Keratinocyte-Derived Cytokine in the Hippocampus Disrupts Extinction of Conditioned Fear Memory in Tumor-Bearing Mice.

While patients with cancer show a higher prevalence of psychiatric disorders than the general population, the mechanism underlying this interaction remains unclear. The present study examined whether tumor-bearing (TB) mice show psychological changes using the conditioned fear paradigm and the role of cytokines in these changes. TB mice were established by transplantation with mouse osteosarcoma AXT cells. These TB mice were then found to exhibit disruption in extinction of conditioned fear memory. Eighteen cytokines in serum were increased in TB mice, among which i.c.v. injection of interleukin (IL)-1ß and IL-6 strengthened fear memory in normal mice. Contents of IL-17 and keratinocyte-derived cytokine (KC) in the amygdala and KC in the hippocampus were increased in TB mice. KC mRNA in both the amygdala and hippocampus was also increased in TB mice, and i.c.v. injection of KC dose-dependently strengthened fear memory in normal mice. In addition, injection of IL-1ß, but not IL-6, increased KC mRNA in the amygdala and hippocampus. In TB mice KC mRNA was increased in both astrocytes and microglia of the amygdala and hippocampus. The microglia inhibitor minocycline, but not the astrocyte inhibitor fluorocitrate, alleviated disruption in extinction of conditioned fear memory in TB mice. Microinjection of KC into the hippocampus, but not into the amygdala, increased fear memory in normal mice. These findings indicate that TB mice show an increase in serum cytokines, including IL-1ß, that increases KC production in microglia of the hippocampus, which then disrupts extinction of fear memory.

Effect of the gut microbiota on the expression of genes that are important for maintaining skin function: Analysis using aged mice.

The gut microbiota changes greatly at the onset of disease, and the importance of intestinal bacteria has been highlighted. The gut microbiota also changes greatly with aging. Aging causes skin dryness, but it is not known how changes in the gut microbiota with aging affects the expression of genes that are important for maintaining skin function. In this study, we investigated how age-related changes in gut microbiota affect the expression of genes that regulate skin function. The gut microbiotas from young mice and aged mice were transplanted into germ-free mice (fecal microbiota transplantation [FMT]). These recipient mice were designated FMT-young mice and FMT-old mice respectively, and the expression levels of genes important for maintaining skin function were analyzed. The dermal water content was significantly lower in old mice than that in young mice, indicating dry skin. The gut microbiota significantly differed between old mice and young mice. The water channel aquaporin-3 (Aqp3) expression level in the skin of FMT-old mice was significantly higher than that in FMT-young mice. In addition, among the genes that play an important role in maintaining skin function, the expression levels of those encoding ceramide-degrading enzyme, ceramide synthase, hyaluronic acid-degrading enzyme, and Type I collagen were also significantly higher in FMT-old mice than in FMT-young mice. It was revealed that the gut microbiota, which changes with age, regulates the expression levels of genes related to skin function, including AQP3.

Placental extract suppresses lipid droplet accumulation by autophagy during the differentiation of adipose-derived mesenchymal stromal/stem cells into mature adipocytes.

OBJECTIVE: Placental extract, which contains various bioactive compounds, has been used as traditional medicine. Many studies have demonstrated additional applications of placental extract and provided a scientific basis for the broad spectrum of its effects. We have previously reported that porcine placental extract (PPE) strongly suppresses adipogenesis in a 3T3-L1 preadipocyte cell line, inhibiting differentiation. This study aimed to examine the effect of PPE on the accumulation of lipid droplets (LD) in adipose-derived mesenchymal stromal/stem cells (ASC). RESULTS: The study findings revealed that PPE decreased the size of LD during the differentiation of ASC into mature adipocytes. RT-qPCR analysis revealed that PPE increased the gene expression of lysosomal acid lipase A (Lipa), a lipolysis-related gene, in ASC-differentiated adipocytes. However, no differences were noted in the adipocyte differentiation markers (Pparg, Cebpa, and Adipoq), or the adipogenesis-related genes (Dgat1, Dgat2, Fasn, Soat1, and Soat2). In addition, PPE promoted autophagosome formation, which was partially co-localized with the LD, indicating that PPE accelerated the degradation of LD by inducing autophagy (termed lipophagy) during the differentiation of ASC into mature adipocytes. These results suggest that the use of PPE may be a potential novel treatment for regulating adipogenesis for the treatment of obesity.

Optimization of QuEChERS Extraction for Determination of Carotenoids, Polyphenols, and Sterols in Orange Juice Using Design of Experiments and Response Surface Methodology.

Several compounds with different physical properties are present in foods, biological components, and environmental samples, and there are cases in which these must be analyzed simultaneously. However, it is difficult to extract compounds with different physical properties from the same sample using a single method. In the present study, we examined the optimal conditions for the QuEChERS extraction of several kinds of compounds from orange juice using design of experiments (DoE) and response surface methodology (RSM) to determine the optimal ratio of organic solvent to sodium chloride. We determined the optimal extraction conditions, which were within the design space, using 100% tetrahydrofuran (THF) as the extraction organic solvent and NaCl:MgSO4 = 75:25 as the salt. The developed LC/MS/MS method using QuEChERS extraction achieved specific detection and precise quantification. Finally, we measured the polyphenols, sterols, and carotenoids in citrus juice using the optimized QuEChERS extraction method before LC/MS/MS analysis. Most of the analytes were quantifiable in orange juice. The optimized method achieved ease of operation, the extraction of analytes from food samples in a short time (within 30 min), minimization of analytical residues, and reliability. The DoE and RSM approach may contribute to better optimization of the extraction conditions for the lowest number of experiments.

Development of a novel drug information provision system for Kampo medicine using natural language processing technology.

BACKGROUND: Kampo medicine is widely used in Japan; however, most physicians and pharmacists have insufficient knowledge and experience in it. Although a chatbot-style system using machine learning and natural language processing has been used in some clinical settings and proven useful, the system developed specifically for the Japanese language using this method has not been validated by research. The purpose of this study is to develop a novel drug information provision system for Kampo medicines using a natural language classifier® (NLC®) based on IBM Watson. METHODS: The target Kampo formulas were 33 formulas listed in the 17th revision of the Japanese Pharmacopoeia. The information included in the system comes from the package inserts of Kampo medicines, Manuals for Management of Individual Serious Adverse Drug Reactions, and data on off-label usage. The system developed in this study classifies questions about the drug information of Kampo formulas input by natural language into preset questions and outputs preset answers for the questions. The system uses morphological analysis, synonym conversion by thesaurus, and NLC®. We fine-tuned the information registered into NLC® and increased the thesaurus. To validate the system, 900 validation questions were provided by six pharmacists who were classified into high or low levels of knowledge and experience of Kampo medicines and three pharmacy students. RESULTS: The precision, recall, and F-measure of the system performance were 0.986, 0.915, and 0.949, respectively. The results were stable even with differences in the amount of expertise of the question authors. CONCLUSIONS: We developed a system using natural language classification that can give appropriate answers to most of the validation questions.

Green tea extract prevents CPT-11-induced diarrhea by regulating the gut microbiota.

Irinotecan (CPT-11) is an anticancer drug with indications for use in treating various cancers, but severe diarrhea develops as a side effect. We investigated the effects of green tea extract (GTE) on CPT-11-induced diarrhea, focusing on ß-glucuronidase and intestinal UGT1A1. When CPT-11 was administered to rats alone, the fecal water content was approximately 3.5-fold higher in this group than in the control group, and diarrhea developed. The fecal water content in the GTE-treated group was significantly higher than that in the control group, but the difference was smaller than that between the group treated with CPT-11 alone and the control group, and diarrhea improved. When CPT-11 was administered alone, the abundances of Bacteroides fragilis and Escherichia coli, which are ß-glucuronidase-producing bacteria, increased and interleukin-6 and interleukin-1ß mRNA levels in the colon increased, but GTE suppressed these increases. CPT-11 decreased colon UGT1A1 and short-chain fatty acid levels; however, this decrease was suppressed in the GTE-treated group. The findings that GTE decreases the abundance of ß-glucuronidase-producing bacteria and increases colon UGT1A1 levels, thereby decreasing the production of the active metabolite SN-38 in the intestinal tract, indicate that GTE ameliorates CPT-11-induced diarrhea.

Effect of partially hydrolyzed guar gum on the expression of aquaporin-3 in the colon.

In recent years, the development of functional foods targeting gastrointestinal disorders has been in progress. Partially hydrolyzed guar gum (PHGG), which is a water-soluble dietary fiber, is known to have a constipation-improving effect. However, many aspects of the mechanism remain unclear. In this study, we investigated the role of aquaporin-3 (AQP3), which regulates the water content of feces in ameliorative effect of PHGG on constipation. Rats were allowed to freely consume a normal diet or a diet containing 5% PHGG for 14 days, and defecation parameters were measured. We also analyzed the expression levels of genes involved in water transport in the colon. The defecation frequency and volume of rats treated with PHGG were not different from those from the control group, but the fecal water content was significantly increased, and soft stools were observed. The expressions of claudin-1, tight junction protein-1, and cadherin-1, which are involved in tight junctions or adherens junctions, were almost the same in the PHGG-treated group and the control group. The expression level of AQP3 in the colon was significantly decreased in the PHGG-treated group. In this study, PHGG decreased the colonic AQP3 expression, thereby suppressing water transport from the luminal side to the vascular side and improving constipation.

Effect of Chimpi, dried citrus peel, on aquaporin-3 expression in HaCaT human epidermal keratinocytes.

BACKGROUND: Chimpi, the dried peel of Citrus unshiu or Citrus reticulata, has various pharmacological effects. Chimpi extract was recently shown to affect the skin, including its inhibitory effect against atopic dermatitis. In this study, we analyzed the effects of Chimpi extract on the functional molecule aquaporin-3 (AQP3), which is involved in water transport and cell migration in the skin. METHODS AND RESULTS: Chimpi extract was added to HaCaT human skin keratinocytes, and the AQP3 expression level was analyzed. A wound healing assay was performed to evaluate the effect of Chimpi extract on cell migration. The components of Chimpi extract and fractions obtained by liquid-liquid distribution studies were added to HaCaT cells, and AQP3 expression was analyzed. Chimpi extract significantly increased AQP3 expression in HaCaT cells at both the mRNA and protein levels. Immunocytochemical staining revealed that Chimpi extract also promoted the transfer of AQP3 to the cell membrane. Furthermore, Chimpi extract enhanced cell migration. Hesperidin, narirutin, and nobiletin did not increase AQP3 levels. Although the components contained in the fractions obtained from the chloroform, butanol, and water layer increased AQP3, the active components could not be identified. CONCLUSIONS: These results reveal that Chimpi extract may increase AQP3 levels in keratinocytes and increase the dermal water content. Therefore, Chimpi extract may be effective for the management of dry skin.

Downregulation of Sparc-like protein 1 during cisplatin-induced inhibition of myogenic differentiation of C2C12 myoblasts.

Patients with cancer often experience muscle atrophy, which worsens their prognosis. Decreased muscle regenerative capacity plays an important role in the complex processes involved in muscle atrophy. Administration of cisplatin, a cancer chemotherapeutic agent, has been implicated as a cause of muscle atrophy. In this study, we examined whether cisplatin affects the differentiation of myoblasts into myotubes. We treated C2C12 myoblasts with a differentiation medium containing cisplatin and its vehicle during for 8 days and observed the changes in the expression of myosin heavy chain (MyHC) and myogenin in the myoblasts. Cisplatin was injected in mice for 4 consecutive days; on Day 5, the mice quadriceps muscles were sampled and examined. The expression of MyHCs increased and that of myogenin decreased after cisplatin treatment. The secretion of acidic cysteine-rich proteins (e.g., Sparc proteins) reportedly promotes C2C12 myoblast differentiation. Therefore, we investigated the Sparc family gene expression during myogenesis in C2C12 myoblasts after cisplatin treatment. Of all the genes investigated, Sparc-like protein 1 (Sparcl1) expression was significantly suppressed by cisplatin on Days 4-8. Simultaneous treatment with recombinant mouse Sparcl1 almost inhibited the cisplatin-induced suppression of total MyHC and myogenin protein levels. Moreover, Sparcl1 expression decreased in the skeletal muscles of mice, leading to cisplatin-induced muscle atrophy. Our results suggest that cisplatin-induced myogenesis suppression causes muscle atrophy and inhibits the expression of Sparcl1, which promotes C2C12 cell differentiation during myogenesis.

Increased 20S Proteasome Expression and the Effect of Bortezomib during Cisplatin-Induced Muscle Atrophy.

Cisplatin is a chemotherapy drug used to treat a variety of cancers. Muscle loss in cancer patients is associated with increased cancer-related mortality. Previously, we suggested that cisplatin administration increases the atrophic gene expressions of ubiquitin E3 ligases, such as atrogin-1 and muscle RING finger-1 (MuRF1), which may lead to muscle atrophy. In this study, C57BL/6J mice were treated with cisplatin (3 mg/kg, intraperitoneally) or saline for 4 consecutive days. Twenty-four hours after the final injection of cisplatin, quadriceps muscles were removed from the mice. The gene expression of Psma and Psmb, which comprise the 20S proteasome, was upregulated by cisplatin administration in the quadriceps muscle of mouse. Systemic administration of cisplatin significantly reduced not only the quadriceps muscle mass but also the diameter of the myofibers. In addition, bortezomib (0.125 mg/kg, intraperitoneally) was administered 30 min before each cisplatin treatment. The co-administration of bortezomib, a proteasome inhibitor, significantly recovered the reductions in the mass of quadriceps and myofiber diameter, although it did not recover the decline in the forelimb and forepaw strength induced by cisplatin. Increased 20S proteasome abundance may play a significant role in the development of cisplatin-induced muscle atrophy. During cisplatin-induced skeletal muscle atrophy, different mechanisms may be involved between loss of muscle mass and strength. In addition, it is suggested that bortezomib has essentially no effect on cisplatin-induced muscle atrophy.

Inhibition of Spred/Sprouty Expression in the Skin of a Contact Dermatitis-Like Model.

We have previously reported that swellings caused by haptens, such as 2,4,6-trinitrochlorobenzene (TNCB), may be associated with the extracellular signal-regulated kinase (ERK)-induced proliferation pathway. However, the involvement of the Spred/Sprouty family as critical negative regulators of the Ras/Raf/ERK signaling pathway at disease sites is not well-established. Thus, in the present study, the effects of hapten-challenge on the expression levels of genes and proteins associated with the Spred/Sprouty family in the ear of mice were investigated. The activation of ERK and epidermal growth factor receptor (EGFR) tyrosine kinase was inhibited by their selective inhibitors, namely, U0126 and PD168393, respectively. Twenty-four hours after the final challenge by the haptens TNCB, 2,4-dinitrofluorobenzene, or oxazolone, ear thickness was augmented by challenge with all haptens and the gene expression levels of Spred1, Spred2, Sprouty1, and Sprouty2 in swelling induced by all haptens were significantly decreased. Furthermore, Spred2, Sprouty1, and Sprouty2 genes were decreased in the epidermis and dermis of the TNCB-challenged ear. In conclusion, it is possible that the mechanism of hapten-challenge-induced skin thickening involves not only the enhancement of cell proliferative functions via the activation of ERK by EGFR tyrosine kinase activation but also the decreases expression of Spred/Sprouty family members.

Copper-Induced Interactions of Caffeic Acid and Sinapic Acid to Generate New Compounds in Artificial Biological Fluid Conditions.

Active ingredients may be ingested through foods, and they can cause several interactions in the human body. Although drug-drug or drug-food interactions are evaluated before the approval of medicines, several functional food interactions are not well-documented because of the wide range of possible combinations of interactions. In this study, we examined the chemical reactions between hydroxycinnamic acids (HCAs), a group of polyphenols, and metal ions in artificial gastric juice or artificial intestinal fluid. Caffeic acid (CaA) and sinapic acid (SA) reacted with copper ions under artificial intestinal fluid conditions and produced new compounds. The triple interactions of CaA or SA with iron and copper ions were also examined. Relative to the initial compounds, CaA and SA derivatives produced by condensation exhibited an increased antioxidant and a decreased prooxidant activity. This study revealed a new food ingredient interaction pattern in which new compounds are produced under biological conditions.

Determination of neurotransmitters in mouse brain using miniaturized and tableted QuEChERS for the sample preparation.

Neurotransmitters (NTs) are important endocrine molecules in the human body that have several characteristics and functions. Therefore, it is necessary to determine the accuracy and precision of the proposed method for the assessment of NTs functions. This study proposes a quick, easy, cheap, effective, rugged, and safe (QuEChERS) tablet for easy sample preparation and application to low-volume samples. We used response surface methodology to design and optimize the QuEChERS tablets. When we measured NTs in the brains of aged mice, whose samples were derivatized by DNS and extracted by QuEChERS, all analytes except VMA, NAS, 5-HTP, DOPA, and A, were detected in the mouse brain samples. Our approach may provide easy sample preparation and/or extraction techniques for small sample volumes, not only in animal samples but also in human samples to avoid invasive collection.

The Mechanism of Pertussis Cough Revealed by the Mouse-Coughing Model.

Pertussis, also known as whooping cough, is a contagious respiratory disease caused by the Gram-negative bacterium Bordetella pertussis. This disease is characterized by severe and uncontrollable coughing, which imposes a significant burden on patients. However, its etiological agent and the mechanism are totally unknown because of a lack of versatile animal models that reproduce the cough. Here, we present a mouse model that reproduces coughing after intranasal inoculation with the bacterium or its components and demonstrate that lipooligosaccharide (LOS), pertussis toxin (PTx), and Vag8 of the bacterium cooperatively function to cause coughing. Bradykinin induced by LOS sensitized a transient receptor potential ion channel, TRPV1, which acts as a sensor to evoke the cough reflex. Vag8 further increased bradykinin levels by inhibiting the C1 esterase inhibitor, the major downregulator of the contact system, which generates bradykinin. PTx inhibits intrinsic negative regulation systems for TRPV1 through the inactivation of Gi GTPases. Our findings provide a basis to answer long-standing questions on the pathophysiology of pertussis cough. IMPORTANCE The Gram-negative bacterium Bordetella pertussis causes a respiratory disease called whooping cough, or pertussis. This disease is characterized by paroxysmal coughing, the mechanism of which has not been intensively studied because of a lack of versatile animal models that reproduce the cough. In this study, we present a mouse model that reproduces coughing after intranasal inoculation with the bacterium or its components. Using this model, we demonstrate that lipooligosaccharide, Vag8, and pertussis toxin of the bacteria cooperatively function to cause coughing. Our results also indicate that bradykinin, an inflammatory mediator, and TRPV1, an ion channel linked to nociceptive signaling, are host factors involved in the coughing mechanism.

Eicosapentaenoic acid suppresses cisplatin-induced muscle atrophy by attenuating the up-regulated gene expression of ubiquitin.

Previously it was shown that cisplatin causes muscle atrophy. Under this condition, cisplatin increased the expression of atorogenes, such as muscle ring finger 1 and atrogin-1 (also known as muscle atrophy F-box protein), in mouse skeletal muscle. It was reported recently that ubiquitin (Ub) and ubiquitinated protein levels in skeletal muscle were also up-regulated in cisplatin-induced muscle atrophy, and cisplatin-induced ubiquitinated proteins were degraded by the 26S proteasome pathway. Eicosapentaenoic acid (EPA) is effective against skeletal muscle atrophy in mice. However, it is unclear how EPA suppresses the Ub-proteasome pathway. In this study, the effect of EPA on cisplatin-induced muscle atrophy in mice was examined. Mice were intraperitoneally injected with cisplatin or vehicle control once daily for 4 d. EPA or its vehicle was orally administered 30 min before cisplatin administration. Cisplatin systemic administration induced decrease in muscle mass, myofiber diameter, and increase in Ub genes and ubiquitinated proteins in mouse skeletal muscle were recovered by co-treatment with EPA. However, weight loss and up-regulated atrogenes induced by cisplatin were not changed by co-treatment with EPA in skeletal muscle. In this study, EPA attenuated cisplatin-induced muscle atrophy via down-regulation of up-regulated Ub gene expression. Although further clinical studies are needed, EPA administration can be effective in the development of muscle atrophy in cisplatin-treated patients.

[Recent Findings on the Mechanism of Cough Hypersensitivity as a Cause of Chronic Cough].

An increasing number of patients complain to medical institutions about a cough that persists for more than 8 weeks, namely chronic cough. The cough observed in patients with chronic cough is not responsive to conventional antitussive agents such as dihydrocodeine and dextromethorphan, and this is a major clinical problem. The most common pathology of chronic cough in Japan is dry cough. Two causes of dry cough are increased sensitivity of cough receptors (cough hypersensitivity) and increased contraction of bronchial smooth muscle. Among these, the mechanisms of cough hypersensitivity are diverse, and understanding these mechanisms is important for the diagnosis and treatment of chronic cough. In this paper I will review the regulatory mechanisms of cough hypersensitivity, especially the regulation of Aδ fiber excitability by C fibers. Furthermore, the central mechanisms involved cough reflex are discussed in relation to central acting antitussives.

Cannabidiol Application Increases Cutaneous Aquaporin-3 and Exerts a Skin Moisturizing Effect.

Cannabidiol (CBD) is a major nonpsychotropic component of Cannabis sativa with various pharmacological activities. In this study, we investigated the skin moisturizing effect of CBD and its mechanism. A 1% CBD solution was applied daily to skin of HR-1 hairless (Seven-week-old, male) for 14 days. The dermal water content in CBD-treated mice was significantly increased compared to that in the control group. Furthermore, no inflammatory reaction in the skin and no obvious skin disorders were observed. The mRNA expression levels of loricrin, filaggrin, collagen, hyaluronic acid degrading enzyme, hyaluronic acid synthase, ceramide degrading enzyme, and ceramide synthase in the skin were not affected by the application of CBD. However, only aquaporin-3 (AQP3), a member of the aquaporin family, showed significantly higher levels in the CBD-treated group than in the control group at both the mRNA and protein levels. It was revealed that CBD has a moisturizing effect on the skin. In addition, it is possible that increased expression of AQP3, which plays an important role in skin water retention, is a contributor to the mechanism. CBD is expected to be developed in the future as a cosmetic material with a unique mechanism.

Exogenous insulin-like growth factor 1 attenuates cisplatin-induced muscle atrophy in mice.

BACKGROUND: A reduction in the skeletal muscle mass worsens the prognosis of patients with various cancers. Our previous studies indicated that cisplatin administration to mice caused muscle atrophy. This is a concern for human patients receiving cisplatin. The insulin-like growth factor 1 (IGF-1)/phosphoinositide 3-kinase (PI3K)/Akt pathway stimulates the rate of protein synthesis in skeletal muscle. Thus, IGF-I can be a central therapeutic target for preventing the loss of skeletal muscle mass in muscle atrophy, although it remains unclear whether pharmacological activation of the IGF-1/PI3K/Akt pathway attenuates muscle atrophy induced by cisplatin. In this study, we examined whether exogenous recombinant human IGF-1 attenuated cisplatin-induced muscle atrophy. METHODS: Male C57BL/6J mice (8-9 weeks old) were injected with cisplatin or saline for four consecutive days. On Day 5, quadriceps muscles were isolated. Mecasermin (recombinant human IGF-1) or the vehicle control was subcutaneously administered 30 min prior to cisplatin administration. A dietary restriction group achieving weight loss equivalent to that caused by cisplatin administration was used as a second control. C2C12 myotubes were treated with cisplatin with/without recombinant mouse IGF-1. The skeletal muscle protein synthesis/degradation pathway was analysed by histological and biochemical methods. RESULTS: Cisplatin reduced protein level of IGF-1 by about 85% compared with the vehicle group and also reduced IGF-1/PI3K/Akt signalling in skeletal muscle. Under this condition, the protein levels of muscle ring finger protein 1 (MuRF1) and atrophy gene 1 (atrogin-1) were increased in quadriceps muscles (MuRF1; 3.0 ± 0.1 folds, atrogin-1; 3.0 ± 0.3 folds, P < 0.001, respectively). The administration of a combination of cisplatin and IGF-1 significantly suppressed the cisplatin-induced downregulation of IGF-1/PI3K/Akt signalling and upregulation of MuRF1 and atrogin-1 (up to 1.6 ± 0.3 and 1.5 ± 0.4 folds, P < 0.001, respectively), resulting in diminished muscular atrophy. IGF-1 showed similar effects in cisplatin-treated C2C12 myotubes, as well as the quadriceps muscle in mice. CONCLUSIONS: The downregulation of IGF-1 expression in skeletal muscle might be one of the factors playing an important role in the development of cisplatin-induced muscular atrophy. Compensating for this downregulation with exogenous IGF-1 suggests that it could be a therapeutic target for limiting the loss of skeletal muscle mass in cisplatin-induced muscle atrophy.

Using Mobile Phone Data to Estimate the Relationship between Population Flow and Influenza Infection Pathways.

This study aimed to analyze population flow using global positioning system (GPS) location data and evaluate influenza infection pathways by determining the relationship between population flow and the number of drugs sold at pharmacies. Neural collective graphical models (NCGMs; Iwata and Shimizu 2019) were applied for 25 cell areas, each measuring 10 × 10 km2, in Osaka, Kyoto, Nara, and Hyogo prefectures to estimate population flow. An NCGM uses a neural network to incorporate the spatiotemporal dependency issue and reduce the estimated parameters. The prescription peaks between several cells with high population flow showed a high correlation with a delay of one to two days or with a seven-day time-lag. It was observed that not much population flows from one cell to the outside area on weekdays. This observation may have been due to geographical features and undeveloped transportation networks. The number of prescriptions for anti-influenza drugs in that cell remained low during the observation period. The present results indicate that influenza did not spread to areas with undeveloped traffic networks, and the peak number of drug prescriptions arrived with a time lag of several days in areas with a high amount of area-to-area movement due to commuting.