MicroRNA-124 conducts neuroprotective effect via inhibiting AK4/ATF3 after subarachnoid hemorrhage.

Spontaneous subarachnoid hemorrhage (SAH) accounts for approximately 5% of all cases of stroke. SAH is correlated with elevated rates of mortality and disability. Despite significant advancements in comprehending the pathogenesis and surgical management, efficacious clinical interventions remain restricted, and the prognosis is yet to be enhanced. MicroRNAs play a crucial role in various pathological processes in organisms. Revealing these regulatory processes is conducive to the development of new treatment methods. MicroRNA-124 is highly expressed in the nervous system and has significant research value for SAH. This study aims to explore the role of miR-124 in the early post-SAH period on neural function and verify whether it is involved in the pathological and physiological processes of SAH. In this study, we used methods such as comparing the expression levels of miR-124 in cerebrospinal fluid, establishing a rat SAH model, and a mouse embryonic primary neuron hemoglobin stimulation model to verify the downstream proteins of miR-124 in SAH. Through transfection techniques, we adjusted the expression of this small RNA in Vitro and in Vivo models using miR-124 inhibitor and mimic in the primary neuron hemoglobin stimulation model and rat SAH model, and observed the phenotype. Finally, by consulting the literature and verifying in Vivo and in Vitro methods, AK4 and downstream molecule ATF3 were identified as downstream targets of miR-124.

Aloe-emodin from Sanhua Decoction inhibits neuroinflammation by regulating microglia polarization after subarachnoid hemorrhage.

ETHNOPHARMACOLOGICAL RELEVANCE: Subarachnoid hemorrhage (SAH) triggers a cascade of events that lead to early brain injury (EBI), which contributes to poor outcomes and appears within 3 days after SAH initiation. EBI involves multiple process including neuronal death, blood-brain barrier (BBB) injury and inflammation response. Microglia are cluster of immune cells originating in the brain which respond to SAH by changing their states and releasing inflammatory molecules through various signaling pathways. M0, M1, M2 are three states of microglia represent resting state, promoting inflammation state, and anti-inflammation state respectively, which can be modulated by pharmacological strategies. AIM OF THE STUDY: After identified potential active ingredients and targets of Sanhua Decoction (SHD) for SAH, we selected aloe-emodin (AE) as a potential ingredient modulating microglia activation states. MATERIALS AND METHODS: Molecular mechanisms, targets and pathways of SHD were reveal by network pharmacology technique. The effects of AE on SAH were evaluated in vivo by assessing neurological deficits, neuronal apoptosis and BBB integrity in a mouse SAH model. Furthermore, BV-2 cells were used to examine the effects of AE on microglial polarization. The influence of AE on microglia transformation was measured by Iba-1, TNF-α, CD68, Arg-1 and CD206 staining. The signal pathways of neuronal apoptosis and microglia polarization was measured by Western blot. RESULTS: Network pharmacology identified potential active ingredients and targets of SHD for SAH. And AE is one of the active ingredients. We also confirmed that AE via NF-κB and PKA/CREB pathway inhibited the microglia activation and promoted transformation from M1 phenotype to M2 at EBI stage after SAH. CONCLUSIONS: AE, as one ingredient of SHD, can alleviate the inflammatory response and protecting neurons from SAH-induced injury. AE has potential value for treating SAH-induced nerve injury and is expected to be applied in clinical practice.

The Relationship between Mastitis and Antimicrobial Peptide S100A7 Expression in Dairy Goats.

S100A7 is an inflammation-related protein and plays an essential role in host defenses, yet there is little research about the relationship between mastitis and S100A7 expression in dairy goats. Here, according to the clinical diagnosis of udders, SCC, and bacteriological culture (BC) of milk, 84 dairy goats were grouped into healthy goats (n = 25), subclinical mastitis goats (n = 36), and clinical mastitis goats (n = 23). The S100A7 concentration in subclinical mastitis goats was significantly upregulated than in healthy dairy goats (p = 0.0056) and had a limited change with clinical mastitis dairy goats (p = 0.8222). The relationship between log10 SCC and S100A7 concentration in milk was positive and R = 0.05249; the regression equation was Y = 0.1446 × X + 12.54. According to the three groups, the log10 SCC and S100A7 were analyzed using the receiver operating characteristics (ROC) curve; in subclinical mastitis goats, the area under the ROC curve (AUC) of log10 SCC was 0.9222 and p < 0.0001, and the AUC of S100A7 concentration was 0.7317 and p = 0.0022, respectively; in clinical mastitis goats, the AUC of log10 SCC was 0.9678 and p < 0.0001, and the AUC of S100A7 concentration was 0.5487 and p = 0.5634, respectively. In healthy goats, S100A7 was expressed weakly in the alveolus of the mammary gland of healthy goats while expressed densely in the collapsed alveolus of mastitis goats. Moreover, S100A7 expression increased significantly in mastitis goats than in healthy dairy goats. In this research, results showed the effects of mastitis on the S100A7 expression in the mammary gland and S100A7 concentration in milk and the limited relationship between SCC and mastitis, which provided a new insight into S100A7's role in the host defenses of dairy goats.

Chitosan-salvianolic acid B coating on the surface of nickel-titanium alloy inhibits proliferation of smooth muscle cells and promote endothelialization.

Introduction: Intracranial stents are of paramount importance in managing cerebrovascular disorders. Nevertheless, the currently employed drug-eluting stents, although effective in decreasing in-stent restenosis, might impede the re-endothelialization process within blood vessels, potentially leading to prolonged thrombosis development and restenosis over time. Methods: This study aims to construct a multifunctional bioactive coating to enhance the biocompatibility of the stents. Salvianolic acid B (SALB), a bioactive compound extracted from Salvia miltiorrhiza, exhibits potential for improving cardiovascular health. We utilized dopamine as the base and adhered chitosan-coated SALB microspheres onto nickel-titanium alloy flat plates, resulting in a multifunctional drug coating. Results: By encapsulating SALB within chitosan, the release period of SALB was effectively prolonged, as evidenced by the in vitro drug release curve showing sustained release over 28 days. The interaction between the drug coating and blood was examined through experiments on water contact angle, clotting time, and protein adsorption. Cellular experiments showed that the drug coating stimulates the proliferation, adhesion, and migration of human umbilical vein endothelial cells. Discussion: These findings indicate its potential to promote re-endothelialization. In addition, the bioactive coating effectively suppressed smooth muscle cells proliferation, adhesion, and migration, potentially reducing the occurrence of neointimal hyperplasia and restenosis. These findings emphasize the exceptional biocompatibility of the newly developed bioactive coating and demonstrate its potential clinical application as an innovative strategy to improve stent therapy efficacy. Thus, this coating holds great promise for the treatment of cerebrovascular disease.

Immunolocalization of antibacterial peptide S100A7 in mastitis goat mammary gland and lipopolysaccharide induces the expression and secretion of S100A7 in goat mammary gland epithelial cells via TLR4/NFκB signal pathway.

The antimicrobial peptide S100A7, with antimicrobial activities for a broad spectrum of bacteria, has attracted more and more attention for the prevention and treatment of mastitis. However, there is little information about the expression and regulation mechanism of S100A7 in mastitis goats. This study revealed that S100A7 was mainly expressed in the stratified squamous epithelium of teat skin and streak canal, and S100A7 was present weakly in the healthy goat alveolus yet densely in the mastitis goat collapsed alveolus. Goat mammary epithelial cells (MECs) were isolated and treated with 2.5, 5, 10 and 20 µg/mL lipopolysaccharide (LPS) respectively for a different time, S100A7 mRNA expression and protein secretion were upregulated significantly with LPS treatment for 3 h, and the secretion level of S100A7 descended after 48 h treatment for all of these four groups. Moreover, after treatment with LPS, the mRNA levels of Toll-like receptor 4 (TLR4) and MyD88 were up-regulated, and the phosphorylation of p65 was up-regulated markedly. However, adding TLR4 inhibitor TAK-242 or/and NF-κB inhibitor QNZ significantly suppressed the phosphorylation of p65, and then inhibited the expression and secretion of S100A7 induced by LPS treatment. In conclusion, LPS induced the expression and secretion of S100A7 in goat MECs via TLR4/NF-κB signaling pathway.

Human umbilical cord mesenchymal stem cells conditioned medium exerts anti-tumor effects on KGN cells in a cell density-dependent manner through activation of the Hippo pathway.

BACKGROUND: The conditioned medium from human umbilical cord mesenchymal stem cells (UCMSCs-CM) provides a new cell-free therapy for tumors due to its unique secretome. However, there are many contradictory reports about the effect of UCMSCs-CM on tumor cells. The loss of contact inhibition is a common characteristic of tumor cells. A relationship between the effect of UCMSCs-CM on tumor cells and contact inhibition in tumor cells is rarely concerned. Whether the effect of UCMSCs-CM on tumor cells is affected by cell density? Here, we explored the effect of UCMSCs-CM on granulosa tumor cell line (KGN) cells at low or high density. METHODS: Growth curve and CCK8 assay were used to assess cell proliferation and viability. Scratch wound and matrigel invasion assay were implicated to detect cell motility of KGN cells. UCMSCs-CM effects on cell cycle, apoptosis and pathway-related proteins were investigated by flow cytometry, TUNEL assay, western blot and immunofluorescence analysis respectively. RESULTS: In growth curve analysis, before KGN cells proliferated into confluence, UCMSCs-CM had no effect on cell proliferation. However, once the cells proliferate to contact each other, UCMSCs-CM significantly inhibited proliferation. Meanwhile, when KGN cells were implanted at high density, UCMSCs-CM could induce cell cycle arrest at G1 phase, inhibit cell migration, invasion and promote apoptosis. While it had no similar effect on KGN cells implanted at low density. In mechanism, the UCMSCs-CM treatment activated the Hippo pathway when KGN cells were implanted at high density. Consistently, the MST1/2 inhibitor, XMU-MP-1, inhibited the activation of the Hippo pathway induced by UCMSCs-CM treatment and accordingly declined the anti-tumor effect of UCMSCs-CM on KGN cells. CONCLUSIONS: The effect of UCMSCs-CM on tumor cells is affected by cell density. UCMSCs-CM exerted anti-tumor effect on KGN cells by activating Hippo pathway to restore contact inhibition. Our results suggest that UCMSCs-CM is a promising therapeutic candidate for GCT treatment.

C-Type Natriuretic Peptide Regulates the Expression and Secretion of Antibacterial Peptide S100A7 in Goat Mammary Gland Through PKG/JNK/c-Jun Signaling Pathway.

During infection, the infected tissue secretes a variety of endogenous peptides to resist further invasion of pathogens. Among these endogenous peptides, the natriuretic peptides and the antimicrobial peptides attracted the most attention. C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor B (NPR-B) were members of the natriuretic peptide system. The antimicrobial peptide S100A7 plays an important role to resist infection of bacteria in mastitis. It is reported that the expression of S100A7 is regulated by an activator protein-1 (AP-1)-responsive promoter. As a subunit of AP-1, c-Jun is a downstream target of CNP/NPR-B signaling pathway. Therefore, it is a hypothesis that the CNP/NPR-B signaling pathway induces the expression and secretion of S100A7 in mammary glands to take part in local mammary gland innate immunity. To verify this hypothesis, goat mammary gland and isolated mammary epithelial cells (MECs) were used to explore the expression of CNP/NPR-B and their physiological roles in goat mammary gland. The results showed that goat mammary gland expressed NPR-B, but not CNP. The expression and secretion of S100A7 in goat MECs were obviously induced by CNP/NPR-B signaling pathway. After treatment with CNP, the cyclic guanosine monophosphate (cGMP) level in goat MECs was significantly upregulated. Along with the upregulation of cGMP level, the phosphorylation levels of c-Jun N-terminal kinase (JNK) and its target c-Jun were also increased gradually. KT5823 is a specific inhibitor for protein kinase G (PKG). KT5823 remarkably inhibited the phosphorylation of JNK and c-Jun induced by CNP. Correspondingly, KT5823 evidently inhibited the expression and secretion of S100A7 induced by CNP. On the other hand, the expression of NPR-B and S100A7 was upregulated in the mastitis goat mammary gland. But, there was no significant difference in expression of CNP between healthy and mastitis goat mammary gland tissues. The goat mastitis model was established in vitro using goat MECs treated by lipopolysaccharide (LPS). LPS treatment also could increase the expression of NPR-B and S100A7. In conclusion, goat mammary gland expressed NPR-B, indicating mammary gland was the target organ for natriuretic peptide system. Moreover, CNP, through NPR-B/JNK/c-Jun signaling pathway to regulate the expression and secretion of S100A7 in MECs, played an important role in mammary gland innate immunity.

Effects of 4-vinylcyclohexene diepoxide on the cell cycle, apoptosis, and steroid hormone secretion of goat ovarian granulosa cells.

4-Vinylcyclohexene diepoxide (VCD) is a potentially hazardous industrial chemical that may enter a goat's body in various ways during industrial breeding. Ovarian granulosa cells (GCs) play a critical role in supporting follicle development and hormone synthesis. However, there are few studies on the effect of VCD on goat ovarian GCs. In this study, goat ovarian GCs were isolated and treated with VCD. The results showed that treatment with VCD increased the proportion of S phase and G2/M cells, but decreased the proportion of G1 phase. VCD treatment significantly inhibited the expression of cyclin A and cyclin-dependent kinase 2 (CDK2). But the expression levels of p21 and p27 were increased. VCD could induce an apparent increase in the proportion of apoptosis and the level of cleaved caspase 3. Treatment with VCD significantly reduced the progesterone and estrogen concentration in the medium in which goat ovarian GCs were cultured. Correspondingly, the expression level of steroidogenic acute regulatory protein (STAR) was significantly downregulated. Treatment with 0.25 and 0.5 mM VCD, the protein expression level of insulin-like growth factor 1 receptor (IGF1R) and Akt were significantly decreased. Moreover, treatment with 0.25 mM VCD significantly inhibited the phosphorylation of Akt. In conclusion, VCD exposure had cytotoxic effects such as decreased cell viability, disordered cell cycle, increased apoptosis, and interference with steroid hormone synthesis on goat GCs. These cytotoxic effects of VCD on goat GCs may be due to the downregulation of IGF1R and the inhibition of IGF1R/Akt signaling pathway.