Many chronic inflammatory diseases, such as autoimmune inflammation, are associated with M1 macrophages, and the key to their treatment is blocking inflammation. Oxymatrine (OMT), a traditional Chinese medicine, has a marked anti-inflammatory effect. However, its anti-inflammatory target and mechanism in M1 cells remain unclear, which limits its clinical application. In this study, we investigated the anti-inflammatory effects of oxymatrine (OMT) on the M1 inflammatory response. We also determined the relationship between OMT treatment and the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) pathway with OMT treatment. To this end, we induced the differentiation of human peripheral blood monocytes (THP-1) into M1 cells. THP-1 cells were induced with a phorbol ester (phorbol-12-myristate-13-acetate (PMA)) and differentiated into naïve M0 macrophages. M0 cells were induced into M1 cells using lipopolysaccharide (LPS). The experimental groups were divided into the M0 macrophage group (NC), M1 inflammatory response group (LPS group), and M1 group treated with different concentrations of OMT (LPS + OMT-L, LPS + OMT-M, LPS + OMT-H). The cells in the OMT-treated groups were treated with OMT for 6 h, followed by LPS for 24 h, and the LPS group was treated with LPS only. The resulting supernatants and cells were collected. The secretion levels of NO were detected by the Griess method and the secretion levels of TNF-α and IL-1β in the supernatants were detected by the ELISA method. The secretion levels of these inflammatory factors were reduced in every OMT-treated group compared to the LPS group (P < 0.01), and the most significant reductions were found in the OMT-H group (P < 0.0001). By western blotting, the protein expression levels of TLR4, NF-κB, NLRP3, and Caspase-1 were all found to be downregulated in the cells of OMT-treated groups compared to the LPS group (P < 0.0001). In situ changes in NLRP3 expression were observed using immunofluorescence. The fluorescence intensity of NLRP3 in M1 cells was weaker in all OMT intervention groups than in the LPS group (P < 0.001). In conclusion, OMT has significant anti-inflammatory effects on the M1 inflammatory responses, and the TLR4/NF-κB/NLRP3 pathway was blocked proportional to the concentration of OMT.

Microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome is a severe neurodevelopmental disorder caused by a deficiency in the X-linked gene calcium/calmodulin-dependent serine protein kinase (CASK). A better understanding of the role of CASK in the pathophysiology of neurodevelopmental disorders may provide insights into novel therapeutic and diagnostic strategies for MICPCH syndrome and other neurodegenerative diseases. To investigate this, we generated CASK knockout (KO) cerebellar granule (CG) cell culture from CASK floxed (CASK^flox/flox) mice by infecting lentiviruses expressing codon-improved Cre recombinase (iCre). We performed RNA-sequencing (RNA-seq) on these cells and found that CASK-KO CG cells underwent apoptosis by activating intracellular Jun N-terminal kinase (JNK) signaling and upregulating reactive oxygen species (ROS)-related gene expression. We also performed mouse gait analysis and limb clasping behavior experiments on trans-heterozygous CASK-KO and Hprt-eGFP (CASK^+/- Hprt^eGFP/+) mice. The CASK^+/- Hprt^eGFP/+ mice exhibited cerebellar ataxic phenotypes as judged by the scores of these experiments compared to the CASK wild-type control (CASK^+/+ Hprt^eGFP/+) mice. Interestingly, the administration of the JNK inhibitor, JNK-IN-8, in CASK-KO CG cell cultures increased CG cell survival by reducing ROS generation. Moreover, injection of JNK-IN-8 into the cerebellum of CASK^+/- Hprt^eGFP/+ mice suppressed CG cell death and alleviated cerebellar ataxic phenotypes in vivo. In conclusion, JNK-IN-8 suppresses the cell death and activation of the ROS pathway in CASK-KO CG cells in both in vitro and in vivo models, suggesting its potential as a therapeutic strategy for cerebellar neurodegeneration in MICPCH syndrome.

Microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome is a severe neurodevelopmental disorder caused by a deficiency in the X-linked gene calcium/calmodulin-dependent serine protein kinase (CASK). A better understanding of the role of CASK in the pathophysiology of neurodevelopmental disorders may provide insights into novel therapeutic and diagnostic strategies for MICPCH syndrome and other neurodegenerative diseases. To investigate this, we generated CASK knockout (KO) cerebellar granule (CG) cell culture from CASK floxed (CASK^flox/flox) mice by infecting lentiviruses expressing iCre. We performed RNA-sequencing (RNA-seq) on these cells and found that CASK-KO CG cells underwent apoptosis by activating intracellular Jun N-terminal kinase (JNK) signaling and upregulating reactive oxygen species (ROS) -related gene expression. We also performed mouse gait analysis and limb clasping behavior experiments on trans-heterozygous CASK-KO and Hprt-eGFP (CASK^+/-Hprt^eGFP/+) mice. The CASK^+/-Hprt^eGFP/+ mice exhibited cerebellar ataxic phenotypes as judged by the scores of these experiments compared to the CASK wild-type control (CASK^+/-Hprt^eGFP/+) mice. Interestingly, the administration of the JNK inhibitor, JNK-IN-8, in CASK-KO CG cell cultures increased CG cell survival by reducing ROS generation. Moreover, injection of JNK-IN-8 into the cerebellum of CASK^+/-Hprt^eGFP/+ mice suppressed CG cell death and alleviated cerebellar ataxic phenotypes in vivo. In conclusion, JNK-IN-8 suppresses the cell death and activation of ROS pathway in CASK-KO CG cells in both in vitro and in vivo models, suggesting its potential as a therapeutic strategy for cerebellar neurodegeneration in MICPCH syndrome.