Protein Expression Levels Research Articles

Targeting TRPV1 signaling: Galangin improves ethanol-induced gastric mucosal injury

Galangin, a bioactive compound extracted from Alpinia officinarum Hance (Zingiberaceae), a plant with significant ethnopharmacological importance, has been used for thousands of years as a spice, condiment, and medicinal agent for various conditions, including gastrointestinal disorders. Although there is evidence suggesting its potential to improve gastric ulcers, the molecular mechanisms underlying its anti-ulcer properties are not fully understood. of the Study: This study aimed to investigate the effects of galangin on ethanol-induced acute gastric mucosal injury (AGMI) in mice and elucidate its molecular mechanisms. Sixty BALB/c mice were randomly assigned into two main groups: a normal control group (n=10) and an ethanol-induced group (n=50). After establishing the AGMI model in mice using a combination of 40% ethanol and anhydrous ethanol, the ethanol-induced group was further subdivided into five subgroups (n=10): an omeprazole control group (20mg/kg), an untreated ethanol group, and three treatment groups receiving high-dose (50mg/kg) or low-dose (25mg/kg) galangin or capsazepine (CPZ, 2mg/kg). The protective effects of galangin were evaluated through mucosal injury indices, hematoxylin and eosin staining, and quantification of inflammatory markers (IL-1β, IL-6, IL-8, and TNF-α). Oxidative stress levels and matrix metalloproteinase activity were measured using specific assay kits. Molecular docking was conducted to assess the binding affinity of galangin to key proteins within the transient receptor potential vanilloid 1 (TRPV1) pathway. Real-time fluorescence quantitative PCR (qPCR) was used to determine mRNA expression levels of TRPV1, calmodulin (CaM), substance P (SP), and CGRP in gastric tissues. Protein expression levels of TRPV1, nerve growth factor (NGF), tropomyosin receptor kinase A (TRKA), transforming growth factor beta (TGF-β), cyclooxygenase-2 (COX-2), and nuclear factor kappa B (NF-κB) were assessed through Western blot analysis. In cellular experiments, Culture of Human Gastric Epithelial Cells (GES-1) were treated with various concentrations of galangin after 7% ethanol induction. Cell proliferation, apoptosis, and migration were evaluated using Hoechst 33258 staining and transwell migration assays. TRPV1 protein expression was detected using immunofluorescence, and the expression levels of Bcl-2, BCL2-Associated X (BAX), and Caspase-3 were quantified by qPCR. Additionally, specific probe kits were used to measure intracellular calcium ions (Ca2+) and mitochondrial membrane potential. The findings indicate that galangin significantly improved mucosal pathology by reducing ulcer indices and inflammatory levels, while enhancing superoxide dismutase (SOD) activity and decreasing malondialdehyde (MDA) concentration. Galangin also reduced matrix metalloproteinase-2 (MMP-2), m metalloproteinase-9 (MMP-9) levels, promoting mucosal repair. At the cellular level, galangin decreased intracellular calcium ion concentration and mitigated the decline in mitochondrial membrane potential, enhance the restoration of mucosal cells, increased migration and proliferation, and reduced apoptosis. Molecularly, galangin demonstrated favorable binding to TRPV1, NGF, TRKA, TGF-β, COX-2, and NF-κB, and reversed the elevated expression of these proteins. Additionally, galangin downregulated the mRNA expression of TRPV1, CaM, SP, CGRP, BAX, and Caspase-3 in gastric tissues/cells, while upregulating Bcl-2 mRNA expression. Galangin mitigates AGMI by inhibiting the overactivation of the TRPV1 pathway, thereby blocking aberrant signal transduction. This study suggests that galangin has therapeutic potential against ethanol-induced AGMI and may be a viable alternative for the treatment of alcohol-induced gastric mucosal injuries.

Botulinum toxin combined with static progressive stretching improves fibrous stiffness of knee joint in rats through TGF-β1/Smad pathway.

Joint stiffness and fibrosis are common complications that affect mobility and quality of life, necessitating effective therapeutic strategies to alleviate these issues. The study aimed to observe the therapeutic effect of static progressive stretching (SPS) combined with botulinum toxin type A (BTX-A) on knee joint stiffness in rats and its effect on the transforming growth factor beta 1 (TGF-β1)/small mother against decapentaplegic (Smad) pathway in the development of joint capsule fibrosis. Forty Sprague Dawley rats were randomly divided into the blank control group, model control group, SPS intervention group, BTX-A intervention group, and SPS combined with BTX-A intervention group. Except for the blank control group, the right knee joints of the other rats were surgically fixed with Kirschner wire internal immobilization in full flexion for four weeks to form joint flexion contracture and cause fibrotic stiffness of the joint. The therapeutic effect of each intervention was assessed by the range of motion (ROM) of the knee joint, joint stiffness, the number of total cells, and collagen deposition in the posterior joint capsule, as well as the protein level expressions of TGF-β1, Smad2, Smad3, Smad4, p-Smad2/3, collagen I and III, and alpha smooth muscle actin (α-SMA) in the posterior joint capsule in the TGF-β1/Smad pathway. SPS combined with BTX-A was more effective in relieving joint fibrosis stiffness, improving the histopathological changes in the posterior joint capsule, and suppressing the high expression of target proteins and the overactivated TGF-β1/Smad pathway. The overactivated TGF-β1/Smad pathway was involved in the formation of knee joint fibrosis stiffness in rats. SPS combined with BTX-A was effective in relieving joint flexion contracture and fibrosis of the joint capsule. Moreover, the inhibition of the overactivated TGF-β1/Smad pathway may be the potential molecular mechanism for its therapeutic effect.

Effect of deletion of protein 4.1R on proliferation, apoptosis and glycolysis of hepatocyte HL-7702 cells

To explore the effects of deletion of protein 4.1R on hepatocyte proliferation, apoptosis, and glycolysis and the molecular mechanisms. A 4.1R-/- HL-7702 cell line was constructed using CRISPR/Cas9 technique, and with 4.1R+/+HL-7702 cells as the control, its proliferative capacity and cell apoptosis were assessed using CCK-8 assay, EdU-488 staining, flow cytometry and Annexin V-FITC/PI staining at 24, 48, 72 h of cell culture. The changes in glucose uptake, lactate secretion, ATP production and pH value of the culture supernatant of 4.1R-/- HL-7702 cells were determined. The mRNA expressions of the key regulatory enzymes HK2, PFKL, PKM2 and LDHA in glycolysis were detected with qRT-PCR, and the protein expressions of AMPK, p-AMPK, Raptor and p-Raptor were determined using Western blotting. Western blotting and sequencing analysis both confirmed the successful construction of 4.1R-/- HL-7702 cell line. Compared with the wild-type cells, 4.1R-/- HL-7702 cells exhibited a lowered proliferative activity with increased cell apoptosis. The deletion of protein 4.1R also resulted in significantly decreased glucose uptake, lactate secretion and ATP production of the cells and increased pH value of the cell culture supernatant. qRT-PCR showed significantly decreased mRNA expressions of the key regulatory enzymes in glycolysis in 4.1R-/- HL-7702 cells. Compared with those in HL-7702 cells, the expression levels of AMPK and Raptor proteins were decreased while the expression levels of p-AMPK and p-Raptor proteins increased significantly in 4.1R-/- HL-7702 cells. Deletion of protein 4.1R in HL-7702 cells results in reduced proliferative capacity, increased apoptosis and suppression of glycolysis, and this regulatory mechanism is closely related with the activation of the downstream AMPK-mTORC1 signaling pathway.

Naturally-derived phenethyl isothiocyanate modulates apoptotic induction through regulation of the intrinsic cascade and resulting apoptosome formation in human malignant melanoma cells

Malignant melanoma is the most aggressive type of skin cancer with increasing incidence rates worldwide. On the other hand, watercress is a rich source of phenethyl isothiocyanate (PEITC), among others, which has been widely investigated for its anticancer properties against various cancers. In the present study, we evaluated the role of a watercress extract in modulating apoptotic induction in an in vitro model of human malignant melanoma consisting of melanoma (A375, COLO-679, COLO-800), non-melanoma epidermoid carcinoma (A431) and immortalized, non-tumorigenic keratinocyte (HaCaT) cells. Moreover, the chemical composition of the watercress extract was characterized through UPLC MS/MS and other analytical methodologies. In addition, cytotoxicity was assessed by the alamar blue assay whereas apoptosis was determined, initially, by a multiplex activity assay kit (measuring levels of activated caspases −3, −8 and −9) as well as by qRT-PCR for the identification of major genes regulating apoptosis. In addition, protein expression levels were evaluated by western immunoblotting. Our data indicate that the extract contains various phytochemicals (e.g. phenolics, flavonoids, pigments, etc.) while isothiocyanates (ITCs; especially PEITC) were the most abundant. In addition, the extract was shown to exert a significant time- and dose-dependent cytotoxicity against all malignant melanoma cell lines while non-melanoma and non-tumorigenic cells exhibited significant resistance. Finally, expression profiling revealed a number of genes (and corresponding proteins) being implicated in regulating apoptotic induction through activation of the intrinsic apoptotic cascade. Overall, our data indicate the potential of PEITC as a promising anti-cancer agent in the clinical management of human malignant melanoma.

Zinc proteinate with moderate chelation strength enhances zinc absorption by up-regulating the expression of zinc and amino acid transporters in primary cultured duodenal epithelial cells of broiler embryos.

Recent study showed that zinc (Zn) and amino acid transporters may be involved in enhancing Zn absorption from Zn proteinate with moderate chelation strength (Zn-Prot M) in the duodenum of broilers. However, the specific mechanisms by which Zn-Prot M promotes the above Zn absorption are unknown. Therefore, in this study, three experiments were conducted to investigate specific and direct effects of Zn-Prot M and Zn sulfate (ZnS) on Zn absorption and expression of related transporters in primary duodenal epithelial cells of broiler embryos so as to preliminarily address possible mechanisms. In experiment 1, cells were treated with 100 μmol Zn/L as ZnS or Zn-Prot M for 20, 40, 60, 80, 100 or 120min. Experiment 2 consisted of 3 sub-experiments. In experiment 2A, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 100 or 200 μmol Zn/L as ZnS or Zn-Prot M for 60min; in experiment 2B, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 200 μmol Zn/L of as the ZnS or Zn-Prot M for 120min; in experiment 2C, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 400 or 800 μmol Zn/L as ZnS or Zn-Prot M for 120min. In experiment 3, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 400 μmol Zn/L as ZnS or Zn-Prot M for 120min. The results of experiment 1 indicated that the minimum incubation time for saturable Zn absorption was determined to be 50.83min using the best fit line. The results in experiment 2 demonstrated that a Zn concentration of 400 μmol/L and an incubation time of 120min were suitable to increase the absorption of Zn from Zn-Prot M compared to ZnS. In experiment 3, Zn absorption across cell monolayers was significantly increased by Zn addition (P < 0.05), and was significantly greater with Zn-Prot M than with ZnS (P < 0.05). Compared to the control, Zn addition significantly decreased Zn transporter 10 and peptide-transporter 1 mRNA expression levels and increased y+L-type amino transporter 2 (y+LAT2) protein abundance (P < 0.05). Moreover, protein expression levels of zrt/irt-like protein 3 (ZIP3), ZIP5 and y+LAT2 were significantly greater for Zn-Prot M than for ZnS (P < 0.05). These findings suggest that Zn-Prot M promote Zn absorption by increasing ZIP3, ZIP5 and y+LAT2 protein expression levels in primary duodenal epithelial cells.

Exploration of the mechanism of Qinglongyi-Buguzhi drug pair in treating vitiligo based on network pharmacology, molecular docking and experimental verification

Ethnopharmacological relevanceThe Qinglongyi-Buguzhi herbal pair (QB) is one of commonly used herbal combinations for treating vitiligo in traditional Chinese medicine, consisting of the exocarp of the immature fruit of Juglans regia L. or Juglans mandshurica Maxim., and dried, mature fruit of Psoralea corylifolia L. However, the active components and potential mechanisms of QB in the treatment of vitiligo are still unclear. Aim of the studyThe purpose of this study is to clarify the effects and mechanisms of QB on vitiligo treatment through integration of network pharmacology and empirical examinations. Materials and methodsThe active components and targets of QB as well as the targets linked to vitiligo were obtained from network databases. Visualization networks were constructed with Cytoscape 3.9.1. GO and KEGG enrichment analysis were conducted to investigate the possible mechanism. Molecular docking was employed to evaluate the binding affinities between the primary active ingredients of QB and essential targets of the PI3K/Akt/Nrf2 pathway. In vivo and in vitro experiments were carried out to confirm the results of network pharmacology. ResultsWe evaluated 44 active compounds and 602 genes from QB, and 107 of these genes linked to vitiligo. GO analysis suggested QB might lessen vitiligo by regulating oxidative stress. KEGG pathway analysis indicated the PI3K/Akt pathway may be crucial for treating vitiligo. Molecular docking results demonstrated the key active ingredients of QB had good binding activity with the major targets in the PI3K/Akt/Nrf2 pathway. In vivo, QB significantly ameliorated vitiligo model mouse's skin pathologies by reducing ROS, elevating CAT and SOD levels. Western blot showed that QB increased the phosphorylation of PI3K and Akt and the expressions of Nrf2 and HO-1 in the skin. In vitro, QB reversed H2O2-induced oxidative injury of melanocytes, enhanced cell survival rate, reduced ROS level, upregulated SOD and CAT activities, and raised the content of melanin. Moreover, QB upregulated the expression levels of Akt, Nrf2, HO-1 mRNA, Akt phosphorylation, HO-1, and nuclear Nrf2 proteins, and also encouraged the nuclear translocation of Nrf2. However, LY294002 treatment significantly reversed the regulatory effect of QB on oxidative damage of melanocytes. ConclusionsThis study revealed that the therapeutic effect of QB on vitiligo was achieved through multiple components, targets and pathways. Experimental investigation demonstrated that QB could improve vitiligo via reducing oxidative stress, which was probably accomplished by activating the PI3K/Akt/Nrf2 signaling pathway.

Analysis of Neuropeptides in the Intestinal Mucus of Patients with Ulcerative Colitis Using RNA Sequencing.

Inflammation in ulcerative colitis (UC) originates in the colorectal mucosa. Transcriptome sequencing analysis of the colorectal mucosa allows the identification of potential neuropeptides related to local neurotransmission. The intestinal mucus lining the surface of the mucosa may harbor biomarkers of mucosal inflammation; however, this has not been sufficiently investigated, given the difficulty in obtaining human samples. We previously reported the feasibility of obtaining mucin samples for proteomic analysis by brushing during colonoscopy. Herein, we aimed to investigate the composition of the intestinal mucus and detect neuropeptides characteristic of UC. Mucus and mucosal samples were collected from patients with UC from the colorectum in areas showing remission or active UC using a brush catheter and biopsy forceps during colonoscopy. RNA sequencing findings of mucus samples of active and remission areas were compared. RNA and protein expression levels of significantly upregulated neuropeptides were analyzed. Of the neuropeptides associated with UC, somatostatin (SST) was significantly elevated in areas of remission, according to RNA sequencing results of mucus and expression levels in mucus RNA and proteins. Conversely, SST expression in the mucosa was increased in the inflamed areas. Flow cytometry revealed that the fluorescence intensity of SST-positive cells in the remission zone was higher in the mucus than in the mucosa. SST expression in the mucus is considered to be an important factor associated with UC activity.

Effect of zinc oxide nanoparticles on hypoxia and pyroptosis of rat neutrophils

Objective: To investigate the effects of zinc oxide nanoparticles (ZnO-NPs) on neutrophil hypoxia and pyroptosis through nucleotide binding of oligomeric domain-like receptor protein 3 (NLRP3) inflammasome, and to analyze the role of pyroptosis on respiratory tract inflammotion induced by ZnO-NPs. Methods: In October 2022, primary cultured neutrophils were obtained from the abdominal aortic blood of SPF adult healthy SD rats. The neutrophils were treated with ZnO-NPs solution (0, 5, 10, 20 μg/ml) at different concentrations, and hypoxia group (5% O(2)) was set up. Hypoxia and reactive oxygen species (ROS) levels were detected by flow cytometry, and the expression levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cleaved Caspase-1 were measured by Western blot. The activity of lactic dehydrogenase (LDH) in the cell supernatant was measured by coloration, and the content of interleukin-1 beta (IL-1β) in cell culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA) . Results: Compared with the control group, hypoxia and ROS levels of neutrophils in hypoxia group and ZnO-NPs groups were significantly increased (P<0.05), and NLRP3, ASC, cleaved Caspase-1 protein expression levels, LDH activity and IL-1β content were significantly increased (P<0.05). Compared with hypoxia group, hypoxia and ROS levels of neutrophils in 5 μg/ml and 10 μg/ml ZnO-NPs groups were significantly decreased (P<0.05), NLRP3, ASC, cleaved Caspase-1 protein expression levels, LDH activity, and IL-1β content were decreased significantly (P<0.05). There were no significant differences in hypoxia, ROS levels, ASC, cleaved Caspase-1 protein expression levels, LDH activity, and IL-1β content between the 20 μg/ml ZnO-NPs group and the hypoxia group (P>0.05) . Conclusion: ZnO-NPs treatment may activate the NLRP3 inflammasome to induce pyroptosis of neutrophils which may be related to ROS and hypoxia.

Expression Patterns of TGF-β1, TβR-I, TβR-II, and Smad2 Reveal Insights into Heterosis for Growth of Hybrid Offspring between Acanthopagrus schlegelii and Pagrus major.

TGF-β1/Smads is a classic signaling pathway, which plays important roles in the development process of organisms. Black porgy Acanthopagrus schlegelii and red porgy Pagrus major are valuable economic fishes, and their hybrid offspring show excellent heterosis traits. Yet the molecular regulation mechanism of the heterosis traits is less clear. Here, we explored the TGF-β1/Smads pathway's molecular genetic information for heterosis in A. schlegelii ♂ × P. major ♀ (AP) and A. schlegelii ♀ × P. major ♂ (PA) in terms of growth and development. The mRNA expression levels of TGF-β1, TβR-I, TβR-II, and Smad2 genes in different developmental stages of A. schlegelii were detected. Furthermore, the expression levels of TGF-β1, TβR-I, TβR-II, and Smad2 genes in different tissues of adult (mRNA level) and larva (mRNA and protein level) of A. schlegelii, P. major, and their hybrids were determined by both real-time quantitative PCR and Western blot techniques. The results indicated the ubiquitous expression of these genes in all developmental stages of A. schlegelii and in all tested tissues of A. schlegelii, P. major, and its hybrids. Among them, the mRNA of TGF-β1, TβR-I, and TβR-II genes is highly expressed in the liver, gill, kidney, and muscle of black porgy, red porgy, and their hybrid offspring. There are significant changes in gene and protein expression levels in hybrid offspring, which indirectly reflect hybrid advantage. In addition, there was no correlation between protein and mRNA expression levels of Smad2 protein. The results provide novel data for the differential expression of growth and development genes between the reciprocal hybridization generation of black porgy and red porgy and its parents, which is conducive to further explaining the molecular regulation mechanism of heterosis in the growth and development of hybrid porgy.

CE9A215 (inotodiol), a lanostane-type oxysterol, mitigates LPS-induced sepsis through multifaceted mechanisms

Dysregulated host response against infection triggers sepsis that leads to multiple organ dysfunction due to uncontrolled inflammatory responses. Despite marked progress in understanding of sepsis, numerous clinical trials for treatment of sepsis have proven daunting and a new therapeutic approach is highly needed. CE9A215 (inotodiol), a fungal secondary metabolite, has been researched for its pharmacological activities and has shown potent anti-allergic effects. In this study, we evaluated the anti-inflammatory activities of CE9A215 upon lipopolysaccharide (LPS) stimulation in vivo and in vitro for the first time. CE9A215 decreased the production of interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), and IL-1β in a concentration-dependent manner in LPS-stimulated RAW264.7 cells. Intriguingly, in human mast cell line LUVA, CE9A215 significantly lowered IL-4 and IL-10, and this effect could be beneficial for the clearance of bacterial infection. In addition, administration of CE9A215 improved the survival rate of LPS-stimulated mice and inhibited the pro-inflammatory cytokines, IL-6, TNF-α, and IL-1β in blood. Moreover, CE9A215 enhanced the expression levels of plasma phospholipid transfer protein (PLTP), apolipoprotein E (ApoE), and ATP-binding cassette transporter (ABCA1) in LPS-stimulated RAW246.7 cells. Liver PLTP level increased significantly in the CE9A215-administered group compared with the control group, which implies that CE9A215 promotes LPS clearance and neutralization by reverse transport of LPS by increasing the expressions of PLTP, ApoE, and ABCA1. Our results highlight CE9A215's potential as a novel therapeutic option for the treatment of sepsis.

Design, synthesis, crystal structure and anticancer activity of organotin(IV)-rhein carboxylates

Two new rhein-organotin complexes Ph3Sn(O2CC14H7O4) (RS01) and Cy3Sn(O2CC14H7O4) (RS02) were synthesized and characterized by elemental analysis, IR, 1H NMR, 13C NMR, 119 Sn NMR, X-ray crystallography and thermo-gravimetric analysis. Both complexes has a triclinic crystal system P-1 space group and one-dimensional supramolecular chain structure. The MTT results demonstrated that rhein's anticancer activity was effectively increased by introducing organotin, RS01 and RS02 exhibited excellent anticancer activity in vitro, significantly superior to cisplatin and mitoxantrone. Interestingly, despite the fact that RS01 and mitoxantrone share the same 9,10-anthraquinone scaffold structure, RS01 not only showed high anticancer activity but also much lower nephrotoxicity than mitoxantrone. Flow cytometry showed that RS01 could significantly induce cell apoptosis and arrest cell cycle in G2 phase. Molecular docking and UV–Vis absorption spectra suggested that RS01 may be intercalation with DNA. Furthermore, western blot results showed that RS01 activated the DNA damage critical protein γ-H2AX and regulated the expression level of the mitochondrial apoptotic pathway critical protein (Bax/Bcl-2). Thus, RS01 induced cell death may be mediated through DNA damage and mitochondrial pathways. As a result, RS01 has significant development and application potential as an anticancer agent.

In vivo evaluation of guide-free Cas9-induced safety risks in a pig model

The CRISPR/Cas9 system has shown great potential for treating human genetic diseases through gene therapy. However, there are concerns about the safety of this system, specifically related to the use of guide-free Cas9. Previous studies have shown that guide-free Cas9 can induce genomic instability in vitro. However, the in vivo safety risks associated with guide-free Cas9 have not been evaluated, which is necessary for the development of gene therapy in clinical settings. In this study, we used doxycycline-inducible Cas9-expressing pigs to evaluate the safety risks of guide-free Cas9 in vivo. Our findings demonstrated that expression of guide-free Cas9 could induce genomic damages and transcriptome changes in vivo. The severity of the genomic damages and transcriptome changes were correlate with the expression levels of Cas9 protein. Moreover, prolonged expression of Cas9 in pigs led to abnormal phenotypes, including a significant decrease in body weight, which may be attributable to genomic damage-induced nutritional absorption and metabolic dysfunction. Furthermore, we observed an increase in whole-genome and tumor driver gene mutations in pigs with long-term Cas9 expression, raising the risk of tumor occurrence. Our in vivo evaluation of guide-free Cas9 in pigs highlights the necessity of considering and monitoring the detrimental effects of Cas9 alone as genome editing via the CRISPR/Cas9 system is implemented in clinical gene therapy. This research emphasizes the importance of further study and implementation of safety measures to ensure the successful and safe application of the CRISPR/Cas9 system in clinical practice.

MMP-3 mediates copper oxide nanoparticle-induced pulmonary inflammation and fibrosis

BackgroundThe increasing production and usage of copper oxide nanoparticles (Nano-CuO) raise human health concerns. Previous studies have demonstrated that exposure to Nano-CuO could induce lung inflammation, injury, and fibrosis. However, the potential underlying mechanisms are still unclear. Here, we proposed that matrix metalloproteinase-3 (MMP-3) might play an important role in Nano-CuO-induced lung inflammation, injury, and fibrosis.ResultsExposure of mice to Nano-CuO caused acute lung inflammation and injury in a dose-dependent manner, which was reflected by increased total cell number, neutrophil count, macrophage count, lactate dehydrogenase (LDH) activity, and CXCL1/KC level in bronchoalveolar lavage fluid (BALF) obtained on day 3 post-exposure. The time-response study showed that Nano-CuO-induced acute lung inflammation and injury appeared as early as day 1 after exposure, peaked on day 3, and ameliorated over time. However, even on day 42 post-exposure, the LDH activity and macrophage count were still higher than those in the control group, suggesting that Nano-CuO caused chronic lung inflammation. The Nano-CuO-induced pulmonary inflammation was further confirmed by H&E staining of lung sections. Trichrome staining showed that Nano-CuO exposure caused pulmonary fibrosis from day 14 to day 42 post-exposure with an increasing tendency over time. Increased hydroxyproline content and expression levels of fibrosis-associated proteins in mouse lungs were also observed. In addition, Nano-CuO exposure induced MMP-3 overexpression and increased MMP-3 secretion in mouse lungs. Knocking down MMP-3 in mouse lungs significantly attenuated Nano-CuO-induced acute and chronic lung inflammation and fibrosis. Moreover, Nano-CuO exposure caused sustained production of cleaved osteopontin (OPN) in mouse lungs, which was also significantly decreased by knocking down MMP-3.ConclusionsOur results demonstrated that short-term Nano-CuO exposure caused acute lung inflammation and injury, while long-term exposure induced chronic pulmonary inflammation and fibrosis. Knocking down MMP-3 significantly ameliorated Nano-CuO-induced pulmonary inflammation, injury, and fibrosis, and also attenuated Nano-CuO-induced cleaved OPN level. Our study suggests that MMP-3 may play important roles in Nano-CuO-induced pulmonary inflammation and fibrosis via cleavage of OPN and may provide a further understanding of the mechanisms underlying Nano-CuO-induced pulmonary toxicity.Graphical

Matrix Attachment Regions Enhance Transgene Expression by Manipulating Position-Dependent Effects in Stably Transfected CHO-K1 Cells.

We previously found that the position of matrix attachment regions (MARs) within the vector significantly affects its ability to enhance transgenic expression in the recombinant protein production. This study aims to systematically investigate the position-dependent impacts of MAR on transgene expression. We observed a significant increase in enhanced green fluores-cent protein (eGFP) expression levels in stably transfected CHO-K1 cells with either MAR 1-68 or MAR X-29 when MARs located upstream of the promoter. This increase was especially evident with MAR flanked the expression cassette. Concurrently, a substantial increase was observed in the percentage of eGFP-expressing cells, with 97.8% and 96.0% in MAR-containing constructs versus 73.7% in MAR-absent constructs. Further analysis of erythropoietin (EPO) expression revealed that constructs with flanking MARs induced the highest EPO productivity. Bioinformatics analysis revealed that certain specific transcription factors are important in modulating the transcription process. In conclusion, vectors harboring both MARs around the expression cassette constitute an optimal construct for enhanced recom-binant protein production in CHO-K1 cells. This insight underscores the importance of strate-gic MAR incorporation in vector design for optimized recombinant protein expression.

The alleviation of difenoconazole-induced kidney injury in common carp (Cyprinus carpio) by silybin: Involvement of inflammation, oxidative stress, and apoptosis

Triazole fungicides, such as difenoconazole (DFZ), are frequently used to control fungus in crops that pollute water. The common carp (Cyprinus carpio) (hereafter referred to as “carp”) is an excellent bio-indicator of water quality. The seeds of the silymarin plant contain a flavonolignan called silybin (SYB), which is used to treat liver disease. To explore SYB's involvement in DFZ-triggered kidney damage in carps, an H&E assay was conducted, and ROS level was also examined. The results demonstrated that SYB alleviated DFZ-induced destruction of kidney tissue structure in carps, as well as alleviating the elevation of kidney ROS level in carps. RT-qPCR and Western blot were used to detect inflammation-, oxidative stress- and apoptosis-related factors at mRNA level and protein level. The experimental findings indicated that relative to the DFZ group, SYB + DFZ co-treatment reduced inflammation-related mRNA level of il-6, il-1β and tnf-α, elevated mRNA level of il-10. It also reduced protein expression levels of NF-κB and iNOS. In addition, SYB + DFZ co-treatment reduced DFZ-induced increase in the oxidative stress-related mRNA indicators sod and cat, and decreased the protein expression levels of Nrf2 and NQO1. SYB reduced the DFZ-induced increase in pro-apoptotic gene Bax mRNA and protein expression levels and the DFZ-induced decrease in anti-apoptotic gene Bcl-2 mRNA and protein expression levels. In summary, SYB potentially mitigates DFZ-induced kidney damage in carp by addressing inflammation, oxidative stress, and apoptosis. Our results establish a theoretical foundation for the clinical advancement of freshwater carp feeds.

Effect of Rab18 on liver injury and lipid accumulation by regulating perilipin 2 and peroxisome proliferator-activated receptor gamma in non-alcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is currently one of the most common chronic liver diseases worldwide, characterized by the presence of lipid droplets. Rab18 is an important lipid droplet protein; however, its effects and mechanisms of action on NAFLD remain unclear. Free fatty acid-stimulated AML-12 cells and high-fat diet (HFD)-fed mice were used as NAFLD models. Lentiviruses overexpressing Rab18 (Rab18-OE) or knockdown (Rab18-KD) were used to generate stable cell lines for genetic analysis. Blood serum levels of alanine aminotransferase, aspartate aminotransferase, total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, glucose, and leptin were measured using a biochemical autoanalyzer. Hematoxylin and eosin staining was performed to detect pathological damage to the liver. Lipid accumulation in the cells was assessed by Oil Red O staining. Target expression was measured using qPCR, western blotting, and immunocytochemistry. Rab18 mRNA and protein expression levels increased in free fatty acid-stimulated AML-12 cells and the livers of HFD-fed mice. Rab18-OE increased lipid accumulation in vitro, which was attenuated by Rab18-KD. In vivo, Rab18-OE augmented liver pathological damage, serum alanine aminotransferase/aspartate aminotransferase activity, and triglyceride, total cholesterol, and low-density lipoprotein levels, whereas Rab18-KD decreased these indicators. Rab18-KD also downregulated blood glucose levels in HFD-fed mice. Mechanistically, Rab18-OE and Rab18-KD regulated the mRNA and protein expression levels of perilipin 2 (PLIN2) and peroxisome proliferator-activated receptor gamma (PPARγ) in vitro and in vivo, respectively. Immunocytochemistry revealed that Rab18 colocalized with PLIN2 and PPARγ in AML-12 cells. Rab18 expression was elevated in vitro and in vivo in the NAFLD mouse model. Rab18 regulates PLIN2 and PPARγ expression to exaggerate liver injury and lipid accumulation in patients with NAFLD. Thus, Rab18 may be a crucial protein in this disease and a potential therapeutic target.

Human umbilical cord mesenchymal stem cells derived-exosomes on VEGF-A in hypoxic-induced mice retinal astrocytes and mice model of retinopathy of prematurity.

To observe the effect of human umbilical cord mesenchymal stem cells (hUCMSCs) secretions on the relevant factors in mouse retinal astrocytes, and to investigate the effect of hUCMSCs on the expression of vascular endothelial growth factor-A (VEGF-A) and to observe the therapeutic effect on the mouse model of retinopathy of prematurity (ROP). Cultured hUCMSCs and extracted exosomes from them and then retinal astrocytes were divided into control group and hypoxia group. MTT assay, flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect related indicators. Possible mechanisms by which hUCMSCs exosomes affect VEGF-A expression in hypoxia-induced mouse retinal astrocytes were explored. At last, the efficacy of exosomes of UCMSCs in a mouse ROP model was explored. Graphpad6 was used to comprehensively process data information. The secretion was successfully extracted from the culture supernatant of hUCMSCs by gradient ultracentrifugation. Reactive oxygen species (ROS) and hypoxia inducible factor-1α (HIF-1α) of mice retinal astrocytes under different hypoxia time and the expression level of VEGF-A protein and VEGF-A mRNA increased, and the ROP cell model was established after 6h of hypoxia. The secretions of medium and high concentrations of hUCMSCs can reduce ROS and HIF-1α, the expression levels of VEGF-A protein and VEGF-A mRNA are statistically significant and concentration dependent. Compared with the ROP cell model group, the expression of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signal pathway related factors in the hUCMSCs exocrine group is significantly decreased. The intravitreal injection of the secretions of medium and high concentrations of hUCMSCs can reduce VEGF-A and HIF-1α in ROP model tissues. HE staining shows that the number of retinal neovascularization in ROP mice decreases with the increase of the dose of hUCMSCs secretion. In a hypoxia induced mouse retinal astrocyte model, hUCMSCs exosomes are found to effectively reduce the expression of HIF-1α and VEGF-A, which are positively correlated with the concentration of hUCMSCs exosomes. HUCMSCs exosomes can effectively reduce the number of retinal neovascularization and the expression of HIF-1α and VEGF-A proteins in ROP mice, and are positively correlated with drug dosage. Besides, they can reduce the related factors on the PI3K/AKT/mTOR signaling pathway.

Rutaecarpine Aggravates Acetaminophen-Induced Acute Liver Injury by Inducing CYP1A2.

In this study, we investigated whether rutaecarpine could aggravate acetaminophen-induced acute liver damage in vivo and in vitro. CCK-8 and apoptosis assays were performed to verify the cytotoxicity of acetaminophen to L02 cells with or without rutaecarpine. The expression levels of the target proteins and genes were determined using Western blotting and qRT-PCR. The liver pathological changes were evaluated with hematoxylin and eosin staining, while the aspartate aminotransferase (AST) and alanine aminotransferase (AST) levels in plasma were measured to assess the liver damage. Our results revealed that pretreatment of the cell and mice with rutaecarpine significantly aggravated the acetaminophen-induced liver damage. Mechanistically, rutaecarpine induces the CYP1A2 protein, which accelerates the metabolism of acetaminophen to produce a toxic intermediate, N-acetyl-p-benzoquinone imine (NAPQI), leading to severe liver inflammation. Rutaecarpine exacerbated the liver damage by upregulating CYP1A2 and proinflammatory factors. These findings highlight the importance of carefully considering the dosage of rutaecarpine when combined with acetaminophen in drug design and preclinical trials.

A protective mechanism of heat inactivation to enhance Levilactobacillus brevis PDD-2 against alcohol-induced chronic liver disease based on proteomic analysis.

A proteomics-based analysis of the effect of heat inactivation on the alleviation of alcoholic liver disease (ALD) using Levilactobacillus brevis PDD-2 is presented, aimed at exploring the potential and mechanisms of postbiotic elements prepared through heat inactivation in the treatment of ALD. It was found that L. brevis PDD-2 and its postbiotic (heat-inactivated L. brevis PDD-2) alleviate chronic ALD via the gut-liver axis. In particular, heat-inactivated L. brevis PDD-2 significantly increased the relative abundance of Erysipelotrichaceae and better facilitated the oxidative stress balance in the liver. The tandem mass tag (TMT)-based quantitative proteomics technique analyses revealed that heat-inactivated L. brevis PDD-2 was associated with up-regulated expression levels of proteins related to the redox system, cellular metabolism, amino acid and oligopeptide transport, and surface proteins with immunomodulatory capacity. These findings provide a theoretical basis for developing novel therapeutic strategies and lay a solid foundation for further revealing its exhaustive mechanisms.

Human umbilical cord mesenchymal stem cell-based gene therapy for hemophilia B using scAAV-DJ/8-LP1-hFIXco transduction

BackgroundHemophilia B is an X-linked bleeding disorder caused by a mutation in the gene responsible for encoding coagulation factor IX (FIX). Gene therapy offers promising potential for curing this disease. However, the current method of relatively high dosage of virus injection carries inherent risks. The purpose of this study was to introduce a novel scAAV-DJ/8-LP1-hFIXco vector transduced human umbilical cord blood derived mesenchymal stem cells (HUCMSCs) as an alternative cell-based gene therapy to conventional gene therapy for Hemophilia B.MethodsThe LP1-hFIXco gene structure was designed by us through searching the literature from NCBI and the scAAV-DJ/8-LP1-hFIXco vector was constructed by a commercial company. The HUCMSCs were cultivated in routine approach and transduced with scAAV-DJ/8-LP1-hFIXco vector. The human FIX activation system was employed for detection of hFIXco activity. The RNA and protein expression levels of the hFIXco were evaluated using PCR and western blot techniques. In animal studies, both NSG and F9-KO mice were used for the experiment, in which clotting time was utilized as a parameter for bleeding assessment. The immunohistochemical analysis was used to assess the distribution of HUCMSCs in mouse tissue sections. The safety for tumorigenicity of this cell-based gene therapy was evaluated by pathological observation after hematoxylin-eosin staining.ResultsThe transduction of HUCMSCs with the scAAV-DJ/8-LP1-hFIXco vector results in consistent and sustainable secretion of human FIXco during 5 months period both in vitro and in mouse model. The secretion level (hFIXco activity: 97.1 ± 2.3% at day 7 to 48.8 ± 4.5% at 5 months) was comparable to that observed following intravenous injection with a high dose of the viral vector (hFIXco activity: 95.2 ± 2.2% to 40.8 ± 4.3%). After a 5-month observation period, no clonal expansions of the transduced cells in tissues were observed in any of the mice studied.ConclusionsWe have discovered a novel and safer HUCMSCs mediated approach potentially effective for gene therapy in hemophilia B.