieving chest pain and heartache. Curcumin as the main active ingredient of turmeric, has been proven to inhibit the abnormal proliferation of diabetic nephropathy. Under the influence of effective nursing care, the patient’s physical condition was improved, which was mainly reflected in the related inflammatory indicators. Studies have shown that fibronectin, which is an important indicator for PACS-2, plays a decisive role in fibrosis. Our testing found that, curcumin significantly reduced the expression of PACS-2 during treatment. Nursing intervention can therefore effectively improve inflammatory response and renal function of diabetic nephropathy through curcumin-mediated miR-449 regulation of PACS-2.

TGF-β3 Restrains Osteoclastic Resorption Through Autophagy

While TGF-β3 promoted defect healing in a primate baboon skull defect model and patients, it remains unclear whether TGF-β3 affects the formation of osteoclasts and bone resorption between osteogenesis and osteolysis. Analysis of the full transcriptome of hPDLSCs (human periodontal ligament stem cells) revealed that the expression of RANKL was significantly up-regulated after TGF-β3 treatment during osteogenesis, which suggests its involvement in clock-controlled autophagy in bone metabolism. TRAP staining and bone resorption lacunae were used to assess the osteoclasts formed from RANKL-induced differentiated BMMs. During osteoclast differentiation, the characteristics of autophagy regulated by TGF-β3 were observed in BMMs through MDC staining, transmission electron microscopy, and LC3 immunofluorescence. The expression of related genes and proteins were detected on the sixth day in mCherry-EGFP-LC3B lentivirus-transfected BMMs using RT-qPCR and WB. Finally, a trans-well co-culture system was used to evaluate the effects of osteogenic differentiated hPDLSCs treated with TGF-β3 on the osteoclastic differentiation of BMMs. The results showed 10 ng/mL of TGF-β3 significantly suppressed osteoclastic differentiation and bone resorption in BMMs (p < 0.05 vs. RANKL). In particular, TGF-β3 augments the expression of LC3-II to stimulate autophagy, consequently restraining osteoclastic resorption. These findings provide a molecular basis and are beneficial to illustrate the potential druggability of TGF-β3 in osteoporotic diseases.

CCL2-CCR2 Axis Inhibition in Osteosarcoma Cell Model: The Impact of Oxygen Level on Cell Phenotype.

Treatment of osteosarcoma is hampered by tumor hypoxia and requires alternative approaches. Although the CCL2-CCR2 axis is indispensable in tumor-induced inflammation and angiogenesis, its blockade has not been effective to date. This study aimed to characterize how CCR2 inhibition affects the crosstalk of osteosarcoma cells with immune cells to better delineate tumor resistance mechanisms that help withstand such treatment. In this study, 143B cells were exposed to healthy donor PBMC supernatants in a transwell assay lacking direct cell-to-cell contact and subjected to different oxygen concentrations. In addition, mice bearing orthotopic 143B tumors were subjected to CCR2 antagonist treatment. Our findings show that hypoxic conditions alter cytokine and cancer- related protein expression on cells and impair CCR2 antagonist effects in the experimental osteosarcoma model. CCL2-CCR2 axis blockade in the 143B xenografts, which are positive for hypoxia marker CAIX, did not slow 143B tumor growth or metastasis but altered tumor microenvironment by VEGFR downregulation and shift in the CD44-positive cell population towards high CD44 expression. This study highlights differential responses of tumor cells to CCR2 antagonists in the presence of different oxygen saturations and expands our knowledge of compensatory mechanisms leading to CCL2-CCR2 treatment resistance.

Transglutaminase 2 regulates endothelial cell calcification via IL-6-mediated autophagy.

Endothelial cell (EC) calcification is an important marker of atherosclerotic calcification. ECs play a critical role not only in atherogenesis but also in intimal calcification, as they have been postulated to serve as a source of osteoprogenitor cells that initiate this process. While the role of transglutaminase 2 (TG2) in cellular differentiation, survival, apoptosis, autophagy, and cell adhesion is well established, the mechanism underlying the TG2-mediated regulation of EC calcification is yet to be fully elucidated. The TG2 gene was overexpressed or silenced by using siRNA and recombinant adenovirus. RT-PCR and WB were used to analyze the relative expression of target genes and proteins. 5-BP method analyzed TG2 activity. mCherry-eGFP-LC3 adenovirus and transmission electron microscopy analyzed EC autophagy level. Calcium concentrations were measured by using a calcium colorimetric assay kit. Alizarin red S staining assay analyzed EC calcification level. Elisa analyzed IL-6 level. Establishing EC calcification model by using a calcification medium (CM). Our findings demonstrated that CM increased TG2 activity and expression, which activated the NF-κB signaling pathway, and induced IL-6 autocrine signaling in ECs. Furthermore, IL-6 activated the JAK2/STAT3 signaling pathway to suppress cell autophagy and promoted ECs calcification. ECs are not only critical for atherogenesis but also believed to be a source of osteoprogenitor cells that initiate intimal calcification. Previous research has shown that TG2 plays an important role in the development of VC, but the mechanism by which it exerts this effect is not yet fully understood. Our results demonstrated that TG2 forms complexes with NF-κB components inhibition of autophagy promoted endothelial cell calcification through EndMT. Therefore, our research investigated the molecular mechanism of EC calcification, which can provide new insights into the pathogenesis of atherosclerosis.

Electroacupuncture of "Jiaji" (EX-B2) inhibits inflammatory response by regulating HMGB1/TLR4/NF-κB signaling pathway in rats with spinal cord injury

To observe the effect of electroacupuncture (EA) of "Jiaji" (EX-B2) on expression of high mobility group box 1 (HMGB1)/Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) related proteins and inflammation-related factors in spinal cord injury (SCI) rats, so as to explore the dynamic process of inhibiting inflammatory response in rats with SCI, and to provide a new idea and theoretical basis of molecular biology for the treatment of SCI. Fifty-four SD rats were randomly divided into sham operation, model, and EA groups (n=18 in each group), which were further divided into 3 d, 7 d and 14 d subgroups, with 6 rats at each time point. The SCI model was established according to the Allen's method. Rats in EA group received EA (1 mA, 100 Hz) intervention at EX-B2 of T9 and T11 24 hours after modeling for 30 min, once a day for 3 d, 7 d, or 14 d, respectively. The motor function of rats' hind limbs was evaluated using BBB scale, the morphological structure of rats' spinal cord tissue was observed by H.E. staining. The contents of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in spinal cord tissue were detected by ELISA. Western blot and immunofluorescence staining were used to detect the relative protein expression and fluorescence positive expression of HMGB1, TLR4 and NF-κB p65 in spinal cord tissue, respectively. Compared with the sham operation group, the BBB scores after modeling and on day 3, 7 and 14 were all decreased (P<0.05), while the contents of iNOS and COX-2 in spinal cord tissue, the protein expression and fluorescence positive expression of HMGB1, TLR4, and NF-κB p65 were increased (P<0.05) in the model group. In comparison with the model group, the BBB scores on day 3, 7 and 14 were obviously increased (P<0.05), while the contents of iNOS and COX-2, the protein expression and fluorescence positive expression of HMGB1, TLR4, and NF-κB p65 were significantly down-regulated in the EA group (P<0.05). EA of "Jiaji" can promote the recovery of SCI neurological function, which may be related to its function in regulating the HMGB1/TLR4/NF-κB signaling pathway, down-regulating the expression of iNOS and COX-2, and inhibiting the neuroinflammatory response after SCI.

Correlation analysis of AVPR1a and AVPR2 with abnormal water and sodium and potassium metabolism in rats.

In clinical practice, an increasing number of patients exhibit concurrent cardiac and renal dysfunction, known as "cardiorenal syndrome," where each condition exacerbates the other, resulting in poorer patient prognosis. Fluid and sodium retention can lead to excessive fluid overload in the body; therefore, correcting fluid and sodium metabolic disorders is crucial for alleviating patient symptoms. This study was to investigate the abnormalities in water and sodium metabolism, as well as the expression levels of arginine vasopressin receptor 1a (AVPR1a) and arginine vasopressin receptor 2 (AVPR2), in a rat model of chronic renal failure-chronic heart failure (CRF-CHF). One hundred male Sprague-Dawley rats were randomly assigned into four groups: the CG group (normal feeding), the CRF group (3/4 nephrectomy using a "two-step surgical method"), the CHF group (subcutaneous injection of isoproterenol at 100 mg/kg), and the CRF-CHF group (3/4 nephrectomy followed by a subcutaneous injection of isoproterenol at 100 mg/kg 1 week later). 4 weeks post-surgery, urine and blood samples were collected to measure 24 h urinary protein, sodium, and potassium levels. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were determined using assay kits. Left ventricular end diastolic pressure (LVEDP) and left ventricular systolic pressure (LVSP) were measured via left ventricular catheterization. The heart was weighed to calculate the left ventricular weight to body weight ratio (LVW/BW). The renal cortex and medulla were isolated to assess the relative mRNA and protein expression levels of AVPR1a and AVPR2. Compared to the CG group, the CRF and CRF-CHF groups exhibited significantly elevated levels of 24 h urinary protein, SCr, BUN, and relative expression levels of AVPR1a and AVPR2 in the renal cortex and medulla. The CHF and CRF-CHF groups showed significant increases in LVEDP and LVW/BW (P < 0.05). Additionally, compared to the CG group, the other three groups had significantly increased urinary sodium and blood potassium levels, and significantly decreased urinary potassium and blood sodium levels (P < 0.05). Compared to the CRF and CHF groups, the CRF-CHF group exhibited significantly higher levels of 24 h urinary protein, SCr, BUN, and relative expression levels of AVPR1a and AVPR2 in the renal cortex and medulla, along with significantly increased LVEDP and LVW/BW, significantly reduced LVSP, significantly increased urinary sodium and blood potassium levels, and significantly decreased urinary potassium and blood sodium levels (P < 0.05). Rats with CRF-CHF experienced exacerbated renal and cardiac failure, characterized by significant disturbances in water and sodium metabolism and abnormal expression of AVPR1a and AVPR2.

MiR-214-3p inhibits LPS-induced macrophage inflammation and attenuates the progression of dry eye syndrome by regulating ferroptosis in cells.

Dry eye disease (DED) is an ocular illness caused by insufficient tear secretion or poor tear quality, and inflammation is a key factor in its pathogenesis. Previous studies have shown that miRNAs are important regulatory factors in DED. The purpose of this study was to explore the potential mechanism by which miR-214-3p influenced the DED process by regulating the macrophage inflammatory response. We induced THP-1 cells to differentiate into M0 macrophages with 100 ng/mL phorbol-12-myristate-13-acetate (PMA) and then added 15 ng/mL lipopolysaccharide (LPS) to induce inflammation. The expression of related genes and proteins was detected via RT‒qPCR, Western blotting, ELISA and immunofluorescence staining; cell viability was measured using the CCK-8 assay; and flow cytometry was used to detect ROS levels. In tear and serum samples from DED patients, the levels of miR-214-3p, IL-10, and Arg1 were decreased, and the levels of IL-6, TNF-α, IL-1β, and iNOS expression were increased. Moreover, the overexpression of miR-214-3p attenuated the effect of LPS and inhibited M1 polarization and inflammation in macrophages. Mechanistically, miR-214-3p inhibited macrophage ferroptosis by downregulating TFRC expression, thereby inhibiting macrophage M1 polarization and inflammation and alleviating the progression of DED. Our study indicated that the upregulation of miR-214-3p expression might be a new target for DED therapy.

The impact of ulinastatin on lymphocyte apoptosis and autophagy in sepsis patients

This study aimed to assess the influence of ulinastatin (UTI) on lymphocyte apoptosis and autophagy in sepsis patients, as well as its effect on inflammatory factors and vital organ function, with the goal of providing insights for improved clinical management of sepsis. A total of 40 sepsis patients were randomly assigned to the UTI group or the control group. The UTI group received standard treatment plus intravenous UTI, while the control group received standard treatment alone. Peripheral blood samples were collected at multiple time points for analysis of lymphocyte apoptosis, autophagy, inflammatory markers, and organ function. Various experimental techniques including Hoechst staining, transmission electron microscopy, and Western blot analysis were utilized to assess lymphocyte apoptosis, autophagy, and related protein expression levels. The study revealed that UTI treatment significantly inhibited lymphocyte apoptosis and promoted autophagy in sepsis patients. The levels of autophagy-related proteins LC3-II and Beclin-1 were substantially elevated, while the ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax was increased following UTI treatment. Furthermore, the levels of inflammatory markers IL-6, procalcitonin, and C-reactive protein were markedly reduced in the UTI group compared to the control group. Additionally, UTI treatment led to improved liver, kidney and cardiac function as evidenced by reduced levels of liver enzymes and creatinine, and cardiac enzymes. The findings of this study demonstrate that UTI exerts a protective effect on septic patients by inhibiting lymphocyte apoptosis, promoting autophagy, and attenuating systemic inflammation. Moreover, UTI treatment was associated with improved liver, kidney, and cardiac function in septic patients. These results contribute to a better understanding of the clinical management of sepsis and underscore the potential of UTI as a therapeutic intervention in septic patients.

Abstract A030: Polycomb repressive complex 2 (PRC2) mediates the epigenetic regulation of cancer-associated fibroblasts in esophageal cancer

Abstract Cancer-associated fibroblasts (CAFs) are a heterogenous cell population that can promote esophageal cancer progression through multiple molecular mechanisms including increased cancer cell proliferation and migration, immunosuppression, and therapeutic resistance. Despite the divergence of CAFs from normal fibroblasts with regards to phenotype and functions, CAFs rarely exhibit widespread genetic alterations. Instead, it is proposed that CAFs are regulated at the epigenetic level. Our objective is to delineate this epigenetic regulation of CAFs in esophageal cancer. Ultimately, we aim to identify the epigenetic regulatory complexes that mediate CAF heterogeneity and pro-tumorigenic functions. To this end, we completed RNA-seq and ATAC-seq on fibroblasts isolated from esophageal squamous cell carcinoma (ESCC), esophageal adenocarcinoma (EAC) and non-cancerous esophageal tissue. Enrichment analysis identified the polycomb repressive complex 2 (PRC2) as a potential regulator of ESCC and EAC CAFs. PRC2 catalyzes the methylation (mono-, di- or tri-) of lysine 27 on histone 3 (H3K27me1/2/3) resulting in transcriptional repression. Inhibition of two key proteins of PRC2 (EZH2 and SUZ12) through either pharmacological or genetic modulation, altered expression of multiple CAF phenotype related proteins and inhibited CAF functions including CAF proliferation. Ongoing studies, using co-cultures of fibroblasts and tumor cells, seek to identify if inhibition of PRC2 can prevent the initial transition of normal esophageal fibroblasts into CAFs. Importantly, we are conducting in vivo studies to determine if selective PRC2 inhibition in CAFs can inhibit esophageal tumor progression with specific focus on primary tumor growth and metastatic spread. In summary, initial studies have identify that PRC2 complex mediates CAF phenotype and functions, and we aim to establish the larger impact of these CAF specific effects on esophageal cancer. Citation Format: Karen J. Dunbar, Raul Navaridas Fernandez de Bobadil, Katherine M. Cunningham, Emily Esquea, Gizem Efe, Anil K. Rustgi. Polycomb repressive complex 2 (PRC2) mediates the epigenetic regulation of cancer-associated fibroblasts in esophageal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A030.

Design, synthesis, and antiproliferative activity evaluation of novel α-mangostin derivatives by ROS/MAPK signaling pathway

Novel hydroxamic acid and 3,6-amide modified α-mangostin derivatives were synthesized and evaluated their antiproliferative activities against KYSE 30 (esophageal cancer), HCT 116 (colon cancer), and HGC 27 (gastric cancer) cell lines. Most of the new derivatives displayed stronger anti-proliferative activities compared to α-mangostin. Among all the derivatives, compound 4a exhibited the most potent activity, with IC50 values of 0.57 ± 0.29 μM, 3.27 ± 0.16 μM, and 2.28 ± 1.02 μM against KYSE 30, HCT 116, and HGC 27 cells, respectively. Subsequent mechanism studies revealed that compound 4a inhibited cancer cells proliferation and colonies formation in a concentration-dependent manner. Additionally, compound 4a caused cell cycle arrest in a p53 dependent manner and induced apoptosis in p53 independent way. Meanwhile, 4a suppressed cell cycle related proteins (Cyclin D1 and cyclin B1) expression, increased pro-apoptotic proteins (cleaved PARP, cleaved caspase-7, and cleaved caspase-9) and decreased anti-apoptotic proteins (Bcl-2) expression. Moreover, 4a increased reactive oxygen species (ROS) levels in KYSE 30 cells and upregulated the expression of proteins related to the ROS related MAPK signaling pathway (p-ERK, p-p38, and p-JNK). These findings suggest that compound 4a holds promising potential as an antiproliferative agent by targeting MAPK signaling pathway to inhibit cell cycle progress, induce apoptosis and produce ROS in cancers.

Study on anti-adhesion effect and mechanism of dynamic and static stress stimulation during early healing process of rat Achilles tendon injury

To investigate the anti-adhesive effect and underlying mechanism of dynamic and static stress stimulation on the early healing process of rat Achilles tendon injury. Achilles tendon tissues of 15 male Sprague Dawley (SD) rats aged 4-6 weeks were isolated and cultured by enzyme digestion method. Rat Achilles tendon cells were treated with tumor necrosis factor α to construct the Achilles tendon injury cell model, and dynamic stress stimulation (dynamic group) and static stress stimulation (static group) were applied respectively, while the control group was not treated. Live/dead cell double staining was used to detect cell activity, ELISA assay was used to detect the expression of α smooth muscle actin (α-SMA), and real-time fluorescence quantitative PCR was used to detect the mRNA expression of collagen type Ⅰ (COL1A1), collagen type Ⅲ (COL3A1), and Scleraxis (SCX). Thirty male SD rats aged 4-6 weeks underwent Achilles tendon suture and were randomly divided into dynamic group (treated by dynamic stress stimulation), static group (treated by static stress stimulation), and control group (untreated), with 10 rats in each group. HE staining and scoring were performed to evaluate the healing of Achilles tendon at 8 days after operation. COL1A1 and COL3A1 protein expressions were detected by immunohistochemical staining, α-SMA and SCX protein expressions were detected by Western blot, and maximum tendon breaking force and tendon stiffness were detected by biomechanical stretching test. In vitro cell experiment, when compared to the static group, the number of living cells in the dynamic group was higher, the expression of α-SMA protein was decreased, the relative expression of COL3A1 mRNA was decreased, and the relative expression of SCX mRNA was increased, and the differences were all significant ( P<0.05). In the in vivo animal experiment, when compared to the static group, the tendon healing in the dynamic group was better, the HE staining score was lower, the expression of COL1A1 protein was increased, the expression of COL3A1 protein was decreased, the relative expression of SCX protein was increased, the relative expression of α-SMA protein was decreased, and the tendon stiffness was increased, the differences were all significant ( P<0.05). Compared with static stress stimulation, the dynamic stress stimulation improves the fibrosis of the scar tissue of the rat Achilles tendon, promote the recovery of the biomechanical property of the Achilles tendon, and has obvious anti-adhesion effect.

Aβ1-42 promotes microglial activation and apoptosis in the progression of AD by binding to TLR4

Alzheimer's disease (AD) is one of the most common age-related neurodegenerative diseases and the most devastating form of senile dementia. It has a complex mechanism and no effective treatment. Exploring the pathogenesis of AD and providing ideas for treatment can effectively improve the prognosis of AD. Microglia were incubated with β-amyloid protein 1-42 (Aβ1-42) to construct an AD cell model. After microglia were activated, cell morphology changed, the expression level of inflammatory factors increased, cell apoptosis was promoted, and the expression of microtubule-associated protein (Tau protein) and related proteins increased. By up-regulating and down-regulating Toll-like receptor 4 (TLR4), the cells were divided into TLR4 knockdown negative control group(Lv-NC group), TLR4 knockdown group(Lv-TLR4 group), TLR4 overexpression negative control group(Sh-NC group), and TLR4 overexpression group(Sh-TLR4 group). The expression of inflammatory factors was detected again. It was found that compared with the Lv-NC group, the expression of various inflammatory factors in the Lv-TLR4 group decreased, cell apoptosis was inhibited, and the expression of Tau protein and related proteins decreased. Compared with the Sh-NC group, the expression of inflammatory factors in the Sh-TLR4 group increased, cell apoptosis was promoted, and the expression of Tau protein and related proteins increased. These results indicate that Aβ1-42 may promote microglial activation and apoptosis by binding to TLR4. Reducing the expression of TLR4 can reduce the occurrence of inflammatory response in AD cells and slow down cell apoptosis. Therefore, TLR4 is expected to become a new target for the prevention and treatment of AD.

Asiatic Acid Alleviates LPS-Induced Pyroptosis and Endoplasmic Reticulum Stress-Mediated Apoptosis via Inhibiting the HMGB1/TLR4/NF-κB Pathway in Broiler Hepatocytes.

Lipopolysaccharides (LPS) is the major compoent of Gram-negative bacteria and an important factor in inducing inflammation, which usually leads to multiple organ failure in broilers, seriously affecting the growth performance of broilers and hindering the development of poultry farming. Under the policy of prohibiting antibiotics in feed, it has become more urgent to find natural drugs to prevent liver damage caused by LPS in broilers. Asiatic acid (AA) is a pentacyclic triterpene that has been proven to have anti-inflammatory and antioxidant effects. However, the protective effects of AA in LPS-induced liver damage in broilers still need to be clarified. This study aims to explore the complete mechanism of AA against LPS-induced acute liver injury (ALI) in broilers. A total of 60 broilers (1 day old) were randomly divided into the control group, LPS group, LPS+AA (15, 30 and 60 mg/kg) group and control+AA (60 mg/kg) group. At 16, 18 and 20 days of age, the broilers were attacked with LPS (0.5 mg/kg) to construct liver injury model. H&E staining assessed liver pathological changes. The mRNA and protein expression levels related tothe HMGB1/TLR4/NF-κB pathway, pyroptosis, and ERS-mediated apoptosis in the liver tissue were detected. Our results founded that intraperitoneal injection of LPS in broilers increased the activities of AST and ALT, as well as raising the related gene and protein expression of the HMGB1/TLR4/NF-κB pathway, pyroptosis, and ERS-mediated apoptosis. Interestingly, AA improved LPS-induced liver damage and decreased the activities of AST and ALT in broilers. Additionally, AA mitigated LPS-induced ALI by reducing the mRNA levels and protein expressions of the HMGB1/TLR4/NF-κB pathway, pyroptosis and ERS-mediated apoptosis. In conclusion, the present study investigated that AA mitigated LPS-induced ALI in broilers by reducing pyroptosis and ERS-mediated apoptosis via inhibition of the HMGB1/TLR4/NF-κB pathway. Therefore, AA may serve as a potential feed additive for the prevention of LPS-induced ALI in broilers.

Anti-inflammatory role of low-intensity pulsed ultrasound in inhibiting lipopolysaccharide-induced M1 polarization of RAW264.7 cells via Wnt2b/AXIN/β-catenin.

Low-intensity pulsed ultrasound (LIPUS) is a special type of low-intensity ultrasound. In periodontal disease, LIPUS is applied as an adjuvant and non-invasive treatment. It has been reported that LIPUS significantly shifts the macrophage phenotype from M1 to M2, but the specific mechanism behind this shift is still unknown. RAW264.7 cells were induced to M1/M2 polarization with lipopolysaccharide (LPS)/interleukin-4 (IL4). LIPUS was performed for 25 min two times, 24 h apart, at an intensity of 45 mW/cm2 to stimulate RAW264.7 cells. PolyA mRNA sequencing was conducted of both the LPS-induced RAW264.7 cells and the LPS-induced RAW264.7 cells with LIPUS treatment. The expression of Wnt2b in RAW264.7 cells was downregulated by siRNA. The macrophage surface markers and downstream inflammatory cytokines were detected using flow cytometry. The relative expression of proteins in the Wnt2b/AXIN/β-catenin pathway was assessed using reverse transcription real-time polymerase chain reaction (RT-qPCR) and Western blot. LIPUS reversed the M1 polarization of RAW264.7 cells, with decreased expression of CD80 and CD86. In addition, LIPUS enhanced the M2 polarization of RAW264.7 cells, with upregulated expression of CD163 and CD206. The polyA mRNA sequencing results indicated that the Wnt signaling pathway participated in the M1 polarization of LIPUS-treated RAW264.7. The results of the RT-qPCR showed a higher expression of Wnt2b in LIPUS-treated and M1- or M2-polarized RAW264.7 cells. Knocking down Wnt2b was shown to reverse the inhibitory effect of LIPUS on M1 polarization and increase the expression of CD80 and CD86. Wnt2b knockdown also regulated downstream AXIN, β-catenin, and inflammatory factors such as tumor necrosis factor alpha (TNFα) and interleukin-6 (IL6). LIPUS plays an anti-inflammatory role by inhibiting LPS-induced M1 polarization of RAW264.7 cells in a Wnt2b/AXIN/β-catenin-dependent way. LIPUS may play a therapeutic role in periodontal diseases by inhibiting inflammation through the regulation of macrophage differentiation.

Comparison of the protein composition of isolated extracellular vesicles from mouse brain and dissociated brain cell culture medium.

Extracellular vesicles (EVs) play a crucial role in intercellular communication. Characterizing EV protein composition is essential to understand EV function(s). Isolating EVs from cell culture medium is a common approach to study EVs, but it remains unclear whether EVs isolated from in vitro conditions accurately reflect physiological conditions of the same source in vivo tissues. Here, we analyzed the protein composition of EVs isolated from freshly dissected mouse forebrain and primary dissociated mouse forebrain culture medium. In total, 3,204 and 3,583 proteins were identified in EVs isolated in vivo and in vitro, respectively. Among the proteins identified from both EV sources, there was substantial overlap (~86%). While the overall proteome compositions were very similar, in vitro EVs were relatively enriched with transmembrane/GPI-anchored membrane and cytosolic proteins (MISEV2023 category 1 and 2) typically associated with EVs. Conversely, while both in vivo and in vitro EVs express likely non-EV proteins (MISEV2023 category 3), the in vivo samples were significantly more enriched with these probable contaminants, specifically ribosomal proteins. Our findings highlight that in vitro EVs may be representative of in vivo EVs when isolated from the same source tissue using similar methodology; however, each population of EVs have differences in both total and, primarily, relative protein expression likely due to differing levels of co-eluting contaminants. Therefore, these points must be considered when interpreting results of EV studies further suggesting that improved methods of isolation to reduce non-EV contaminants should be further investigated.