Forsythiaside A attenuates lipopolysaccharide-induced mouse mastitis by activating autophagy and regulating gut microbiota and metabolism.

Mastitis is an inflammatory disease of the mammary gland with a high incidence in lactating animals, significantly impacting their health and breastfeeding. Moreover, mastitis adversely affects milk quality and yield, resulting in substantial economic losses for the dairy farming industry. Forsythiaside A (FTA), a phenylethanol glycoside analog extracted from Forsythia, exhibits notable anti-inflammatory and antioxidant properties. However, its protective effects and specific mechanisms against mastitis remain unclear. In this study, a lipopolysaccharide (LPS)-induced mouse mastitis model was used to investigate the protective effect of FTA on LPS-induced mastitis and its potential mechanism using histological assays, Western blot, qRT-PCR, FITC-albumin permeability test, 16s rRNA gene sequencing analysis and non-targeted metabolomics assays to investigate the protective effect of FTA on LPS-induced mastitis model and its potential mechanism. The results demonstrated that FTA significantly mitigated LPS-induced mouse mastitis by reducing inflammation and apoptosis levels, modulating the PI3K/AKT/mTOR signaling pathways, inducing autophagy, and enhancing antioxidant capacity and the expression of tight junction proteins. Furthermore, FTA increased the abundance of beneficial microbiota while decreasing the levels of harmful microbiota in mice, thus counteracting the gut microbiota disruption induced by LPS stimulation. Intestinal metabolomics analysis revealed that FTA primarily regulated LPS-induced metabolite alterations through key metabolic pathways, such as tryptophan metabolism. This study confirms the anti-inflammatory and antioxidant effects of FTA on mouse mastitis, which are associated with key metabolic pathways, including the restoration of gut microbiota balance and the regulation of tryptophan metabolism. These findings provide a novel foundation for the treatment and prevention of mammalian mastitis using FTA.

[The role and the molecular mechanism of abietic acid in the proliferation, invasion and migration of cisplatin-resistant nasopharyngeal carcinoma cells].

Objective:To investigate the effects and molecular mechanisms of abietic acid in the cell proliferation, invasion and migration of cisplatin-resistant nasopharyngeal carcinoma cells. Methods:①Cisplatin-resistant C666/DDP cell line was constructed by increasing drug concentration method. ②The effects of abietic acid on proliferation, invasion and migration of C666/DDP cells were investigated by CCK-8 method, reactive oxygen species（ROS） and mitochondrial membrane potential（MMP） level assay and subcutaneous tumorigenesis assay in nude mice to detect the effects of abietic acid on proliferation and apoptosis of C666/DDP cells in vitro and in vivo. The effect of abietic acid on the proliferation and apoptosis of C666/DDP cells in vitro and in vivo was measured by Transwell assay. ③Western blot and IHC method to detect the expression of PI3K/AKT/mTOR pathway related proteins. Results:①The IC50 of cisplatin cytotoxicity to C666-1 was about 25 µmol/L. RI=25 µmol/L /4 µmol/L=6.25, resistance was obtained, and the C666-1-DDP resistant strain was successfully constructed. ②Abietic acid promoted apoptosis and inhibited proliferation of C666/DDP cells, and showed G2/M phase block; transwell showed that abietic acid inhibited C666/DDP cell migration and invasion, increased ROS level of C666/DDP cells and decreased MMP. Transwell showed that abietic acid inhibited the migration and invasion ability of C666/DDP cells, increased the ROS level of C666/DDP cells and decreased MMP. ③Animal experiments showed that abietic acid inhibited the proliferation of cisplatin-resistant nasopharyngeal carcinoma in vivo in a concentration gradient and suppressed the expression of PI3K/AKT/mTOR signaling pathway-related proteins. Conclusion:Abietic acid inhibits proliferation, invasion and migration of cisplatin-resistant nasopharyngeal carcinoma cells by a mechanism related to inhibition of PI3K/AKT/mTOR signaling pathway.

Forsythiaside A attenuates mastitis via PINK1/Parkin-mediated mitophagy.

BACKGROUND: Bovine mastitis is the most common animal production disease in the global dairy industry, which affects the health of dairy cows. When bovine mastitis occurs, the mitochondrial metabolism of breast tissue increases, and the relationship between inflammation and mitophagy has become a hot topic for many scholars. The abuse of antibiotics leads to the increase of resistance to bovine mastitis. FTA is one of the main effective components of Forsythia suspensa, which has anti-inflammatory, anti-infection, anti-oxidation and anti-virus pharmacological effects, and has broad application prospects in the prevention and treatment of bovine mastitis. However, the relationship between the anti-inflammatory effects of FTA and mitophagy is still unclear. PURPOSE: This study mainly explores the anti-inflammatory effect of FTA in bovine mastitis and the relationship between mitophagy. METHODS: MAC-T cells and wild-type mice were used to simulate the in vitro and in vivo response of mastitis. After the pretreatment with FTA, CsA inhibitors and siPINK1 were used to interfere with mitophagy, and the mitochondrial function impairment and the expression of inflammatory factors were detected. RESULTS: It was found that pre-treatment with FTA significantly reduced LPS induced inflammatory response and mitochondrial damage, while promoting the expression of mitophagy related factors. However, after inhibiting mitophagy, the anti-inflammatory effect of FTA was inhibited. CONCLUSION: This study is the first to suggest the relationship between the anti-inflammatory effect of FTA and mitophagy. PINK1/Parkin-mediated mitophagy is one of the ways that FTA protects MAC-T cells from LPS-induced inflammatory damage.

Esculetin improves murine mastitis induced by streptococcus isolated from bovine mammary glands by inhibiting NF-κB and MAPK signaling pathways.

Cow mastitis, caused by Streptococcus infection of the mammary glands, is a common clinical disease that can lead to decreased milk quality and threaten animal welfare and performance. Esculetin (ESC) is a coumarin with anti-inflammatory and anti-asthmatic effects. However, whether ESC has therapeutic effects on mastitis remains unexplored. This study was conducted to investigate the protective effect of ESC against murine mastitis caused by Streptococcus isolated from bovine mammary glands and elucidate the underlying mechanisms. Streptococcus uberis was used to construct a mouse model of mastitis. The results showed that the mice exhibited edema and thickening of the acinar wall with inflammatory infiltration after S. uberis treatment. Intraperitoneal injection of ESC significantly reduced inflammatory cell infiltration, restored normal physiological function, and inhibited the production of the inflammatory cytokines interleukin-1ß, interleukin-6, and tumor necrosis factor-α. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot analysis revealed that ESC reduced P38 phosphorylation, further inhibited the influence of mammary Streptococcus on cytoplasmic translocation of nuclear factor-κB (P65), and inhibited the transcriptional activation of P65, thus inhibiting the generation of inflammatory cells. Collectively, ESC may inhibit mitogen-activated protein kinase and nuclear factor-κB, thereby highlighting its potential for the treatment and prevention of mastitis.

Pulvinar Response Profiles and Connectivity Patterns to Object Domains.

Distributed cortical regions show differential responses to visual objects belonging to different domains varying by animacy (e.g., animals vs tools), yet it remains unclear whether this is an organization principle also applying to the subcortical structures. Combining multiple fMRI activation experiments (two main experiments and six validation datasets; 12 females and 9 males in the main Experiment 1; 10 females and 10 males in the main Experiment 2), resting-state functional connectivity, and task-based dynamic causal modeling analysis in human subjects, we found that visual processing of images of animals and tools elicited different patterns of response in the pulvinar, with robust left lateralization for tools, and distinct, bilateral (with rightward tendency) clusters for animals. Such domain-preferring activity distribution in the pulvinar was associated with the magnitude with which the voxels were intrinsically connected with the corresponding domain-preferring regions in the cortex. The pulvinar-to-right-amygdala path showed a one-way shortcut supporting the perception of animals, and the modulation connection from pulvinar to parietal showed an advantage to the perception of tools. These results incorporate the subcortical regions into the object processing network and highlight that domain organization appears to be an overarching principle across various processing stages in the brain.SIGNIFICANCE STATEMENT Viewing objects belonging to different domains elicited different cortical regions, but whether the domain organization applied to the subcortical structures (e.g., pulvinar) was unknown. Multiple fMRI activation experiments revealed that object pictures belonging to different domains elicited differential patterns of response in the pulvinar, with robust left lateralization for tool pictures, and distinct, bilateral (with rightward tendency) clusters for animals. Combining the resting-state functional connectivity and dynamic causal modeling analysis on task-based fMRI data, we found domain-preferring activity distribution in the pulvinar aligned with that in cortical regions. These results highlight the need for coherent visual theories that explain the mechanisms underlying the domain organization across various processing stages.

m6A modification-mediated BATF2 suppresses metastasis and angiogenesis of tongue squamous cell carcinoma through inhibiting VEGFA.

The aim is to explore the underlying mechanism of basic leucine zipper ATF-like transcription factor 2 (BATF2) in tongue squamous cell carcinoma (TSCC). The expression of BATF2 in TSCC tissues and corresponding adjacent normal TSCC tissues, human TSCC cell lines (SCC-15 and CAL-27) and human normal tongue epithelial cells NTEC was detected. Then, SCC-15 cells with stable BATF2 knockdown and CAL-27 cells with BATF2 overexpression were established to investigate the functional effect of BATF2 on TSCC. Thereafter, the effect of BATF2 on TSCC angiogenesis and BATF2 m6A methylation was also examined. BATF2 was significantly downregulated in TSCC tissues and cell lines, and BATF2 overexpression could suppress growth, metastasis and angiogenesis of TSCC. Mechanistically, vascular endothelial growth factor A (VEGFA) was identified as a downstream gene of BATF2, and it was confirmed that BATF2 suppressed growth, metastasis and angiogenesis of TSCC via inhibiting VEGFA. In addition, the N6-methyladenosine (m6A) modification of BATF2 mRNA mediated by METTL14 suppressed its expression in TSCC. METTL14/BATF2 axis could serve as a novel promising therapeutic candidate against angiogenesis for TSCC.

Brain intrinsic connection patterns underlying tool processing in human adults are present in neonates and not in macaques.

Tool understanding and use are supported by a dedicated left-lateralized, intrinsically connected network in the human adult brain. To examine this network's phylogenetic and ontogenetic origins, we compared resting-state functional connectivity (rsFC) among regions subserving tool processing in human adults to rsFC among homologous regions in human neonates and macaque monkeys (adolescent and mature). These homologous regions formed an intrinsic network in human neonates, but not in macaques. Network topological patterns were highly similar between human adults and neonates, and significantly less so between humans and macaques. The premotor-parietal rsFC had most significant contribution to the formation of the neonatal tool network. These results suggest that an intrinsic brain network potentially supporting tool processing exists in the human brain prior to individual tool use experiences, and that the premotor-parietal functional connection in particular offers a brain basis for complex tool behaviors specific to humans.

[Comparison of acellular dermal allograft and tragus cartilage perichondrium in type â tympanoplasty under otoendoscopy].

Objective:To compare the therapeutic effect of otoendoscopic tympanoplasty with acellular dermal allograft（AlloDerm） and tragus cartilage perichondrium. Method:121 patients who underwent type Ⅰ tympanoplasty under otoscope were retrospectively analyzed. According to the grafts used, they were divided into two groups: AlloDerm group （56 cases） and tragus cartilage perichondrium group （65 cases）. The operative time, postoperative tympanic membrane healing rate, and hearing recovery were compared between two groups. The follow-up time was twelve months. Result:The operative time in the AlloDerm group were lower than those in the tragus cartilage perichondrium group（P<0.05）. The successful closure rates between the acellular dermal allograft group and tragus cartilage perichondrium group at 1-month follow-up were 92.86% and 92.31% respectively, while the closure rates between two groups at 6-month follow-up were drop to 91.07% and 90.77% respectively, the closure rates between two groups at 12-month follow-up were also 91.07% and 90.77% respectively,the was no statistically difference between two groups（P>0.05）. The difference in pre-and post-operative air bone gap（ABG） values between two groups was no statistically significant（P>0.05）. Conclusion:Both the AlloDerm and the tragus cartilage perichondrium tympanoplasty can achieve satisfactory healing rate of the tympanic membrane and audiologic improvement. However, AlloDerm has a short operation time, no need to obtain materials and less trauma, and is worth of promotion and application.

Decreased expression of Beclin­1 is significantly associated with a poor prognosis in oral tongue squamous cell carcinoma.

The autophagy-related gene Beclin-1 is critical in the regulation of tumourigenesis and progression, but its role in oral tongue squamous cell carcinoma (OTSCC) has not yet been reported. This study aimed to investigate Beclin­1 expression and its significance in OTSCC. Beclin­1 expression was assessed by reverse transcription­quantitative polymerase chain reaction or western blot analysis in 14 OTSCC tissues and matched adjacent noncancerous tissues as well as in 5 OTSCC cell lines and a normal tongue epithelial cell line. Beclin­1 protein expression was examined by immunohistochemistry in 133 OTSCC specimens, and the correlation between Beclin­1 expression and clinicopathological features was investigated. Furthermore, MTT and colony formation assays were performed to investigate the effect of Beclin­1 on the proliferation and clonogenicity of OTSCC cells. It was demonstrated that Beclin­1 expression was significantly decreased in the majority of the 14 OTSCC tissues and the 5 OTSCC cell lines relative to the matched non­cancerous tissues and the normal tongue epithelial cell line, respectively. Immunohistochemistry analysis revealed that decreased Beclin­1 expression was significantly correlated with poor differentiation, lymph node metastasis, advanced clinical tumour­node­metastasis stage, and a poor prognosis in patients with OTSCC. The in vitro assays indicated that the overexpression of Beclin­1 significantly inhibits the proliferation and clonogenicity of OTSCC cells. These results demonstrate that Beclin­1 acts as a tumour suppressor in the development or progression of OTSCC and that Beclin­1 may represent a novel prognostic marker for patients with OTSCC.

[The expression and clinical significance of BATF2 in oral tongue squamous cell carcinoma].

OBJECTIVES: To investigate the expression and clinical significance of BATF2 in the oral tongue squamous cell carcinoma (OTSCC). METHODS: Expression of BATF2 mRNA and protein in 16 paired OTSCC tissues and adjacent non-tumor mucosa were examined using quantitative PCR, western blotting analysis and immunohistochemistry assays, and the relation between BATF2 expression and clinical pathologic factor and prognosis was analyzed. RESULTS: In 16 paired tissues, expression of BATF2 mRNA in 13 OTSCC tissues and expression of BATF2 protein in 14 OTSCC tissues were significantly lower than that in adjacent non-tumor mucosa. In 202 paraffin-embedded OTSCC samples, BATF2 was not expressed in 20 cases (9.9%), low expressed in 104 cases (51.5%) and highly expressed in 78 (38.6%). BATF2 expression level was significantly correlated with histological differentiation (P = 0.002). Patients with low BATF2 expression had significantly poorer overall survival and disease-free survival than those with high BATF2 expression (P < 0.001). CONCLUSIONS: BATF2 was low expressed in OTSCC and related to tumor differentiation and prognosis and may serve as a prognostic biomarker and potential therapeutic target for this disease.

Decreased expression of BATF2 is significantly associated with poor prognosis in oral tongue squamous cell carcinoma.

BATF2, also called SARI, is associated with several cancer types, and loss of BATF2 expression is frequently detected in aggressive and metastatic cancers. The expression of BATF2 was previously shown to slow the growth rate of malignant tumor cells injected into athymic nude mice, and decreased expression of BATF2 has been correlated to poor prognosis in hepatocellular carcinoma. However, the functional role of BATF2 in oral tongue squamous cell carcinoma (OTSCC) remains unknown. In the present study, we examined BATF2 expression in 16 fresh, paired OTSCC and adjacent non-tumor tissues, as well as in a normal tongue epithelial cell line and in 5 OTSCC cell lines by quantitative PCR and western blot analysis. We also evaluated BATF2 expression in 202 paraffin­embedded OTSCC and 30 adjacent non-tumor samples by immunohistochemistry, and its relationship with clinicopathological features and prognosis was investigated. We found that BATF2 expression was significantly reduced in the majority of the 16 OTSCC tumor tissues and the 5 OTSCC cell lines when compared with the non-tumor tissues and the normal tongue epithelial cell line, respectively. Consistent with these results, our immunohistochemistry analysis revealed that decreased BATF2 expression was present in 124 of the 202 cases and was significantly correlated with poor tumor differentiation (P=0.002). Patients with decreased BATF2 expression showed reduced survival when compared to those with high expression (P<0.001). Multivariate analysis revealed that BATF2 expression is an independent predictor of overall survival (P=0.001). These results demonstrate that BATF2 plays a tumor-suppressor role in the development of OTSCC and that BATF2 may serve as a prognostic biomarker and potential therapeutic target for this disease.

TRB3 overexpression due to endoplasmic reticulum stress inhibits AKT kinase activation of tongue squamous cell carcinoma.

Our investigation aims to evaluate the significance of TRB3, an endoplasmic reticulum stress (ERS)-inducible gene, and explore its relationship with AKT in oral tongue squamous cell carcinoma (OTSCC). Expression of TRB3 and phosphorylated AKT (p-AKT) in OTSCC tissues and adjacent normal tissues were assessed by RT-PCR, Western blot and immunohistochemistry assay. Correlation of TRB3 and AKT was validated by TRB3 adenovirus plasmid (Ad-TRB3) transfection and short hairpin RNA (shRNA) inhibition. The mRNA expression of TRB3 was significantly higher than adjacent noncancerous tissues by RT-PCR in 15 of 18 specimens of OTSCC (83.3%, P<0.01). Both of TRB3 and AKT were highly expressed in 13 of 18 (72.2%) specimens of OTSCC comparing with adjacent noncancerous tissues by Western blot assay (P<0.05). TRB3 was significantly elevated in 49.2% (63/128) of pathologically confirmed specimens and 13.3% (4/30) of adjacent noncancerous specimens by immunohistochemical analysis (P<0.01). TRB3 overexpression was closely correlated with tumor pathological T stage, lymph node metastasis and tumor recurrence. In addition, both mRNA and protein expression of TRB3 was increased under thapsigargin (TG) or tunicmycin (TU)-induced ERS in Tca8113 and CAL-27 cells. Moreover, expression of p-AKT protein decreased when Ad-TRB3 was transected with OTSCC Tca8113 cells. However, expression of p-AKT protein increased when TRB3 was inhibited by TRB3 shRNA inhibition. TRB3 expression was closely correlated with OTSCC prognosis. Under ERS, TRB3 was up-regulated, resulting in inhibiting the activation of AKT in OTSCC.