The protective effects of Ninjin'yoeito against liver steatosis/fibrosis in a non-alcoholic steatohepatitis model mouse.

Non-alcoholic steatohepatitis (NASH) is a progressive fibrotic form of non-alcoholic fatty liver disease. Liver fibrosis leads to liver cancer and cirrhosis, and drug therapy for NASH remains lacking. Ninjin'yoeito (NYT) has shown antifibrotic effects in a model of liver fibrosis without steatosis but has not been studied for NASH. Therefore, we evaluated the efficacy of NYT in mice fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) as a NASH model. Compared with the normal diet group, mice fed CDAHFD showed decreased body weight and increased white adipose tissue, liver weight, and triglyceride content in the liver. Furthermore, a substantial increase in the hepatic concentration of hydroxyproline, expression of α-smooth muscle actin (α-SMA), and transforming growth factor-ß was observed in CDAHFD-fed mice. Masson's trichrome and Picro-Sirius red staining revealed a remarkable increase in collagen fiber compared with the normal diet group. Compared with mice that received CDAHFD alone, those supplemented with NYT exhibited reduced hepatic triglyceride and hydroxyproline levels and α-SMA expression. Additionally, compared with the group fed CDAHFD alone, the stained liver tissues of NYT-treated mice exhibited a reduction in Masson's trichrome- and Picro-Sirius red-positive areas. Locomotor activity was significantly reduced in the CDAHFD-fed group compared with the normal diet group. In the NYT-treated group, the CDAHFD-induced decrease in locomotor activity was significantly suppressed. The findings indicate that NYT inhibited fatty and fibrotic changes in the livers of NASH mice and alleviated the decrease in locomotor activity. Therefore, NYT may serve as a novel therapeutic approach for NASH.

Effects of Ninjin'yoeito on Human CYP3A and Mouse CYP3A Activity.

Ninjin'yoeito (NYT) is widely used clinically for the management of patients with frailty and other multiple symptoms. NYT is often administered with other drugs; however, little information is available on its drug interactions. Previous studies using human liver microsomes have reported that constituents of NYT either inhibit (schisandra fruit, cinnamon bark, glycyrrhiza, and poria sclerotium) or induce (schisandra fruit and glycyrrhiza) CYP3A4 expression. Herein, we conducted in vitro and in vivo studies targeting human CYP3A and mouse CYP3A to elucidate the effects of NYT coadministration with other drugs on hepatic drug metabolism. In an inhibition study using human liver microsomes, NYT showed concentration-dependent reversible inhibition and time-dependent inhibition. Furthermore, in an induction study using frozen human hepatocytes, the addition of 0.01-0.1 mg/mL NYT resulted in a concentration-dependent increase in CYP3A gene expression. Contrarily, no significant changes in CYP3A substrate blood concentrations were observed between untreated mice and mice that received either a single dose of NYT or repeated doses for 15 days. These results demonstrate that NYT has inhibitory and inductive effects on hepatic CYP3A in vitro, but orally administered NYT does not affect drug metabolism mediated by hepatic CYP3A in vivo in the mouse model. Although there is a little information about drug interactions of NYT, this study provides new evidence for that.

Ninjin'yoeito ameliorated PPE-induced pulmonary emphysema and anxiety/depressive-like behavior in aged C57BL/6J mice.

The prevalence of chronic obstructive pulmonary disease (COPD) is increasing in the elderly. COPD is a chronic respiratory disease characterized by airway remodeling and alveolar emphysema. COPD patients are also at high risk for mental illnesses such as depression and anxiety. Ninjin'yoeito (NYT) is prescribed to patients with conditions such as post-illness and postoperative weakness, fatigue, poor appetite, skin rash, cold hands and feet, and anemia. In addition to traditional uses, NYT is also prescribed as a therapeutic drug for poor functioning of the digestive organs, respiratory organs, and urinary organs. NYT is also known to have an antioxidant effect. The objective of this study was to investigate whether NYT could ameliorate COPD-induced lung injury and anxiety/depression in aged C57BL/6J mice exposed to porcine pancreatic elastase (PPE). While intratracheal administration of PPE induced emphysema in elderly mice, long-term administration of NYT suppressed the pathology. NYT was also found to suppress the apoptosis and oxidative stress caused by PPE. In addition, long-term administration of NYT was found to ameliorate PPE-induced depressive-like behavior in three different behavioral studies. These results suggest that NYT has a therapeutic effect on emphysema and the behavioral abnormalities caused by PPE.

[Effects of Yokukansankachimpihange on Memantine-induced Dizziness in Mice with Memory Impairment].

Memantine (Mem) is a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist used in the treatment of Alzheimer's disease. However, side effects, including dizziness, headache and confusion, have been reported. Therefore, although it reduces the symptoms of dementia, such as memory loss, its use is limited by side effects for patients at risk of injury. In the present study, we investigated the effects of the Japanese Kampo medicine yokukansankachimpihange (YKSCH) on Mem-induced dizziness in a mouse model of memory impairment. Mem (20 mg/kg B.W., p.o.) reduced the performance score during the beam balance test and walking time on a rotarod, confirming the disrupted sense of balance and motor coordination. In contrast, YKSCH (750 mg/kg B.W., p.o.) significantly suppressed this disruption of balance and motor coordination in mice. Moreover, YKSCH did not attenuate the ameliorative effects of Mem on learning and memory impairment in the Y-maze test or step-through passive avoidance task. Spatial learning and memory significantly recovered in the Mem-treated group and Mem plus YKSCH-treated group, suggesting no pharmacological interaction between Mem and YKSCH in mice. Therefore, YKSCH may be effective at alleviating the Mem-induced equilibrium disturbance in mice with memory impairment without reducing its memory disorder improvement effects. Our study may be useful for future studies on the combined use of Mem and YKSCH in the treatment of Alzheimer's disease.

Neuron-enriched phosphatase and actin regulator 3 (Phactr3)/ nuclear scaffold-associated PP1-inhibiting protein (Scapinin) regulates dendritic morphology via its protein phosphatase 1-binding domain.

Phosphatase and actin regulator 3/nuclear scaffold-associated protein phosphatase 1-inhibiting protein (Phactr3/Scapinin) is an actin- and protein phosphatase 1 (PP1)-binding protein known to negatively regulate axon elongation. In this study, we examined the expression pattern of Phactr3/Scapinin in several tissues and investigated the effect of Phactr3/Scapinin on dendritic morphology of cortical neurons. Results showed that Phactr3/Scapinin expression was up-regulated in the developing brain and enriched in neurons and in the postsynaptic density fraction, but not in astrocytes. Overexpression of wild type or mutant Phactr3/Scapinin, which lacked actin-binding activity, resulted in increased dendritic complexity and percentage of spines with a mushroom or stubby shape, as well as a decrease in spine density. However, overexpression of mutant Phactr3/Scapinin that lacked PP1-binding activity did not. Taken together, these findings suggest that Phactr3/Scapinin expression is neuronal and might contribute to synaptic formation via distinct actin- and PP1-binding domains involved in dendritic and axonal morphology, respectively.

Involvement of SRF coactivator MKL2 in BDNF-mediated activation of the synaptic activity-responsive element in the Arc gene.

The expression of immediate early genes (IEGs) is thought to be an essential molecular basis of neuronal plasticity for higher brain function. Many IEGs contain serum response element in their transcriptional regulatory regions and their expression is controlled by serum response factor (SRF). SRF is known to play a role in concert with transcriptional cofactors. However, little is known about how SRF cofactors regulate IEG expression during the process of neuronal plasticity. We hypothesized that one of the SRF-regulated neuronal IEGs, activity-regulated cytoskeleton-associated protein (Arc; also termed Arg3.1), is regulated by an SRF coactivator, megakaryoblastic leukemia (MKL). To test this hypothesis, we initially investigated which binding site of the transcription factor or SRF cofactor contributes to brain-derived neurotrophic factor (BDNF)-induced Arc gene transcription in cultured cortical neurons using transfection and reporter assays. We found that BDNF caused robust induction of Arc gene transcription through a cAMP response element, binding site of myocyte enhancer factor 2, and binding site of SRF in an Arc enhancer, the synaptic activity-responsive element (SARE). Regardless of the requirement for the SRF-binding site, the binding site of a ternary complex factor, another SRF cofactor, did not affect BDNF-mediated Arc gene transcription. In contrast, chromatin immunoprecipitation revealed occupation of MKL at the SARE. Furthermore, knockdown of MKL2, but not MKL1, significantly decreased BDNF-mediated activation of the SARE. Taken together, these findings suggest a novel mechanism by which MKL2 controls the Arc SARE in response to BDNF stimulation.

Synaptic localisation of SRF coactivators, MKL1 and MKL2, and their role in dendritic spine morphology.

The megakaryoblastic leukaemia (MKL) family are serum response factor (SRF) coactivators, which are highly expressed in the brain. Accordingly, MKL plays important roles in dendritic morphology, neuronal migration, and brain development. Further, nucleotide substitutions in the MKL1 and MKL2 genes are found in patients with schizophrenia and autism spectrum disorder, respectively. Thus, studies on the precise synaptic localisation and function of MKL in neurons are warranted. In this study, we generated and tested new antibodies that specifically recognise endogenously expressed MKL1 and MKL2 proteins in neurons. Using these reagents, we biochemically and immunocytochemically show that MKL1 and MKL2 are localised at synapses. Furthermore, shRNA experiments revealed that postsynaptic deletion of MKL1 or MKL2 reduced the percentage of mushroom- or stubby-type spines in cultured neurons. Taken together, our findings suggest that MKL1 and MKL2 are present at synapses and involved in dendritic spine maturation. This study may, at least in part, contribute to better understanding of the molecular mechanisms underlying MKL-mediated synaptic plasticity and neurological disorders.