Signaling Pathway-related Factors Research Articles

The bta-miR-22-3p can alleviate LPS-induced inflammatory response in yak endometrial epithelial cells by targeting KSR2

Bovine endometritis is a common reproductive system disease in dairy cows that leads to decreased milk production and reproductive performance, causing significant economic losses for farmers. Research has shown that microRNAs (miRNAs) play a significant role in regulating the expression of biological genes and are closely related to the occurrence of inflammation, including bta-miR-22-3p. However, the specific molecular mechanisms by which miRNAs regulate bovine endometritis remain unclear. To investigate the regulatory mechanism of bta-miR-22-3p in yak endometritis, uterine tissues were collected from three healthy bos grunniens and three bos grunniens with endometritis, approximately 21 days postpartum. Various methods were employed, including real-time quantitative polymerase chain reaction (RT-qPCR), Western blot (WB), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF). The results demonstrated that overexpression of bta-miR-22-3p led to a significant decrease (P < 0.05) in factors related to the mitogen-activated protein kinase (MAPK) signaling pathway and associated inflammatory factors, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (P38), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β). Furthermore, dual-luciferase assays confirmed that the kinase suppressor of the Ras 2 (KSR2) gene is a downstream target of bta-miR-22-3p. Overexpression of bta-miR-22-3p inhibited the expression of KSR2. When KSR2 was inhibited, the levels of MAPK signaling pathway-related factors and inflammation also significantly decreased (P < 0.05). Thus, bta-miR-22-3p suppresses the activation of the MAPK signaling pathway through the inhibition of KSR2, resulting in a reduction of inflammatory factors. In conclusion, this study demonstrated that bta-miR-22-3p targets the KSR2 gene to alleviate LPS (Lipopolysaccharide)-induced inflammatory damage.

ADAM8 promotes alcoholic liver fibrosis through the MAPK signaling pathway

The effect and molecular regulatory mechanism of A Disintegrin and Metalloproteinase 8 (ADAM8) were explored in alcoholic liver fibrosis (ALF). C57BL/6N male mice were randomly divided into control, alcohol, and ADAM8-sgRNA3 plasmid groups. The control group received control liquid diet, while the alcohol and ADAM8-sgRNA3 plasmid groups were given alcohol liquid feed diet combined with ethanol gavage treatment for 8 weeks to induce ALF modeling. In addition, the ADAM8-sgRNA3 plasmid group was injected with the effective ADAM8-sgRNA3 plasmid, while the alcohol and control group mice were injected with an equivalent amount of physiological saline. LX-2 human hepatic stellate cells were divided into control, alcohol, si-ADAM8-2, and si-ADAM8-NC groups and induced for 48 h for model establishment in vitro. Serological detection, pathological staining, Western blotting, qRT-PCR and CCK8 assay were performed for experiments. Compared with the alcohol group, ADAM8 mRNA, protein and, positive area rate, serological indicators, pathological changes, and the expression of liver fibrosis marker and MAPK signaling pathway-related factors in the ADAM8-sgRNA3 plasmid group significantly decreased in vivo. Compared with the alcohol group, ADAM8 mRNA and protein expression, cell viability, and the expression of liver fibrosis markers and MAPK signaling pathway-related factors (p-ERK1/2, PCNA, Bcl-2, p-c-Jun, TGFβ1, p–p38 MAPK and HSP27) reduced significantly in the si-ADAM8-2 group. Therefore, ADAM8 promotes ALF through the MAPK signaling pathway, a promising target for treating ALF.

O96 NICD LACTYLATION OF NOTCH SIGNALING IMPROVES MICROVASCULATION RAREFACTION IN HYPERTENSION

Background and Objective: Microvascular rarefaction is a key pathophysiologic basis of hypertension-related cardiovascular disease. Exercise decreases blood pressure and promotes angiogenesis related to inhibition of NOTCH signaling. The activation of the NOTCH pathway depends on the transcriptional regulation of its intracellular active fragment NICD after nuclear translocation. The post-translational modification status of proteins plays a crucial role in the nuclear translocation effect of transcription factors. Lactylation is influenced by the level of lactate in the body. Exercise can promote glycolysis in endothelial cells, and increase intracellular lactate levels. Therefore, we explored whether the beneficial effects of Exercise were mediated by promoting NICD lactylation, inhibiting the NOTCH signaling, and promoting angiogenesis. Methods: To test our hypotheses, WKYs (normotensive rats) and SHRs (spontaneously hypertensive rats) were given exercise for 12 weeks. Blood pressure was determined by the tail-cuff method, and cardiac function was assessed by echocardiography. Using rats’ retina, gastrocnemius, and myocardium detected microvascular density. The expression levels of NOTCH signaling pathway related proteins and angiogenesis related factors were measured by Western Blotting. The level of lactate was measured by using a lactate assay kit. Protein modification omics was used to detect the modification types and levels of NICD in microvascular endothelial cells, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to detect the lactylation sites of NICD in microvascular endothelial cells. Results: Exercise in SHRs resulted in reduced blood pressure and concentric hypertrophy. Exercise promoted angiogenesis and improved microcirculation rarefaction. Exercise also reduced the production of NOTCH pathway related proteins and increased the expression level of angiogenesis related factors. The lactate level increased after exercise, and the lactylation level of NICD increased. Conclusions: In summary, Exercise decreases hypertension and improves cardiac function in SHRs. Our research results indicated that exercise increases lactate production and induces NICD lactylation, thereby inhibiting the NOTCH signaling, upregulating the expression of angiogenic factors, promoting angiogenesis, and lowering blood pressure.

A Novel Scaffold of Icariin/Porous Magnesium Alloy-Repaired Knee Cartilage Defect in Rat by Wnt/β-Catenin Signaling Pathway.

Cartilage defects caused by joint diseases are difficult to treat clinically. Tissue engineering materials provide a new means to promote the repair of cartilage defects. The purpose of this study is to design a novel scaffold of porous magnesium alloy loaded with icariin and sustained release in order to explore the effect and possible mechanism of this scaffold in repairing SD rat knee articular cartilage defect. We constructed a novel type of icariin/porous magnesium alloy scaffold, observed the structure of the scaffold by electron microscope, detected the drug release of icariin in the scaffold and the biological safety, and established an animal model of cartilage defect in the femoral intercondylar fossa of the knee joint in rats; the scaffold was placed in the defect. After 12 weeks of repair, the rat knee articular cartilage repair was evaluated by gross specimens and micro-CT, HE, safranin O-fast green, and toluidine blue staining combined with the modified Mankin's score. The protein expressions of the Wnt/β-catenin signaling pathway-related factors (β-catenin, Wnt5a, Wnt1, sFRP1) and chondrogenic differentiation-related factors (Sox9, Aggrecan, Col2α1) were detected by immunohistochemical staining. We found that the novel scaffold of icariin/porous magnesium alloy can release icariin slowly and has biosafety in rats. Compared with other groups, icariin/porous magnesium alloy can significantly promote the repair of cartilage defects and the expressions of β-catenin, Wnt5a, Wnt1, Sox9, Aggrecan, and Col2α1 (P < 0.05). This novel scaffold can promote the repair of rat knee cartilage defects, and this process may be achieved by activating the Wnt/β-catenin signaling pathway.

The silent information regulator 1 agonist SRT1720 reduces experimental intracerebral hemorrhagic brain injury by regulating the blood-brain barrier integrity.

Intracerebral hemorrhage (ICH) is a significant public health matter that has no effective treatment. ICH-induced destruction of the blood-brain barrier (BBB) leads to neurological deterioration. Astrocytic sonic hedgehog (SHH) alleviates brain injury by maintaining the integrity of the BBB after ICH. Silent information regulator 1 (SIRT1) is neuroprotective in several central nervous system diseases via BBB regulation. It is also a possible influential factor of the SHH signaling pathway. Nevertheless, the role of SIRT1 on BBB and the underlying pathological process associated with the SHH signaling pathway after ICH remain unclear. We established an intracerebral hemorrhagic mouse model by collagenase injection. SRT1720 (a selective agonist of SIRT1) was used to evaluate the effect of SIRT1 on BBB integrity after ICH. SIRT1 expression was reduced in the mouse brain after ICH. SRT1720 attenuated neurobehavioral impairments and brain edema of ICH mouse. After ICH induction, SRT1720 improved BBB integrity and tight junction expressions in the mouse brain. The SHH signaling pathway-related factors smoothened and glioma-associated oncogene homolog-1 were increased with the intervention of SRT1720, while cyclopamine (a specific inhibitor of the SHH signaling pathway) reversed these effects. These findings suggest that SIRT1 protects from ICH by altering BBB permeability and tight junction expression levels. This process is associated with the SHH signaling pathway, suggesting that SIRT1 may be a potential therapeutic target for ICH.

The miR-15b-5p/miR-379-3p-FOXO axis regulates cell cycle and apoptosis in scleral remodeling during experimental myopia

BackgroundMyopia is one of the most common eye diseases in children and adolescents worldwide, and scleral remodeling plays a role in myopia progression. However, the identity of the initiating factors and signaling pathways that induce myopia-associated scleral remodeling is still unclear. This study aimed to identify biomarkers of scleral remodeling to elucidate the pathogenesis of myopia.MethodsThe gene expression omnibus (GEO) and comparative toxicogenomics database (CTD) mining were used to identify the miRNA-mRNA regulatory network related to scleral remodeling in myopia. Real-time quantitative PCR (RT-qPCR), Western blot, immunofluorescence, H&E staining, Masson staining, and flow cytometry were used to detect the changes in the FOXO signaling pathway, fibrosis, apoptosis, cell cycle, and other related factors in scleral remodeling.ResultsmiR-15b-5p/miR-379-3p can regulate the FOXO signaling pathway. Confirmatory studies confirmed that the axial length of the eye was significantly increased, the scleral thickness was thinner, the levels of miR-15b-5p, miR-379-3p, PTEN, p-PTEN, FOXO3a, cyclin-dependent kinase (CDK) inhibitor 1B (CDKN1B) were increased, and the levels of IGF1R were decreased in Len-induced myopia (LIM) group. CDK2, cyclin D1 (CCND1), and cell cycle block assessed by flow cytometry indicated G1/S cell cycle arrest in myopic sclera. The increase in BAX level and the decrease in BCL-2 level indicated enhanced apoptosis of the myopic sclera. In addition, we found that the levels of transforming growth factor-β1 (TGF-β1), collagen type 1 (COL-1), and α-smooth muscle actin (α-SMA) were decreased, suggesting scleral remodeling occurred in myopia.ConclusionsmiR-15b-5p/miR-379-3p can regulate the scleral cell cycle and apoptosis through the IGF1R/PTEN/FOXO signaling pathway, thereby promoting scleral remodeling in myopia progression.Graphical

Targeting the ZMIZ1-Notch1 signaling axis for the treatment of tongue squamous cell carcinoma

Zinc finger MIZ-type containing 1 (ZMIZ1) is a transcriptional coactivator related to the protein inhibitors of activated STATs (PIAS) family. Mounting evidence suggests that ZMIZ1 plays a crucial role in the occurrence and development of cancers. The function of ZMIZ1 in tongue squamous cell carcinoma (TSCC) and the mechanisms underpinning its role in this disease have not been fully clarified. We performed qualitative ZMIZ1 protein expression analyses using immunohistochemistry in 20 patient-derived, paraffin-embedded TSCC tissue sections. We used RNAi to knock down ZMIZ1 expression in the CAL-27 TSCC cell line and quantified the impact of ZMIZ1 knock down on proliferation, migration and apoptosis via CCK-8, scratch assay and flow cytometry, respectively. We used qRT-PCR and western blotting to investigate the role of ZMIZ1 in this cell line. Finally, we established a model of lung metastasis in nude mice to replicate the in vitro results. ZMIZ1 protein was significantly more abundant in TSCC case tissue samples. ZMIZ1 knockdown reduced the invasion and metastases of TSCC tumor cells and promoted apoptosis. ZMIZ1 knockdown was associated with the down-regulation of Notch signaling pathway related factors Jagged1 and Notch1, and invasion and metastasis related factors MKP-1, SSBP2 and MMP7 in vitro and in vivo, at the mRNA level. In vitro and in vivo data suggest that knock down of ZMIZ1 may inhibit TSCC invasion and metastasis by modulating Notch signaling. ZMIZ1 inhibition may therefore represent a new therapeutic target for TSCC.

PCV2 Induced Endothelial Derived IL-8 Affects MoDCs Maturation Mainly via NF-κB Signaling Pathway.

Porcine circovirus type 2 (PCV2) infection can cause immunosuppressive diseases in pigs. Vascular endothelial cells (VECs), as the target cells for PCV2, play an important role in the immune response and inflammatory regulation. Endothelial IL-8, which is produced by porcine hip artery endothelial cells (PIECs) infected with PCV2, can inhibit the maturation of monocyte-derived dendritic cells (MoDCs). Here, we established a co-culture system of MoDCs and different groups of PIECs to further investigate the PCV2-induced endothelial IL-8 signaling pathway that drives the inhibition of MoDC maturation. The differentially expressed genes related to MoDC maturation were mainly enriched in the NF-κB and JAK2-STAT3 signaling pathways. Both the NF-κB related factor RELA and JAK2-STAT3 signaling pathway related factors (IL2RA, JAK, STAT2, STAT5, IL23A, IL7, etc.) decreased significantly in the IL-8 up-regulated group, and increased significantly in the down-regulated group. The expression of NF-κB p65 in the IL-8 up-regulated group was reduced significantly, and the expression of IκBα was increased significantly. Nuclear translocation of NF-κB p65 was inhibited, while the nuclear translocation of p-STAT3 was increased in MoDCs in the PCV2-induced endothelial IL-8 group. The results of treatment with NF-κB signaling pathway inhibitors showed that the maturation of MoDCs was inhibited and the expression of IL-12 and GM-CSF at mRNA level were lower. Inhibition of the JAK2-STAT3 signaling pathway had no significant effect on maturation, and the expression of IL-12 and GM-CSF at mRNA level produced no significant change. In summary, the NF-κB signaling pathway is the main signaling pathway of MoDC maturation, and is inhibited by the PCV2-induced up-regulation of endothelial-derived IL-8.

Effect of Shenlingyigan decoction on inflammatory factors related to liver injury regulated by TLR3 signaling pathway

BackgroundTo investigate the therapeutic effect of Shenlingyigan decoction on acute liver injury. Further explored the mechanisms involved in the therapeutic properties of Shenlingyigan decoction by test several key proteins (TLR3, TRIF, TBK1, IRF3, IFNβ, IL-1 and IL-6) within the TLR3 signaling pathway. MethodsThe mouse acute liver injury model group was established by pretreatment with D-GalN and Poly (I:C) induction. The acute liver injury mouse treatment groups were gavage with different doses of Shenlingyigan decoction for 3 days. The therapeutic effects of Shenlingyigan decoction were preliminarily evaluated using organ indices, tissue images, and HE staining. Furthermore, potential associated signaling pathways and target effects were predicted through network pharmacology. Western blot experiments were conducted to examine the expression of relevant proteins (TLR3, TRIF, TBK1, IRF3, IL-1, and IL-6). In addition, immunofluorescence assays were performed to assess the localization of IRF3 and IFNβ expression in the cytoplasm and nucleus. Finally, the effects of Shenlingyigan decoction on the expression of TLR3, TRIF, TBK1 and IRF3 genes were further studied by QT-PCR. ResultsThe liver organ index, the tissue photos and HE staining showed that Shenlingyigan decoction could reduce inflammation by decreasing the presence of inflammatory cells and downregulating the expression of IL-1 and IL-6. The result of network pharmacology showed 709 potential drug and disease overlapping targets. Toll-like receptor signaling pathway was related with these targets through KEGG analysis. Besides, TLR3, TBK1, IRF3, IL6, were important targets associated with viral hepatitis. Westernblot and Immunofluorescence analysis showed that Shenlingyigan decoction reduced the expression of TLR3 and TBK1 in mice with liver injury, while increasing the expression of IRF3. Shenlingyigan decoction does not significantly affect the expression of TRIF and IFNβ; however, it enhances the expression of IRF3 in the nucleus, consequently leading to increased expression of IFNβ in the nucleus. The results of QT-PCR showed that Shenlingyigan decoction could down-regulate the expression of TLR3, TRIF and TBK1 genes, and up-regulate the expression of IRF3 gene. ConclusionsShenlingyigan decoction participated in immune responses by effecting the expression of TLR3 signaling pathway-related factors to treat the acute liver injury.

Chanling Gao suppresses colorectal cancer via PI3K/Akt/mTOR pathway modulation and enhances quality of survival.

The Chinese medicine formula Chanling Gao (CLG) exhibits significant tumor inhibitory effects in colorectal cancer (CRC) nude mice. However, the detailed mechanisms remain elusive. CRC in situ nude mouse models were treated with CLG. Small animal magnetic resonance imaging (MRI) tracked tumor progression, and overall health metrics such as food and water intake, body weight, and survival were monitored. Posttreatment, tissues and blood were analyzed for indicators of tumor inhibition and systemic effects. Changes in vital organs were observed via stereoscope and hematoxylin-eosinstaining. Immunohistochemistry quantified HIF-1α and P70S6K1 protein expression in xenografts. Double labeling was used to statistically analyze vascular endothelial growth factor (VEGF) and CD31 neovascularization. Enzyme-linked immunosorbent assay was used to determine the levels of VEGF, MMP-2, MMP-9, IL-6, and IL-10 in serum, tumors, and liver. Western blotting was used to assess the expression of the PI3K/Akt/mTOR signaling pathway-related factors TGF-β1 and smad4 in liver tissues. CLG inhibited tumor growth, improved overall health metrics, and ameliorated abnormal blood cell counts in CRC nude mice. CLG significantly reduced tumor neovascularization and VEGF expression in tumors and blood. It also suppressed HIF-1α, EGFR, p-PI3K, Akt, p-Akt, and p-mTOR expression in tumors while enhancing PTEN oncogene expression. Systemic improvements were noted, with CLG limiting liver metastasis, reducing pro-inflammatory cytokines IL-6 and IL-10 in liver tissues, decreasing MMP-2 in blood and MMP-2 and MMP-9 in tumors, and inhibiting TGF-β1 expression in liver tissues. CLG can enhance survival quality and inhibit tumor growth in CRC nude mice, likely through the regulation of the PI3K/Akt/mTOR signaling pathway.

Molecular Characteristics of Everolimus-resistant Renal Cell Carcinoma Cells Generated by Continuous Exposure to Everolimus.

Mammalian target of rapamycin (mTOR) inhibitors represent the standard of care for metastatic renal cell carcinoma (RCC). However, treatment outcomes are relatively poor, suggesting a potential problem with tolerating mTOR inhibitors. The aim of this study was to establish everolimus-resistant sublines and to compare their molecular characteristics with those of their counterparts. Human-derived RCC, Caki-2, and 786-O cells were continuously exposed to everolimus at 1 μM, and the established resistant sublines were designated as Caki/EV and 786/EV, respectively. Cellular characteristics were compared between both cells. Caki/EV and 786/EV cells showed a decrease in sensitivity to everolimus as well as other mTOR inhibitors. Expression of mTOR and its effectors exhibited no alteration in resistant sublines and their counterparts. However, phosphorylation of S6K, an index of mTOR activity, decreased in resistant sublines. PCR array analysis of mTOR signaling pathway-related factors indicated that the expression of INSR, TP53, and IGFBP3 increased in Caki/EV cells, whereas that of TELO2, HRAS, and SGK1 was up-regulated in 786/EV cells. The levels of DDIT4, DEPTOR, HIF1A, and PLD1 mRNAs decreased in both cell lines. The novel everolimus-resistant Caki/EV and 786/EV cells exhibited cross-resistance to other mTOR inhibitors and decreased mTOR activity. Furthermore, down-regulation of DDIT4, DEPTOR, HIF1A, and PLD1 may contribute to everolimus resistance.

Herbal cake-separated moxibustion relieves aspirin induced gastric mucosal injury by activating Nrf2/ARE/HO-1 signaling in rats

To investigate whether herbal cake-separated moxibustion can activate nuclear factor erythroid 2-related factor 2 （Nrf2） / antioxidant responsive element （ARE） /hemeoxygenase-1 （HO-1） signaling pathway to repair aspirin induced gastric mucosal injury （GMI） in rats. SD rats （half male and half female） were randomly divided into blank control, model, moxibustion, inhibitor of HO-1 （inhibitor）, model+inhibitor, moxibustion + inhibitor groups, with 20 rats in each group. The GMI model was established by gavage of aspirin 150 mg/kg（1 mL/100 g）. Herbal cake-separated moxibustion was alternatively applied to bilateral "Zusanli" （ST36） and "Zhongwan" （CV12） and bilateral "Pishu" （BL20） and "Weishu" （BL21） for 30 min, once daily for 8 days. The rats in the three inhibitor groups received intraperitoneal injection of HO-1 inhibitor zinc protoporphyrin （5 mg/kg）. The rats' behavior score, emotional response score, skin hair score, diet score and stool state score were given. The GMI index was calculated according to Guth's methods. Histopathological changes of gastric mucosa were observed by H.E. staining. The activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） in the gastric mucosal tissue and serum were detected by ELISA. The levels of Nrf2/ARE/HO-1 signaling pathway-related factors Keap1, Nrf2, HO-1, CAT, GST, and NQO1 in the gastric mucosal tissue were detected by quantitative real-time PCR and Western blot, separately. Compared with the blank control group, the behavior score, emotional response score, skin hair score, diet score and stool state score, GMI index, MDA contents of gastric mucosal tissue and serum, expression le-vels of Keap1 mRNA and protein were significantly increased （P<0.01）, while the activity of gastric mucosal and serum SOD, the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins were considerably decreased （P<0.01） in the model group. Compared with the model group, moxibustion obviously reversed the increase of emotional response score, skin hair score, stool state score, GMI index, MDA levels of gastric mucosal tissue and serum, and expression levels of Keap1 mRNA and protein （P<0.01, P<0.05）, and the decrease of activity of SOD of gastric mucosal and serum, and the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. After administration of antagonist of HO-1, the effects of moxibustion were eliminated or weakened pronouncedly in reducing skin hair score, GMI index, contents of gastric mucosal and serum MDA, and expression of Keap1 mRNA and protein, and in up-regulating gastric mucosal and serum SOD, and expression of HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. Herbal-cake separated moxibustion can improve the GMI in rats, which may be associated with its effects in reducing oxidative stress and activating Nrf2/ARE/HO-1 signaling pathway.

Herbal cake-separated moxibustion relieves aspirin induced gastric mucosal injury by activating Nrf2/ARE/HO-1 signaling in rats

To investigate whether herbal cake-separated moxibustion can activate nuclear factor erythroid 2-related factor 2 （Nrf2） / antioxidant responsive element （ARE） /hemeoxygenase-1 （HO-1） signaling pathway to repair aspirin induced gastric mucosal injury （GMI） in rats. SD rats （half male and half female） were randomly divided into blank control, model, moxibustion, inhibitor of HO-1 （inhibitor）, model+inhibitor, moxibustion + inhibitor groups, with 20 rats in each group. The GMI model was established by gavage of aspirin 150 mg/kg（1 mL/100 g）. Herbal cake-separated moxibustion was alternatively applied to bilateral "Zusanli" （ST36） and "Zhongwan" （CV12） and bilateral "Pishu" （BL20） and "Weishu" （BL21） for 30 min, once daily for 8 days. The rats in the three inhibitor groups received intraperitoneal injection of HO-1 inhibitor zinc protoporphyrin （5 mg/kg）. The rats' behavior score, emotional response score, skin hair score, diet score and stool state score were given. The GMI index was calculated according to Guth's methods. Histopathological changes of gastric mucosa were observed by H.E. staining. The activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） in the gastric mucosal tissue and serum were detected by ELISA. The levels of Nrf2/ARE/HO-1 signaling pathway-related factors Keap1, Nrf2, HO-1, CAT, GST, and NQO1 in the gastric mucosal tissue were detected by quantitative real-time PCR and Western blot, separately. Compared with the blank control group, the behavior score, emotional response score, skin hair score, diet score and stool state score, GMI index, MDA contents of gastric mucosal tissue and serum, expression le-vels of Keap1 mRNA and protein were significantly increased （P<0.01）, while the activity of gastric mucosal and serum SOD, the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins were considerably decreased （P<0.01） in the model group. Compared with the model group, moxibustion obviously reversed the increase of emotional response score, skin hair score, stool state score, GMI index, MDA levels of gastric mucosal tissue and serum, and expression levels of Keap1 mRNA and protein （P<0.01, P<0.05）, and the decrease of activity of SOD of gastric mucosal and serum, and the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. After administration of antagonist of HO-1, the effects of moxibustion were eliminated or weakened pronouncedly in reducing skin hair score, GMI index, contents of gastric mucosal and serum MDA, and expression of Keap1 mRNA and protein, and in up-regulating gastric mucosal and serum SOD, and expression of HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. Herbal-cake separated moxibustion can improve the GMI in rats, which may be associated with its effects in reducing oxidative stress and activating Nrf2/ARE/HO-1 signaling pathway.

Key Roles of p53 Signaling Pathway-Related Factors GADD45B and SERPINE1 in the Occurrence and Development of Gastric Cancer.

p53 can function as an independent and unfavorable prognosis biomarker in cancer patients. We tried to identify the key factors of the p53 signaling pathway involved in gastric cancer (GC) occurrence and development based on the genotype-tissue expression (GTEx) and the Cancer Genome Atlas (TCGA) screening. We downloaded gene expression data and clinical data of GC included in the GTEx and TCGA databases, followed by differential analysis. Then, the key factors in the p53 signaling pathway were identified, followed by an analysis of the correlation between key factors and the prognosis of GC patients. Human GC cell lines were selected for in vitro cell experiments to verify the effects of key prognostic factors on the proliferation, migration, invasion, and apoptosis of GC cells. We found 4,944 significantly differentially expressed genes (DEGs), of which 2,465 were upregulated and 2,479 downregulated in GC. Then, 27 DEGs were found to be involved in the p53 signaling pathway. GADD45B and SERPINE1 genes were prognostic high-risk genes. The regression coefficients of GADD45B and SERPINE1 were positive. GADD45B was poorly expressed, while SERPINE1 was highly expressed in GC tissues, highlighting their prognostic role in GC. The in vitro cell experiments confirmed that overexpression of GADD45B or silencing of SERPINE1 could inhibit the proliferation, migration, and invasion and augment the apoptosis of GC cells. Collectively, the p53 signaling pathway-related factors GADD45B and SERPINE1 may be key genes that participate in the development of GC.

Electroacupuncture ameliorates pain in cervical spondylotic radiculopathy rat by inhibiting the CaMKII/CREB/BDNF signaling pathway and regulating spinal synaptic plasticity.

Central sensitization is one of the important mechanisms underlying neuropathic and radicular pain due to cervical spondylotic radiculopathy (CSR). Recent studies have shown that the calmodulin-dependent protein kinase II (CaMKII)/cAMP-response element binding protein (CREB)/brain-derived neurotrophic factor (BDNF) signaling pathway mediates central sensitization through its involvement in spinal cord synaptic plasticity. Our group has previously found that electroacupuncture (EA) has a good analgesic effect on CSR. However, the central analgesic mechanism of EA for CSR is not yet clear. The rats were randomly divided into Blank group, Sham-operated group, CSR group, and EA group. We prepared the CSR rat model using the fish wire extrusion method. The behavioral and mechanical pain thresholds of the rats in each group were measured 5 days after successful modeling and 7 days after the intervention. The first intervention was started 5 days after successful modeling, and the EA group was treated by acupuncture at the bilateral LI4 and LR3 points on the same side as one group, connected to a G6805-I electroacupuncture apparatus with continuous waves at 1.5Hz. The remaining groups were not subjected to EA intervention. The treatment was administered once a day for 7 consecutive days and then executed. We used WB, immunofluorescence, and qRT-PCR to detect the expression of CaMKII/CREB/BDNF signaling pathway-related factors in the synaptic of rat spinal cord in each group. EA improved pain threshold and motor function in CSR rats, inhibited the expression of BDNF, P-TrkB, CAMKII, and P-CREB in spinal cord synapses, reduced the expression of pain factor c-fos and postsynaptic membrane protein molecule neuroligin2, exerted a modulating effect on spinal cord synaptic plasticity in CSR rats, and suppressed the overactive synaptic efficacy. EA mediates central sensitization and exerts analgesic effects on CSR by modulating spinal synaptic plasticity, which may be related to the inhibition of CaMKII/CREB/BDNF signaling pathway.

Knockdown of protein interacting with C α kinase 1 aggravates sepsis-induced acute liver injury by regulating the TLR4/NF-κB pathway

Acute liver injury (ALI) may manifest at any phase of sepsis, yet an explicit therapeutic approach remains elusive. In this study, LPS and cecum ligation and puncture (CLP) were utilized to establish an inflammatory cell model and a murine model of sepsis-induced liver injury, respectively, aiming to explore the potential protective effect of protein interacting with C α kinase 1 (PICK1) on sepsis-induced ALI and its underlying mechanisms. In both the cell supernatant and the murine whole blood, the concentrations of inflammatory factors were quantified by ELISA, while the protein and mRNA expressions of PICK1, cleaved-PARP-1, caspase1, TLR4, IκBα, and NF-κB were assessed via western blot and qRT-PCR. The outcomes revealed that the knockdown of PICK1 increased the levels of inflammatory factors and apoptosis, alongside activation of TLR4/NF-κB signaling pathway-related factors in both in vivo and in vitro models. Moreover, the murine liver samples were subjected to Hematoxylin–Eosin (HE) staining for assessment of histopathological morphology. The HE staining and liver injury scoring results manifested a markedly exacerbated hepatic damage in PICK1 knockout mice as compared to WT mice following CLP. Furthermore, the liver macrophages were isolated from murine livers, and the expression and activity of the factors associated with the TLR4/NF-κB signaling pathway were verified through RT-qPCR and western blot, and EMSA assay demonstrated an augmented NF-κB activity subsequent to PICK1 knockout. Finally, the expression and localization of PICK1 in macrophages were further scrutinized via immunofluorescence, and the interaction between PICK1 and TLR4 was identified through co-immunoprecipitation. In conclusion, the knockdown of PICK1 appeared to modulate inflammatory factors by activating the TLR4/NF-κB signaling pathway, thereby exacerbating hepatic damage induced by sepsis.

Glycyrrhizic Acid Protects Glomerular Podocytes Induced by High Glucose by Modulating SNARK/AMPK Signaling Pathway.

Diabetic nephropathy is one of the most important microvascular complications of diabetes, which mainly refers to glomerular capillary sclerosis. Podocytes are an important part of glomerular capillaries. Previous clinical and basic studies have shown that fibrosis is the main factor of diabetic nephropathy. This study aimed to assess the protective mechanism of glycyrrhizic acid (GA) on glomerular podocytes induced by high glucose as we hypothesized that GA may have antifibrotic and anti-inflammatory effects on podocytes through regulation of the adenosine 5'-monophosphate-activated protein kinase (AMPK)/sucrose nonfermenting AMPK-related kinase (SNARK) signaling pathway. SNARK siRNA was used to transfect podocytes. Real-time quantitative polymerase chain reaction and immunofluorescence staining assays were used for molecular and pathological analysis. The expression levels of key pathway proteins (including TGF-β1, α-SMA, SITR1, AMPKα, LKB1, PGC-1α, NF-κB, IL-6, and TNF-α) were verified by Western blotting. The expression of inflammatory factors in podocytes was detected by ELISA. We demonstrated that GA decreased the expression of podocyte fibrosis signaling pathway-related factors by upregulating the AMPK pathway and its related factors. However, after transfection of podocytes with SNARK siRNA, there was an increased expression of fibrosis-related factors and inflammation-related factors. GA can protect podocytes and alleviate fibrosis and inflammation induced by high glucose, which is related to the AMPK signaling pathway. Meanwhile, knockdown of SNARK protein can inhibit the AMPK signaling pathway, aggravate fibrosis, and increase inflammation.

LAMA5: A new pathogenic gene for non-syndromic cleft lip with or without cleft palate

BackgroundThis study aimed to investigate the effect of LAMA5 on palatal development in mice. MethodsThe palatine process of C57BL/6 J fetal mice on the embryonic day 13.5 (E13.5) was cultured in vitro via the rotating culture method. The LAMA5-shRNA adenovirus vector was constructed, then transfected into the palatal process of E13.5 for 48 h in vitro. A fluorescence microscope was used to visualize the fusion of palates. The expression of LAMA5 was also detected. The expression of ki67, cyclin D1, caspase 3, E-cadherin, vimentin and SHH signaling pathway-related signaling factors in the blank control group, the negative control group, and the LAMA5 interference group were detected after virus transfection. ResultsThe bilateral palates in the LAMA5 interference group were not fused after virus transfection. PCR and WB showed that the mRNA and protein expressions of LAMA5 were decreased in the LAMA5 interference group. Furthermore, the mRNA and protein expressions of ki67, cyclin D1 and gli1 were decreased in the LAMA5 interference group, while the mRNA and protein expressions of caspase 3 were increased. However, the mRNA and protein expression of E-cadherin, vimentin, Shh and ptch1 did not significantly change in the LAMA5 interference group. ConclusionsLAMA5 silencing causes cleft palate by inhibiting the proliferation of mouse palatal cells and promoting apoptosis, which may not be involved in EMT. LAMA5 silencing can also cause cleft palate by interfering with the SHH signaling pathway.

Lithium chloride promotes diabetic corneal epithelial wound healing by activating the Wnt/β‑catenin signaling pathway.

Corneal epithelial abnormality is a common manifestation of diabetic keratopathy and leads to delayed epithelial wound healing. The Wnt/β-catenin signaling pathway participates in the development, differentiation and stratification of corneal epithelial cells. The present study compared the expression of Wnt/β-catenin signaling pathway related factors, including Wnt7a, β-catenin, cyclin D1 and phosphorylated (p-) glycogen synthase kinase 3 β (Gsk3b) between normal and diabetic mouse corneas, by reverse transcription-quantitative PCR, western blotting and immunofluorescence staining. It was found that the expression of the Wnt/β-catenin signaling pathway related factors was downregulated in diabetic corneas. Upon corneal epithelium scraping, the wound healing rate was significantly increased in diabetic mice after topical treatment with lithium chloride. After further investigation, significantly upregulated levels of Wnt7a, β-catenin, cyclin D1 and p-Gsk3b were found in the diabetic group 24 h after treatment, accompanied by β-catenin nuclear translocation observed by immunofluorescence staining. These results suggest that active Wnt/β-catenin pathway can promote diabetic corneal epithelial wound healing.

MAP4K4 is involved in the neuronal development of retinal photoreceptors

Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is a potential regulator of photoreceptor development. To investigate the mechanisms underlying MAP4K4 during the neuronal development of retinal photoreceptors, we generated knockout models of C57BL/6j mice in vivo and 661 W cells in vitro. Our findings revealed homozygous lethality and neural tube malformation in mice subjected to Map4k4 DNA ablation, providing evidence for the involvement of MAP4K4 in early stage embryonic neural formation. Furthermore, our study demonstrated that the ablation of Map4k4 DNA led to the vulnerability of photoreceptor neurites during induced neuronal development. By monitoring transcriptional and protein variations in mitogen-activated protein kinase (MAPK) signaling pathway-related factors, we discovered an imbalance in neurogenesis-related factors in Map4k4 −/− cells. Specifically, MAP4K4 promotes jun proto-oncogene (c-JUN) phosphorylation and recruits other factors related to nerve growth, ultimately leading to the robust formation of photoreceptor neurites. These data suggest that MAP4K4 plays a decisive role in regulating the fate of retinal photoreceptors through molecular modulation and contributes to our understanding of vision formation.