Streptococcus suis, a major zoonotic pathogen that causes problems in human health and in the global swine industry, is an elongated and ellipsoid bacterium that undergoes successive parallel splitting perpendicular to its long axis. Studies on cell cycle processes in this bacterium are limited. Here, we report that MsmK (multiple sugar metabolism protein K), an ATPase that contributes to the transport of multiple carbohydrates, has a novel role as a cell division protein in S. suis. MsmK can display ATPase and GTPase activities, interact with FtsZ via the N terminus of MsmK, and promote the bundling of FtsZ protofilaments in a GTP-dependent manner in vitro. Deletion of the C-terminal region or the Walker A or B motif affects the affinity between MsmK and FtsZ and decreases the ability of MsmK to promote FtsZ protofilament bundling. MsmK can form a complex with FtsZ in vivo, and its absence is not lethal but results in long chains and short, occasionally anuclear daughter cells. Superresolution microscopy revealed that the lack of MsmK in cells leads to normal septal peptidoglycan walls in mother cells but disturbed cell elongation and peripheral peptidoglycan synthesis. In summary, MsmK is a novel cell division protein that maintains cell shape and is involved in the synthesis of the peripheral cell wall.Bacteria of different shapes have adopted distinct mechanisms to faithfully coordinate morphogenesis and segregate their chromosomes prior to cell division. Despite recent focuses and advances, the mechanism of cell division in ovococci remains largely unknown. IMPORTANCE Bacterial cell division is a highly ordered process regulated in time and space and is a potential target for the development of antimicrobial drugs. Bacteria of distinct shapes depend on different cell division mechanisms, but the mechanisms used by ovococci remain largely unknown. Here, we focused on the zoonotic pathogen Streptococcus suis and identified a novel cell division protein named MsmK, which acts as an ATPase of the ATP-binding cassette-type carbohydrate transport system. MsmK has GTPase and ATPase activities. In vitro protein assays showed that MsmK interacts with FtsZ and promotes FtsZ protofilament bundling that relies on GTP. Superresolution microscopy revealed that MsmK maintains cell shape and is involved in peripheral peptidoglycan synthesis. Knowledge of the multiple functions of MsmK may broaden our understanding of known cell division processes. Further studies in this area will elucidate how bacteria can faithfully and continually multiply in a constantly changing environment. Bacillus subtilis and Escherichia coli) or spherical  microorganisms, the molecular basis underlying morphogenesis in ovococci is poorly studied  at midcell (Peptidoglycan (PG) is the major constituent of the bacterial cell wall , and cel midcell . The rin midcell . Unlike  midcell . Periphe midcell . Accordi midcell . If sept midcell . DespiteStreptococcus suis is a major zoonotic pathogen that affects humans and swine worldwide  in cell division for the first time. As a component of the carbohydrate transport system, MsmK binds to FtsZ in vitro and in vivo. MsmK can promote FtsZ protofilament bundling in vitro. The deletion of MsmK in S. suis increases the number of cells in the chain and leads to abnormal cell morphology. On the basis of observation of PG synthesis pattern, MsmK is a novel element that maintains cell shape and is involved in the synthesis of the peripheral cell wall.Here, we aimed to identify novel cell division proteins in S. suis was constructed through homologous protein mapping. Predicted results showed that MsmK, an ATPase belonging to the ATP-binding cassette (ABC)-type sugar transport system, contributes to multiple-carbohydrate utilization , the dissociation constant (dK) or equilibrium dissociation constant (KD) of the MsmK-FtsZ complex is lower than that obtained from the N_MsmK-FtsZ complex. This result revealed a high-affinity interaction between MsmK and FtsZ and a weak binding of N_MsmK to FtsZ.A bacterial two-hybrid assay was conducted. The fusion strains carrying full-length not grow . The fingth MsmK  or its Ngth MsmK  producedgth MsmK  or buffeDK values, the affinity among the four FtsZ-interacting MsmK constructs was in the order A_MsmK > B_MsmK > MsmK > N_MsmK . As shown in The polymerization characteristics of MsmK and its derivatives in the presence of KTEM was performed to investigate the morphological changes of FtsZ during protofilament formation and bundling. FtsZ assembled into an extensive network of long and thin polymers in the presence of GTP but not ATP . As show10.1128/mSphere.00119-21.5FIG S2FIG S2, TIF file, 0.2 MB.Electron microscopic examination of the C_MsmK and FtsZ polymerization reaction. A sample of 0.6 μM FtsZ was incubated with 0.6 μM C_MsmK. Reactions were started by adding 2 mM GTP. Download Copyright © 2021 Tan et al.2021Tan et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Ultracentrifugation was used to sediment FtsZ protofilaments and any bound proteins . The add10.1128/mSphere.00119-21.4FIG S1FIG S1, TIF file, 2.1 MB.Results of sedimentation assays. Diverse sedimentation results of FtsZ in the presence of MsmK (A), MsmK and ATP (B), N_MsmK (C), C_MsmK (D), A_MsmK (E), and B_MsmK (F). Lane M represents molecular weight markers. Polymerization was induced by addition of 2 mM GTP. If needed, 1 mM ATP was added. Polymers were recovered through ultracentrifugation and analyzed by Coomassie blue staining of electrophoresis gels. Download Copyright © 2021 Tan et al.2021Tan et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. suis strain SC19 (serotype 2) was isolated from a diseased pig during an epidemic outbreak in 2005 in Sichuan, China (msmK strain) was previously prepared  slightly decreased at the late logarithmic phase (Wild-type (WT) n, China . The eryprepared . The E. entation . During ic phase . The proic phase .msmK expression cassette with a coding sequence for a His tag at the C terminus was constructed and electroporated into the ΔmsmK strain, which produced the strain CΔmsmK-His, to study in vivo complexes containing MsmK and FtsZ. Exponentially growing cells of CΔmsmK-His were cross-linked using formaldehyde and then subjected to immunoprecipitation experiments on nickel-nitrilotriacetic acid (Ni-NTA) resins. The input mixed bacterial proteins and the final eluted protein fractions were detected by Western blotting analysis by using antibodies to His tag, MsmK, and FtsZ. Control mock pulldowns were run on cross-linked cells of CΔmsmK expressing MsmK instead of the His-tagged MsmK and were used as the background control. The prey protein, endogenous FtsZ of S. suis, was attached to the bait protein  at the C terminus    and TEM py (SEM)  were accWT cells . AnalysimsmK cells (n = 370), and the cells with lengths of 1.0 to 1.1 μm represented the highest proportion. The lengths of the ΔmsmK cells were distributed in the range 0.4 to 1.3 μm (n = 352), and the cells with lengths of 0.8 to 0.9 μm represented the highest proportion. The proportions of WT cells with lengths of >1.0, 0.8 to 1.0, and <0.8 μm were 67.57% (250/370), 32.43% (120/370), and 0% (0/370), respectively. In comparison, the proportions of ΔmsmK cells with lengths of >1.0, 0.8 to 1.0, and <0.8 μm were 30.68% (108/352), 42.33% (149/352), and 26.99% (95/352), respectively. On average, the cells lacking MsmK were significantly shorter (0.89 ± 0.20 μm) than the WT cells (1.05 ± 0.14 μm)  was also significantly thinner than that in WT cells   . The widWT cells . TherefoWT cells . Calculan = 370) . No diffn = 370) .msmK cells still displayed symmetrical cell division sites and equal-sized daughter cells either in chains or in diplococcal morphology  with an average value of 0.98 ± 0.15 μm (msmK cells was 24.96 ± 0.15 nm (n = 380). Therefore, msmK complementation fully restored the mutant morphology and size, and the above defective phenotypes of the ΔmsmK cells were caused by msmK deletion. In summary, lack of MsmK leads to cell rounding, suggesting that this protein is involved in the maintenance of S. suis cell shape.The recombinant plasmid with the  mutants , and CΔmWT cells . Analysi 0.15 μm . On averS. pneumoniae, produces similar-looking chains of nearly spherical cells. The decreased length-to-width ratio in ΔmsmK cells suggests that MsmK might be involved in peripheral cell wall synthesis. Here, the morphology of nascent biosynthesized PG was examined by using TAMRA-d-lysine   and subjected to structured illumination microscopy to achieve colocalization. Superresolution micrographs of the WT cells displayed a typical pattern, with cell wall synthesis labeled at the midcell and equators of future daughter cells  . S. suiser cells . As expeer cells , therebyer cells , and minS. suis strains were grown in the presence of HCC-amino-d-alanine (HADA), a fluorescent hydroxycoumarin derivative of d-alanine  are control elements in ovococci that coordinate several key processes over the cell cycle . The S. and MapZ . After iragments , thereby10.1128/mSphere.00119-21.6FIG S3in vitro. Purified MsmK and its fragments were subjected to phosphorylation by kinase STK in the presence of [γ-32P]ATP. Samples were resolved by SDS-PAGE, stained with Coomassie blue, and exposed to a sensitive screen. The image on the right shows the radioactive signal. Myelin basic protein (MBP) was used as a positive control to test the kinase activity of STK. Download FIG S3, TIF file, 1.3 MB.Kinase assay Copyright © 2021 Tan et al.2021Tan et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. suis  and FtsX (SSUSC84_1270). Moreover, the chain phenotype of the mutant suggests that the PcsB function is affected in the absence of MsmK. As the ATPase subunit in S. suis  was isolated from a sick pig during an epidemic outbreak of S. suis infections in Sichuan Province, China  with 5% (vol/vol) fetal bovine serum . E. coli strains were grown in lysogeny broth (Difco) at 37°C. When necessary, the following antibiotics were added, as appropriate: erythromycin (90 μg/ml), streptomycin (20 μg/ml), spectinomycin (100 μg/ml), kanamycin (25 μg/ml), chloromycetin (25 μg/ml), and tetracycline (12.5 μg/ml). Unless otherwise specified, all antibiotics, chemicals, and substrates were purchased from Biosharp .Bacterial strains and plasmids are listed in e, China . The msmK strain . For themK cells . The con10.1128/mSphere.00119-21.1TABLE S1Table S1, DOC file, 0.07 MB.Bacterial strains and plasmids used in this study. Download Copyright © 2021 Tan et al.2021Tan et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00119-21.3TEXT S1Text S1, DOC file, 0.10 MB.Supplemental materials and methods. Download Copyright © 2021 Tan et al.2021Tan et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Oligonucleotides used are listed in 10.1128/mSphere.00119-21.2TABLE S2Table S2, DOC file, 0.05 MB.Primers used in this work. Download Copyright © 2021 Tan et al.2021Tan et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In vivo interaction assays were performed as described previously  buffer at 4°C. A 10% paraformaldehyde solution was added for cross-linking to a final concentration of 0.1% (vol/vol). Mixtures were incubated at 37°C for 1 h. Exactly 1.0 M glycine was added and incubated at 25°C for 10 min to abolish cross-linking reactions. The cells were then collected, washed, and resuspended with lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mg/ml lysozyme, 1% Triton X-100 (vol/vol), and 1% phenylmethylsulfonyl fluoride (vol/vol). After sonication, bacterial debris was removed through centrifugation at 14,000 × g for 10 min at 4°C. The supernatant of each strain was incubated with Ni-NTA columns  at 4°C for 2 h in binding buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 25 mM imidazole. The beads were washed four times with 80 mM imidazole for 5 min each. His-tagged protein was eluted by incubating with 250 mM imidazole for 1.5 h at 4°C. The original supernatant (input) and the final elution of each strain were separated by polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Invitrogen). The membrane was then probed with mouse antihistidase antibody , in-house-prepared anti-MsmK serum , or in-house-prepared anti-FtsZ serum . This experiment was performed in triplicate.eviously  with fewg at 4°C. Proteins (0.6 μM each) were incubated with 2 mM GTP and/or 1 mM ATP for 10 min at 37°C in buffer P  were incubated at 37°C for 2 min in buffer P to analyze FtsZ polymerization in vitro. Afterward, 2 mM GTP and/or 1 mM ATP was added into the mixtures, which were continuously incubated for 10 min at 37°C. Mixtures were then withdrawn and applied to glow-discharged carbon-coated grids. The grids were air-dried and visualized using an H-7650 TEM .All purified proteins were precentrifuged for 10 min at 16,000 × buffer P  to obser600 of 0.5 and fixed overnight with 2.5% glutaraldehyde at 4°C to observe the bacterial morphology under TEM. For SEM, the cell cultures were grown in TSB to an OD600 of 0.5 and spotted onto poly-l-lysine-coated coverslips, followed by washing with PBS buffer. The cells were fixed overnight with 2.5% glutaraldehyde at 4°C. Subsequent dehydration steps were performed using ethanol as previously described  and the goat anti-mouse IgG (heavy plus light chain [H+L])-Cy3 conjugate  for 1 h at 37°C. Cells were transferred onto microscope slides and covered by 1% agarose gel. Superresolution images were obtained using a Nikon structured illumination microscope  equipped with an electron-multiplying charge-coupled device camera  and a total internal reflection fluorescence objective . Images were captured with multiple phases and angles of illumination pattern before direct reconstruction by using the Nikon NIS-Elements AR software.The immunofluorescence microscopy of cytoplasmic proteins was performed as previously described , with a msmK, and CΔmsmK strains were grown to an OD600 of 0.5 and then centrifuged at 9,000 × g for 5 min. After washing with PBS, the cells were resuspended in 1 ml of PBS containing 5 μl of TDL and 5 μl of AF 647. The mixtures were incubated for 1 h at 37°C in the dark. The cells were then washed with PBS and stained with 1.5 μg/ml DAPI for 5 min in the dark at room temperature. Following the same incubation and pretreatment steps, the cells were incubated in 1 ml of PBS containing 5 μl of HADA for 30 min at 37°C in the dark to observe the cell wall contours.TDL and HADA  were synt tests in GraphPad Prism 6.Cell sizes were measured with the automated MicrobeTracker software  by using


The physiological and pathological significance of the arachnoid membrane (AM) is still unknown. In this study, we investigated various characteristics of the AM, focusing on the influence of inflammation and fibrosis.Small pieces of AM sample were obtained during neurosurgical procedures from 74 cases. The clinical and pathological characteristics of the hyperplastic AM group (≥ 50 μm) and the non-hyperplastic AM group (< 50 μm) were compared. Then, potential correlations between AM thickness and clinical characteristics were analyzed. Moreover, VEGFα, TGFβ, and TGFα levels were quantitated by real time PCR. Then, the potential correlations between AM thickness and these inflammatory or anti-inflammatory markers, and the influence of the original disease were calculated.+ cells, CD86+ cells, and CD206+ cells in the hyperplastic AM group was significantly higher than that in the non-hyperplastic AM group. The AM thickness was significantly correlated to age and number of fibroblasts, CD68+ cells, CD86+ cells, and CD206+ cells. The thickness of the AM was significantly correlated to the messenger RNA expression levels of VEGFα (ρ = 0.337), and the VEGFα expression levels were significantly correlated with TGFβ and TNFα.The median age of the patients in hyperplastic AM group was significantly older than that of the non-hyperplastic AM group. Moreover, the number of fibroblasts, CD68The AM hyperplasia was influenced by aging and could be a result of inflammation and fibrosis through cytokine secretion from the inflammatory cells and fibroblasts in the AM. The arachnoid membrane (AM) is a tissue named “choroid meninx” by Herophilus in 3 B.C. . PresentRecently, low-level and chronic systematic inflammation has been found to influence the onset and recurrence of various lifestyle diseases –7. This 2) of the sample were obtained. Using the obtained samples, we assessed the following: (1) pathological characteristics of the AM, (2) messenger RNA expression of the cytokines.This study was approved by the Ethics Committee of Sapporo Medical University Hospital (no. 292–128), the Ethics Committee of Sapporo City General Hospital (H30–057-449), the Ethics Committee of Shinsapporo Neurosurgical Hospital (2018–1), the Ethics Committee of Oji General Hospital (OGH2018–16) and the Ethics Committee of Hakodate Shintoshi Hospital (2018001), and was performed in accordance with the ethical standards of the 1964 Declaration of Helsinki and its later amendments. This study was a prospective and observational study. Five institutes including our institute with sufficient surgical experience participated in the study. All patients provided informed consent before participating. From August 2017 to August 2019, the AM was obtained during neurosurgical procedure. The study included all cases in which informed consent was provided within a specified period without limiting disease specificity. In the patients with vascular disease, only the AM was obtained, whereas cerebral cortex samples were obtained along with AM in patients with brain tumor, cerebral infarction, and severe trauma. AM sampling was performed above the middle frontal gyrus, and was avoided around the Sylvian fissure, superior sagittal sinus, posterior fossa, and basal cistern because these parts are physiologically hyperplastic. The AM above the cortical artery  was dissected under a microscope, and small pieces  as a 1.0 cm square cube sample. For flattening the sample, 4 points of the corners of the AM were sewn on a silicon sheet  under a microscope using 10–0 nylon. After sewing onto the silicon sheet, the sample was infiltrated in formalin-fixation liquid for 5 days, and then embedded in 6.5% Sabouraud agar. Finally, the Sabouraud agar-embedded sample was set in paraffin. The paraffin-embedded specimens were sectioned to a thickness of 3 μm.+ cells, CD86+, and CD206+ cells were counted in 3 areas using immunohistochemical staining, and the mean value was similarly calculated.Infiltration of inflammatory cells and fibrosis was assessed by H-E and immunohistochemical staining. CD68s (macrophage markers), CD86s (type I [M1] macrophage markers), and CD206s (type II [M2] macrophage markers) were used to assess inflammation. Antibodies against the following antigens were used for assessment: CD68 ; rabbit anti-CD86 ; rabbit anti-mannose receptor antibody . In each sample with H-E stain, the thickness of AM was measured in 3 places and the mean value was calculated . The number of fibroblasts was then counted in 3 areas of each glass slide , and the mean value was calculated. In addition, the local existence of fibroblasts was assessed. Next, the number of CD68Patient clinical data were stratified based on the following 11 clinical variables: (1) age; (2) sex; (3) disease; (4) smoke; (5) systolic blood pressure; (6) total cholesterol; (7) high density lipoprotein (HDL) cholesterol; (8) low-density lipoprotein (LDL) cholesterol; (9) glucose metabolism disorders; (10) a family history of coronary artery disease; and (11) degree of atherosclerosis. The degree of atherosclerosis was determined using the ASCVD score and Suita score , 19. TheOnly AM was used to assess mRNA expression. Samples with adhered blood components or brain tissue were excluded in this analysis. First, RNA extraction was performed using the RNeasy Fibrous Tissue Mini Kit  according to the manufacturer’s instructions. The total RNA concentration and the A260/A280 ratio were measured using a NanoDrop Lite spectrophotometer . Samples in which the A260/A280 ratio was less than 1.30 were excluded. GoScript™ Reverse Transcription Kit  was used to reverse-transcribe 500 ng of total RNA. PCR was performed in a 10 μL volume with 2 μL of cDNA diluted 1:5. TaqMan Universal Master Mix II with UNG and TaqMan Gene Expression Assays for glyceraldehyde 3-phosphate dehydrogenase , vascular endothelial growth factor A , transforming growth factor beta 1 , and tumor necrosis factor  were purchased from Thermo Fisher Scientific Inc. qRT-PCR was performed in duplicate on the Applied Biosystems StepOnePlus™ . The thermal cycling protocol consisted of 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The calibration curve of a reference sample with a known level of mRNA expression was used to calculate the expression level of each mRNA. Based on relative quantification, the expression of each mRNA was revised as the ratio to the reference mRNA expression (target mRNA expression/GAPDH expression). Using the mRNA expression data, correlations between the thickness of the AM and mRNA expression levels of TNFα, TGFβ, and VEGFα were investigated.p < 0.05.Data are expressed as median (interquartile range). The Mann-Whitney U test was used for comparison between the hyperplastic AM and non-hyperplastic AM groups. In addition, the male-to-female ratio and presence of clinical characteristics  were compared between the hyperplastic AM and non-hyperplastic AM groups using Fisher’s exact probability test. Simple regression analysis was used to compare the thickness of the AM and the potential factors or mRNA expression levels of cytokines. Spearman’s correlation coefficient was used to calculate the correlation coefficients. A correlation coefficient (ρ) larger than 0.7 indicated a strong correlation, a ρ value between 0.5 and 0.7 indicated moderate correlation, and a ρ < 0.5 indicated weak correlation. All statistical analyses were performed using the SPSS software package , and statistical significance was set at This study included 98 cases operated at our Hospital and an affiliated hospital from September 2017 to April 2019. Twenty-three cases could not be measured because the specimens were over-minute, or due to thermal denaturation during the preparation process. One case was excluded because the AM could not be separated from the meningioma. Consequently, 74 cases could be used to measure the thickness of the AM. Of these 74 cases, 17 cases had partially missing clinical data. The messenger RNA (mRNA) expression levels were measured completely in 57 patients. Of the 74 patients, 44 cases suffered from cerebrovascular disease, 21 cases had brain tumor, and 9 cases were of other diseases. Of the 44 cases of cerebrovascular disease, 14 cases were unruptured aneurysms, 13 cases were of moyamoya disease, 10 cases were of atherosclerotic steno-occlusive disease, 4 cases were of cerebral hemorrhage, and 3 cases were of cerebral arteriovenous malformation. Of the 21 cases of brain tumor, 7 cases were high grade glioma, 5 cases were meningioma, 3 cases were malignant lymphoma, 2 cases were low grade glioma, 1 case was ependymoma, 1 case was craniopharyngioma, 1 case was pituitary adenoma, and 1 case was metastatic carcinoma. The median (interquartile range) age of the patients at the time of surgery was 54.0 years (39.0–68.0). Thirty-one patients were male and 43 were female. The AM was collected from the frontal lobe in 29 cases, the temporal lobe in 35 cases, parietal lobe in 3 cases, occipital lobe in 4 cases, and from the cerebellum in 3 cases. The median Atherosclerotic Cardiovascular Disease (ASCVD) score and Suita score was 6.0 (2.1–24.3) % and 41.0 (23.0–51.3), respectively.+, CD86+, CD206+ staining showed the infiltration of macrophages, M1 macrophages and M2 macrophages respectively, in the AM  μm. The median number of fibroblasts counted by Hematoxylin-Eosin (H-E) stain was 88.48 (41.14–125.97) × 10− 3/μm2. A total of 46 cases were stained positively for CD68, and the median number of CD68+ cells was 2.85 (0.00–12.56) × 10− 3/μm2. Of these 46 cases stained positively for CD68, 13 cases were stained positively for CD86 as well, and the median number of CD86+ cells was 0.00 (0.00–0.00) × 10− 3/μm2. Additionally, 32 cases were stained positively for CD206, and the median number of CD206+ cell was 0.00 (0.00–10.24) × 10− 3/μm2.H-E staining showed fibroblasts, fibers, and the barrier cell layer in the AM. Fibroblasts tend to exist at the inward pial side, and the fibroblast decreased at the outward dural side being replaced by fibers Fig. a, b. In p = 0.020). The AM thickness was significantly correlated to age  . With regards to other clinical factors including disease, smoke, systolic blood pressure, total cholesterol, HDL cholesterol, LDL cholesterol, glucose metabolism disorders, and familial history of coronary artery disease, there was no significant difference between hyperplastic AM group and non-hyperplastic AM group. The median ASCVD score in the hyperplastic AM group [11.1 (2.2–27.2) %] was higher than that in the non-hyperplastic AM group [4.3 (1.2–16.4) %], though it was not significant (p = 0.176). The median Suita score in the hyperplastic AM group [45.0 (31.00–54.00)] was higher than that in the non-hyperplastic AM group [40.0 (− 1.50–48.50)] and was also not significant (p = 0.153). The median number of fibroblasts in the hyperplastic AM group and non-hyperplastic AM group were 110.16 (69.83–138.35) × 10− 3/μm2 and 65.84 (19.28–97.01) × 10− 3/μm2, respectively, with a significant difference between the 2 groups (p = 0.004). The AM thickness was significantly correlated to the number of fibroblasts   × 10− 3/um2 and 0.00 (0.00–5.75) × 10− 3/μm2, respectively, with a significant difference (p = 0.023). The AM thickness was significantly correlated to the number of CD68+ cells   × 10− 3/μm2 and 0.00 (0.00–0.00) × 10− 3/μm2, respectively, with a significant difference (p = 0.005). The AM thickness was significantly correlated to the number of CD86+ cells   × 10− 3/μm2 and 0.00 (0.00–2.34) × 10− 3/μm2, respectively, with a significant difference (p = 0.020). The AM thickness was significantly correlated to the number of CD206+ cells  . VEGFα mRNA expression levels were significantly correlated with the thickness of the AM  . The median value of VEGFα mRNA expression levels was 1.30 (0.29–2.85) in the hyperplastic AM group, and 0.23 (0.07–1.60) in the non-hyperplastic AM group (p = 0.936 and 0.445 respectively), VEGFα mRNA expression was significantly correlated with TGFβ mRNA expression   (Fig. Next, TNFα and TGFβ were compared with the thickness of the AM and VEGFα expression, because these are representative inflammatory mediators that influence VEGFα. Although the median values of TNFα and TGFβ mRNA expression levels were not significantly different between the hyperplastic AM group and the non-hyperplastic AM group (+ cells, CD86+ positive cells, and CD206+ cells were observed, suggesting the presence of chronic inflammation focusing on macrophages in the AM. Macrophages are a key regulator of chronic inflammation and fibrosis in various disorders such as pericarditis, and hepatic cirrhosis and arteriosclerotic plaque [+ cells. In the AM, inflammatory and anti-inflammatory cells should be derived from the cerebrospinal fluid of the subarachnoid space, and/or the cerebral arteries and brain tissue. The macrophages are divided into two phenotypes varying in their physiological roles: M1 and M2 macrophages [+ cells and 43.2% were CD206+ cells, and the M2 marker was superior. The M2 macrophages facilitate healing and repairing of the inflammatory process, show anti-inflammatory action, and promote fibrosis [In the AM, hyperplasia due to fibroblast proliferation, and infiltration of CD68c plaque , and in rophages , 24. Therophages . On the rophages . The disrophages . In thisfibrosis ; it is tfibrosis –29.Fibroblast proliferation is observed in the regeneration process of chronic inflammation due to anti-inflammatory cytokines . Our resThere are some limitations to this study. First, this study included various kinds of diseases, and the degree of the disease was different in each case. For instance, local factors might depend on the stage of the original disease especially in extra-axial tumors. On the other hand, it was considered that the AM could be affected widely in chronic ischemic disease, though the degree of the ischemia was different in each case. This study did not exclude the influences of disease specificity. Secondly, the quantity of specimens was not sufficient for analysis by real-time qRT-PCR. Samples in which the A260/A280 ratio was set less than 1.30 were excluded in this study, though their value was relatively low. Third, there were problems in terms of AM fixation. Because AM is a very thin and delicate tissue, thermal denaturation might have occurred in the Sabouraud agar fixation process. Although, an alternative technique is needed to assess the cerebral environment, this could not be achieved to find pathological significance.In conclusion, AM hyperplasia was influenced by aging. Pathologically, fibroblasts and M2 macrophages proliferated in the inward pia side especially around the arteries. AM hyperplasia was correlated with VEGFα and might be a result of anti-inflammatory changes and fibrosis. These alterations might be affected by cerebral ischemia, especially in moyamoya disease. Further analysis of the AM in various diseases And more number of cases will provide us with answers to some of these hypothesis.In conclusion, AM hyperplasia was influenced by aging. Pathologically, fibroblasts and M2 macrophages proliferated in the inward pia side especially around the arteries. AM hyperplasia was correlated with VEGFα and might be a result of anti-inflammatory changes and fibrosis. Further analysis of the AM in various diseases and more number of cases will provide us with answers to some of these hypothesis.


In the real world, many relationships between events are uncertain and probabilistic. Uncertainty is also likely to be a more common feature of daily experience for youth because they have less experience to draw from than adults. Some studies suggest probabilistic learning may be inefficient in youths compared to adults, while others suggest it may be more efficient in youths in mid adolescence. Here we used a probabilistic reinforcement learning task to test how youth age 8-17 (N = 187) and adults age 18-30 (N = 110) learn about stable probabilistic contingencies. Performance increased with age through early-twenties, then stabilized. Using hierarchical Bayesian methods to fit computational reinforcement learning models, we show that all participants’ performance was better explained by models in which negative outcomes had minimal to no impact on learning. The performance increase over age was driven by 1) an increase in learning rate ; 2) a decrease in noisy/exploratory choices. In mid-adolescence age 13-15, salivary testosterone and learning rate were positively related. We discuss our findings in the context of other studies and hypotheses about adolescent brain development. Adolescence is a time of great uncertainty. It is also a critical time for brain development, learning, and decision making in social and educational domains. There are currently contradictory findings about learning in adolescence. We sought to better isolate how learning from stable probabilistic contingencies changes during adolescence with a task that previously showed interesting results in adolescents. We collected a relatively large sample size (297 participants) across a wide age range (8–30), to trace the adolescent developmental trajectory of learning under stable but uncertain conditions. We found that age in our sample was positively associated with higher learning rates and lower choice exploration. Within narrow age bins, we found that higher saliva testosterone levels were associated with higher learning rates in participants age 13–15 years. These findings can help us better isolate the trajectory of maturation of core learning and decision making processes during adolescence. In the everyday world, perfectly predictable outcomes are rare. Yet, we need to track important events and their relationships to other events and actions. For example, we might want to learn where the best place to obtain food is, or where a potential mate likes to hang out—this might help us decide where to go, expecting a positive outcome to occur frequently, but not always. Our ability to learn about these probabilistic relationships is crucial for our daily life and decision making.better mean in this context? Most learning mechanisms are subject to tradeoffs between speed and stability. Fast learning may be suitable for a highly certain environment with deterministic relationships/statistics, but can lead to impulsive behavior in a more uncertain environment with probabilistic relationships/statistics  for participant j was drawn from a normal distribution Normal truncated at 0 and 1. Individual participants’ parameters were then used in the likelihood of each participant’s actions on each trial (a[j][t]) according to the αβ model, where j and t indicate participant number and trial number, respectively.We fitted all RL models using hierarchical Bayesian methods  jointly The hierarchical model made the likelihood intractable , but it matstanlib library , used to compute the likelihood of participant j’s choices, followed Normal, T , where age[j] was the z-scored age of participant j, and αintercept, αlinear were regression coefficients for which we set weakly informative priors. To probe quadratic effects, we just further included αquadratic * age[j]2.Same as before, we assumed that the model parameters for individual participants followed a truncated normal distribution with a group-level standard deviation, but now we replace the prior on the group-level mean with a regression statement with respect to age. For example, to probe linear effects of age on αlinear) and quadratic (αquadratic) regression coefficients were significantly different from 0.To test for effects of age on the model parameters, we examined whether the posterior distribution of all 16000 samples for the linear  = 8.5, p < 0.0001; block: F = 136, p < 0.0001). There was no interaction between age group and block  = 1, p = 0.49). This shows that participant’s performance improved as the experiment progressed, and older participants generally outperformed younger participants.All age groups exhibited learning over the course of the experiment. Specifically, we found a significant main effect of age group and block on participants’ performance  = 3.5, p = 0.0001), and significantly lower than of 18–25 year-olds  = 2.5, p = 0.01). However, there was no significant difference  = 0.2, p = 0.8) between the performance of 25–30 year-olds and 18–25 year-olds  was significantly higher than of 8–13 year-olds  = 7.6, p = 0.006). The regression analysis revealed linear and quadratic effects of age on performance . There was no effect of sex or its interaction with age .To examine the continuous relationship between participants’ performance and age, we ran a regression analysis using age to predict performance . We founβage = 0.11, z = 5.24, p < 0.0001, and after which βage = 0, z = 0.14, p = 0.9. This indicates that performance linearly increased with age through adolescence and stabilized in early adulthood, showing that the quadratic effect reflected a linear increase then stable process rather than an inverse U-shape.To identify whether the quadratic effect was indicative of an inverse U shape, we conducted the two-line regression . We founβage = −0.01, 95% CI = ). This suggests that participants reacted faster with age, confirming previous results ). Two-line regression revealed a break point at 19 years old, before which we found βage = −0.05, z = −4.15, p < 0.0001, and after which we found βage = 0.03, z = 1.88, p = 0.06. This indicates that the variability in reaction time decreased with age, and this decrease stabilizes in early adulthood and might even invert, consistent with previous findings , p = 0.18; T1: βT1 = 0.14, 95% CI = , p = 0.05). PDS, but not T1, had a marginal linear effect on reaction time . When controlling for age in the regression, the effect of T1 on performance disappeared  = 0.53, p(age) = 0.001). However, for the effect of PDS on reaction time, adding age did not improve model fit  = 0.1, p = 0.76). Note that these results are generally consistent with our previous findings relating age and behavioral measures (p’s > 0.16).Regarding the behavioral measures , we founmeasures , as expemeasures , albeit α+ , and no effect on the forgetting parameter f . There was a linear effect of PDS, but not T1, on β . However, the effects of PDS and T1 on α+ and β disappeared when adding age into the regression , while age remained the only significant predictor . There was no effect of sex in any of the regressions .For the fitted model parameters , we founα+ . This remained significant when correcting for multiple comparisons (two parameters by four groups). This T1 effect remained when controlling for age in the regression  = 0.006, p(age) = 0.95), as T1 and age were decorrelated in this age group  as well as inverse temperature (β), a parameter indicating a decrease in noise or exploration in choice.Consistent with the age effects observed in previous work using tasks with probabilistic  and deteα+ and inverse temperature β increase with age and pubertal development during adolescence are generally consistent with previous work using the deterministic learning task RLWM, tested in the same participants as shown here [t(168) = 2.3, p = 0.02). Because our 18–25 year-olds were differently recruited than the rest of our sample, we additionally compared the 13–18-year-old adolescents to N = 54 25–30-year-old adults (i.e. not including the undergraduate participants between 20–25 years old). We still found significantly better performance in adults  = 2.2, p = 0.03).Our observations that learning rate own here , and a pown here . Howeverown here  Fig 3A)α+ and inown here , which lα+ in our sample was 0.18, whereas in [The finding in  was inteereas in , the meaereas in ). In higereas in , an incrα+0βf did not learn from negative feedback at all. A high α− can also result in worse asymptotic performance in the Butterfly task  on the α and β parameters as on the α+ and β parameters in the winning model α+0βf.Moreover, we modeled learning from positive and negative feedback asymmetrically , as oppotask see , resultiTherefore, while we found a similar trend as in  that leaThe incentivization for undergraduate participants (course credit) and community participants (monetary) was also different. Furthermore, participants’ performance  was not translated into real-life reward such as money. While there have been studies showing that the human brain treats primary and secondary reinforcers similarly –52, the β (random/noisy behavior), which could only strengthen our findings. Finally, since the study is cross-sectional rather than longitudinal, age could also be confounded by birth year.Another potential limitation of our sample is that the majority of the excluded participants were under 18, which could contain meaningful variance. This might be due to the fact that younger participants were a bit more likely to be distracted during the task/had a more difficult time understanding the task logistics, although most (about 90%) of our younger participants were able to understand and engage well in the task to pass this criterion. In particular, we excluded participants who, for a given butterfly, were more likely to switch than stay after positive feedback , whereas in a volatile task setting [While we found that performance increased during adolescence and stabilized in early adulthood in this stable probabilistic learning task, a probabilistic switching task in the same sample of participants  found a  setting , negativ setting  gave theWhile we found that random effects of delay on performance  became more pronounced with age , computaα+ within the third age group of 13–15 years  and a decrease in exploration. These data and models help explain why learning and decision making differ during development and why a ‘one-size-fits-all’ approach may not equally serve youth at different stages.S1 Text(PDF)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Figα+0βf model per participant, represented by ΔBIC on the y-axis. Color represents 6 age groups. Results show that the α+0βf model is the winning model in all groups consistently.We calculated the difference between the BIC for each of the six models with (TIF)Click here for additional data file.S7 Figα+ (y-axis) and β (x-axis), where each subplot corresponds to f = 0 − 0.2 from left to right. Black rectangle highlights the local maximum within each column of each subplot , whereas the red rectangle highlights the global maximum.Overall simulated performance changes with respect to (TIF)Click here for additional data file.S8 Figα+ (y-axis) and f (x-axis), where each subplot corresponds to β = 5 − 15 from left to right. Black rectangle highlights the local maximum within each column of each subplot , whereas the red rectangle highlights the global maximum.Overall simulated performance changes with respect to (TIF)Click here for additional data file.S9 Figα− (x-axis), where each subplot corresponds to a combination of  values. The vertical bar corresponds to the α+ value. The error bars show standard error across 100 simulations.Overall simulated performance (y-axis) changes with respect to (TIF)Click here for additional data file.S10 Figαβ model. We found positive linear effect of age on α and β, and negative quadratic effect of age on β. The model with quadratic age effect on α failed to converge.We directly incorporated age-related parameters into MCMC sampling to test within the hierarchical Bayesian modeling framework whether age had a linear or quadratic effect on the fitted parameters from the (TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S12 Figα+. (D) T1 vs. α+.(A) PDS vs. median reaction time. (B) T1 vs. overall performance. (C) PDS vs. (TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S3 TableThe rows indicate the model where the dataset was generated from, whereas the columns indicate the model used for recovery. Each entry indicates protected exceedance probability.(TIF)Click here for additional data file.S4 TableWithin each of the age group under 18, we calculated the variance of pubertal measures (PDS and T1) and their correlations to age.(TIF)Click here for additional data file.


While the transport of 100 and 200 μg·mL−1 PUR presents a strong directionality, and this directionality can be inhibited by verapamil and cyclosporine. When PUR and GAS were used in combination, GAS could increase the absorption of PUR while PUR had no obvious influence on GAS. Therefore, the compatibility of PUR and GAS is reasonable, and GAS can promote the transmembrane transport of PUR, the effect of which is similar to that of verapamil.Puerarin (PUR) and gastrodin (GAS) are often used in combined way for treating diseases caused by microcirculation disorders. The current study aimed to investigate the absorption and transportation mechanism of PUR and GAS and their interaction via Caco-2 monolayer cell model. In this work, the concentration in Caco-2 cell of PUR and GAS was determined by HPLC method. The bidirectional transport of PUR and GAS and the inhibition of drug efflux including verapamil and cyclosporine on the transport of these two components were studied. The mutual influence between PUR and GAS, especially the effect of the latter on the former of the bidirectional transport were also investigated. The transport of 50 μg·mL Gas has a variety of pharmacological effects such as sedation and sleeping, promoting intelligence, protecting neurons, lowering blood pressure, antioxidant, and improving microcirculation, etc. It is extensively used to treat dizziness, migraine, high blood pressure, stroke and epilepsy [Puerarin A, PUR, cii Benth ,2,3. PURii Benth ,6,7. Howii Benth ,9,10. Hoepilepsy ,13,14,15In the treatment of cardiovascular and cerebrovascular diseases, PUR and GAS are often used in combination. In addition, many Chinese medicine prescriptions usually contain these two components ,17. In oTo our knowledge, there have been some studies on the intestinal absorption mechanism of PUR. With regard to GAS, for its good oral bioavailability, few studies were concerned to the absorption of GAS. Liang  found thThe Caco-2 cell line was derived from human colon adenocarcinoma and its morphology and function are similar to those of human intestinal epithelial cells, and it contains enzymes related to the brush border epithelium of small intestine . Caco-2 Thus, in this study, based on the clinical application and combination of PUR and GAS, Caco-2 cells was used to study the absorption and transportation mechanism of PUR and GAS and their mutual influence, so as to lay an experimental foundation for their feasibility of the compatibility.−1) -streptomycin (100 μg·mL−1) double antidotic solution, trypsin, and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. . Nonessential amino acids (NEAA) were obtained from Corning Cellgro . Hank’s equilibrium salt solution  was purchased from Beijing Solarbio Science and Technology Co., Ltd. . Ethylene diamine tetraacetic acid (EDTA) was obtained from Merck and Co., Inc. . Acetonitrile was bought from Thermo Fisher Scientific .PUR and GAS were purchased from Wei Keqi Biotechnology Co., Ltd.  qualifying as administration drug and from National Institutes for food and drug control , the purity >98%. P-hydroxy phenylethyl alcohol  was purchased from Aladdin reagent limited company , the purity >98%. Verapamil hydrochloride was obtained from National Institutes for food and drug control . Cyclosporin was bought from Shanghai Jingke Chemical Technology Co., Ltd. . The Dulbecco’s modified Eagle’s medium (DMEM), and heat-inactivated fetal bovine serum (FBS) were obtained from Invitrogen. Penicillin (100 IU·mL2 at 37 °C in DMEM supplemented 10% (v/v) heat-inactivated FBS, 1% non-essential amino acids, 1% L-glutamine, and penicillin (100 IU·mL−1)-streptomycin (100 μg·mL−1) double antidotic solution.Caco-2 cell line were initially obtained from the American Type Culture Collection (ATCC) and maintained in our laboratory. Caco-2 cells were maintained under humidity with 5% CO18 column  equipped with an Agilent analytical guard column .The analysis was performed using the Shimadzu HPLC system . Analytes were separated on an Agilent ZORBAX SB-Aq C2O (B) and 0.05% phosphoric acid as modifier, was used to analyze the samples. The gradient elution method was as follows: 0–4.5 min: 3% A; 4.5–8 min: 3% A→15% A, 8–15 min: 15% A→30% A; 15–20 min: 30% A→30% A; 20–25 min: 3% A.The gradient mobile phase system, consisting of ACN (A)-H−1 PUR and GAS mixed solution, respectively. The above mixed solution was then diluted with HBSS solution to 0.08, 0.17, 0.33, 0.83, 1.67, 3.33, 8.33, 16.67, 25, 33.33, 50, 100, 166.67, and 333.33 μg·mL−1 working solution, respectively. Accurately weigh a certain amount of P-hydroxy phenylethyl alcohol (IS), and dilute to a concentration of 22.60 μg·mL−1 with HBSS solution.10 mg PUR and GAS were precisely weighed and diluted with HBSS solution to obtain 1 mg·mL−1, respectively.The calibration standard samples were prepared by transferring 30 μL of the each working solution into an Eppendorf tube (EP tube), to which 10 μL of IS and 60 μL methyl alcohol were added. The concentrations of calibration standard samples were 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 7.5, 10, 15, 30, 50, and 100 μg·mLg·min−1 for 10 min. Store the supernatant at 4 °C for subsequent analysis.Transfer 30 μL of cell sample, 10 μL of p-hydroxy phenylethyl alcohol (IS) and 60 μL of methanol to an EP tube in turn, and vortex for 2 min. Then centrifuged at 12,000× −1.The specificity of the method was assessed by comparing the chromatograms of blank plasma, blank plasma spiked with internal standard/analyte, and cell samples. Calibration curves were established based on the peak area ratio versus nominal concentration. Intra-day and inter-day precision and accuracy are expressed by evaluating the measurement results of QC samples at 0.1, 5, and 100 μg·mLThe retention time for PUR, GAS, and p-hydroxy phenylethyl alcohol (IS) were 16.5, 8.6, and 14.8 min, respectively, and there were little interferential substances with the analytes and IS in the HBSS solution and cells. Representative chromatogram of analytes and IS in Caco-2 cell was shown in −1, and the high linear rang is 2.5–100 μg·mL−1. For GAS, the low linear rang is 0.1–5 μg·mL−1, and the high linear rang is 5–100 μg·mL−1. The calibration curves of PUR and GAS exhibited good linearity, and the regression equations with correlation coefficients and linear range were listed in Due to the wide range of concentration span, two standard curves of the analytes are made. For PUR, the low linear rang is 0.025–2.5 μg·mL−1. Each of the six samples were processed in parallel. Precisions were expressed by the relative standard deviation . The intra-day and inter-day precision R.S.D. values of PUR and GAS were all less than 10%.Transfer 30 μL of each working solutions, and the samples were prepared in the same way as the cell samples at concentration of 0.1, 5, 100 μg·mL−1) concentrations were 104.44%, 105.01%, and 100.17%, respectively; the recoveries for low, medium, and high concentrations of GAS were 108.71%, 107.16%, and 99.87%, respectively.The accuracy (%), i.e., relative recovery, compare the measured concentration with the actual concentration to obtain the method recovery rate, the difference value is the relative recovery rate. The recovery rates of PUR for low, medium and high  was calculated according to Equation (1).1 (Ω) is the measured value of the cell group, R0 (Ω) is the measured value of the blank group (without inoculated cells); in the transport experiment, R1 (Ω) is the measured value of the administered cell group, R0 (Ω) is the measured value of the blank cell group (before administration), and A (cm2) is the area of the polycarbonate membrane per hole (The area of Transwell membrane used in this experiment is 1.12 cm2).Before the transport experiment, Rapp) of the Caco-2 cell model refers to the data processing reported by Artursson [app of the Caco-2 cell monolayer, see Equation (2). The larger the Papp value, the higher the permeability.The calculation of the apparent permeability coefficients  (μg·mL−1); A is the same as the meaning in the Equation (1); C0 is the initial mass concentration of the drug in the supply pool (μg·mL−1).ΔQ is the accumulative transport amount of drug (μg); ΔQ/Δapp(BL→AP) to Papp(AP→BL) to the apparent permeability coefficient of the drug transported from the AP side to the BL side.The drug efflux ratio (ER) is the ratio of Papp(BL→AP) is the Papp of drug transported from the BL side to the AP side, and Papp(AP→BL) is the Papp of drug transported from the AP side to the BL side. It is generally believed that when Papp(BL→AP) is close to Papp(AP→BL), the drug is absorbed by passive diffusion. When Papp(BL→AP)/Papp(AP→BL) > 1.5, it is indicated that the drug may involve in active transport mechanism.PSince the HBSS blank solution must be supplemented after each sampling, which is equivalent to a dilution effect on the penetration of the drug, the cumulative transport amount (ΔQ) (μg) of the drug can be corrected by the following formula:nth sample (μg·mL−1); −1).Mean ± SD.SPSS statistic 17.0  was used for statistical analysis of the data, and the results were expressed as −1, the bidirectional transport amount increases linearly with time, indicating that within this concentration range, the absorption mode of PUR is just passive diffusion  of PUR was reduced, while the ΔQ(AP→BL) was basically unchanged, that is, the addition of verapamil can reduce the efflux of PUR and finally promote the absorption of PUR. In addition, the absorption of PUR increased (Papp(AP→BL) increased from 1.285 to 1.413), the efflux decreased significantly (Papp(BL→AP) decreased from 4.539 × 10−6 cm·s−1 to 3.004 × 10−6 cm·s−1), and the ER dropped from 3.531 to 2.126 (a decrease of 39.79%) (It can be seen from  39.79%) .−1 verapamil, the ΔQ(AP→BL) increased, and there was no significant difference in the apparent permeability coefficient between the two groups  of PUR increased, while the ΔQ(BL→AP) was basically constant in the concentration range of 50–200 μg·mL−1. That is, after adding cyclosporin, it appears to increase the absorption of PUR. In additional, the Papp(AP→BL) increased significantly from 1.285 × 10−6 cm·s−1 to 4.759 × 10−6 cm·s−1, while Papp(BL→AP) decreased from 4.539 × 10−6 cm·s−1 to 4.014 × 10−6 cm·s−1, a decrease of 76.11%  and Papp(AP→BL)  D.−1 PUR is combined with 100 μg·mL−1 GAS, the biodirectional ΔQ were shown in (BL→AP) of PUR decreased, while there was no obvious change with the ΔQ(AP→BL) (app(AP→BL) of PUR increased  decreased significantly from 4.539 × 10−6 cm·s−1 to 3.108 × 10−6 cm·s−1, and the ER decreased from 3.531 to 2.181, a decrease of 38.22% (When 100 μg·mLQ(AP→BL) A. In addf 38.22% .(BL→AP) of GAS was significant increased, while the ΔQ(AP→BL) was basically unchanged  significantly, which indicates that GAS can improve the PUR’s intestinal absorption. For intestinal absorption is an important index reflecting the process of drug absorption, so the poor absorption of PUR may closely relate to the intestinal efflux.In the present study, the rationality of the compatibility of PUR and GAS was verified from the perspective of Caco-2 cell transport. It is found that GAS enhanced the P−1 PUR in Caco-2 cells has no obvious directionality, and the transport rate is basically constant, indicating that the transport of PUR within this concentration range is a passive transport process. The transmembrane transport of 100 and 200 μg·mL−1 PUR in Caco-2 cells present a strong directionality, with ER both greater than 1.5, and this tropism can be inhibited by verapamil and cyclosporin. So, it is speculated that PUR is not only a substrate of P-gp, but also a substrate of MRP2. Cyclosporin is more obvious than verapamil to promote transmembrane transport of PUR. Moreover, in addition to passive diffusion, there is also an active transport mode for PUR across the membrane. This result is consistent with the research of Cheng et al. [−1 PUR was 3.531 and 2.654, respectively, which was consistent with the previous research [−1 GAS in Caco-2 cells has no obvious directionality, and the transport profile could not be altered by verapamil and cyclosporine, suggesting that the transport of GAS is a pure passive process. However, as GAS has no oral malabsorption, thus we just investigated 100 μg·mL−1 GAS, did not explore the bidirectional transport of different concentrations of GAS in Caco-2 cells, and the Papp(BL→AP) experiment of GAS was also omitted.P-gp and MRP, its subfamily MRP2, are both expressed in the Caco-2 monolayer cell model, and they can efflux drugs from the inside of the cell to the outside of the cell ,32. Verag et al. . In addig et al. . In our research . The traapp(AP→BL) increased from 1.285 × 10−6 cm·s−1 to 1.425 × 10−6 cm·s−1, the efflux index Papp(BL→AP) decreased significantly from 4.539 × 10−6 cm·s−1 to 3.108 × 10−6 cm·s−1, and the ER decreased by 38.22%. This is consistent with our previous pharmacokinetic results in rats, which showed that the combined administration of GAS and PUR could increase the absorption, decrease the clearance rate, and prolong the mean retention time, and the bioavailability of the two components when compared with the single-administered group [When GAS and PUR were used in combination, for PUR, the absorption index Ped group . It is sapp values of PUR and GAS were 1.766 × 10−6 cm·s−1 and 2.866 × 10−6 cm·s−1, respectively. The Papp of PUR is smaller than that of GAS, which is consistent with the determination of the oil-water partition coefficient in our previous study [app > 1 × 10−6 cm·s−1), and that of drugs with incomplete absorption is low (Papp < 1 × 10−7 cm·s−1) [app for passive diffusion drugs. However, for active transport drugs, the Papp obtained from Caco-2 cell experiments can only be used as a qualitative rather than quantitative indicator of in vivo absorption.In this study, the Pus study . Studiesus study  have sho cm·s−1) ,39. It c cm·s−1) . This isThe absorption and transport mechanism of PUR and GAS and their interaction were studied and we try to clarify the rationality of their absorption mechanism and compatibility application from the cellular level. The research results show that the compatibility of PUR and GAS is reasonable, and GAS can promote the transmembrane transport of PUR, the effect of which is similar to that of verapamil, which may provide a certain theoretical basis for its combined application.


Under continuous UV-B irradiation at 0.1 W m−2 for 3 or 6 h, leaf color changed from red to green and leaf fresh weight decreased, compared with the control treatment. No leaf color change was observed under the 3-h treatment with UV-B radiation at 0.05 W m−2, wherein leaf fresh weight was similar to that of the control. Furthermore, RA concentration under continuous UV-B irradiation at 0.05 W m−2 for 3 h increased two-fold compared to that under control treatment, while PA concentration was not affected by UV-B irradiation. Thus, our data showed that continuous UV-B irradiation at 0.05 W m−2 for 3 h could effectively produce RA-rich perilla leaves without reducing in phenotypic quality or productivity. However, a 6-h intermittent illumination inhibited flowering without altering phytochemical concentration.In Japan, red perilla leaves are used in the food and coloring industries, as well as in crude medicine. Perilla leaves contain a high concentration of phytochemicals such as perillaldehyde (PA) and rosmarinic acid (RA). The effects of UV-B radiation intensity (0.05–0.2 W m As consumers continue to become health-conscious, interest in vegetable phytochemicals has increased. In addition to being recognized as nutrients, phytochemicals are defined as bioactive non-nutrient plant compounds present in fruits, vegetables, grains, and other plant foods . In recePerilla frutescens) is an annual plant of the family Lamiaceae that has been used in food and coloring industries, and in crude medicine in Japan. Red perilla leaves are rich in phytochemicals such as perillaldehyde (PA) and rosmarinic acid (RA). PA is a characteristic aroma component of red perilla with neuropharmacological [Red perilla  under the 0.1-W m−2 treatment, at 5 weeks after treatment initiation . In the present study, no growth inhibition via subjection to UV-B irradiation was observed under the same conditions. However, leaf fresh weight decreased upon subjection to UV-B radiation intensities beyond 0.1 W m−2. Therefore, for plant growth to remain uninhibited by exposure to 3 h of UV-B irradiation, the light intensity should not exceed 0.05 W m−2. In our experiment using intermittent lighting for 6 h daily (experiment 3), plant growth was suppressed under conditions of subjection to irradiation at 0.05 W m−2. Therefore, the effect of intermittent lighting on growth inhibition seemed to be more influenced by daily exposure dose rather than by UV-B intensity, and indicated that the exposure dose for normal plant growth should be less than 0.54 kJ m−2 day−1. Intermittent lighting was expected to confer better protection to plant growth against UV damage during the non-UV irradiation period compared with continuous lighting. However, the results of fresh weight upon subjection to treatment with 2.2 kJ m−2 day−1 of UV-B, as described in experiments 1–3, showed that intermittent lighting was not effective in preventing damage to leaf growth caused by exposure to UV-B light.As UV-B light sources are being used as artificial lighting to improve phytochemical concentration and to control flowering, it is desirable to provide illumination to an extent that does not impair growth rate and phenotype quality. Using the same light source as that used in the present study, Ota et al.  did not −2 irradiation, we observed no change in leaf color under the same conditions. The effect of light at the end of the dark period on perilla flowering is reportedly greater than that at the beginning of the dark period. In this study, lighting was provided from 02:00 to 05:00, before the beginning of the natural light period. In contrast, Ota et al. [−2 day−1 UV-B exposure treatments between experiments 1, 2, and 3, 0.1 W m−2 with 1 h of exposure to intermittent lighting was observed to suppress growth but did not cause greening of the leaves. Therefore, it was suggested that the UV-B irradiation interval, as well as the daily exposure dose, exerted an effect on leaf greening.It has been reported that UV-B irradiation decreases the concentration of anthocyanins in red perilla leaves and causes a change in their color to green ,21,24. Ia et al. . In factRecently, a study using Arabidopsis as a model plant, reported that UV-B light could affect flowering via the photoreceptor UVR8 and expression of related genes . However−2 UV-B radiation. Takimoto and Ikeda [−2 s−1 converted to photosynthetic photon flux density (PPFD) of daytime sunlight) in the latter half of the dark period. The PPFD was 0.17 µmol m−2 s−1 at 0.1 W m−2  seeds were dipped in tap water until rooting and then sown on saturated urethane sponges. Seeds were grown in a controlled-environment room equipped with white fluorescent lamps . The environmental conditions were as follows: 25/20 °C (light/dark period) air temperature, 70% relative humidity, and 16 h/8 h day/night photoperiod. The PPFD from the light source was set to 150 µmol m−2 s−1 at the canopy level using a quantum sensor . Three weeks after sowing, plants were transplanted into hydroponic containers equipped with air pumps. A commercial nutrient solution  with EC at 0.9 dS m−1 was used. Seedlings were grown under these conditions until the leaves at the 5th node (from bottom to top) were unfolded. The leaves at the 1st–3rd nodes were fully expanded; however, the leaves at the 4th node were not.Perilla . We used UV-B fluorescent lamps . The spectrum of the UV-B lamp was measured using a spectroradiometer  (BE) proposed by Caldwell [Seedlings were transplanted into hydroponic containers equipped with air pumps in an N-S oriented greenhouse. A commercial nutrient solution  with EC 0.9 at dS m, Japan) . Table 5Caldwell  was calcCaldwell .−2 (UV-BBE: 0.083 W m−2) by adjusting the distance from the light sources to each plant material. The distance was adjusted on a weekly basis. The integrated UV-B energy at 0.1 W m−2 was 2.2 kJ m−2 day−1. Perilla seedlings were grown with or without (control) subjection to UV-B light treatment. Seedlings were irradiated for 6 h from 23:00 to 05:00 h in all UV-B light treatments. Experiment 1 was performed from August 18, 2020, to September 22, 2020 (5 weeks). UV-B intensity at the top of the plant canopy was set to 0.1 W m−2  by adjusting the distance from the light source to each plant material. The distance was adjusted on a weekly basis. The integrated UV-B energy at 0.05, 0.1 and 0.2 W m−2 was 0.54, 1.0 and 2.2 kJ m−2 day−1, respectively. Based on the results of experiment 1, physiological disorders such as greening of leaves were observed at 0.1 W m−2. Therefore, we conducted a 0.05-W m−2 treatment in experiment 2. Additionally, we also performed a 0.2-W m−2 treatment with the same integrated UV-B energy (2.2 kJ m−2 day−1) as that at 0.1 W m−2 conducted in experiment 1.Seedlings were irradiated for 3 h from 02:00 to 05:00 in all UV-B light treatments. Experiment 2 was performed from September 29, 2020, to November 3, 2020 (5 weeks), at three different levels of UV-B intensity, using UV-B lamps. The UV-B radiation intensity at the plant canopy level was set to 0.05, 0.1, or 0.2 W m−2  by adjusting the distance from the light source to each plant material. The distance was adjusted on a weekly basis. The integrated UV-B per night at 0.05 or 0.1 W m−2 was 1.1 or 2.2 kJ m−2 day−1, respectively. Seedlings were irradiated for 1 or 2 h every 1 or 2 h for 6 h, from 18:00 to 06:00 . Experim−2. To evaluate the effect of the visible light of UV-B lamps on flowering, we covered the lamp with a UV cut film . The spectrum was measured using a spectroradiometer   . SeedlinThe phyllotaxis of perilla plants is decussate. Thus, each node has two leaves at the same growth stage. Leaves at the 4th node counted from the bottom of the main stem were harvested 1 week after treatment initiation; thereafter, leaves at the 5th, 6th, and 7th nodes were sampled from the same plants at 2, 3, and 4 weeks after treatment initiation. One leaf at each node was used to measure the PA and RA content, and the other leaf was used to calculate the dry matter ratio by dividing leaf dry weight by leaf fresh weight. The fresh weights of both leaves were measured immediately after harvesting. Thereafter, one leaf for the measurement of the PA and RA contents was placed in a polyethylene bag, snap frozen in liquid nitrogen, and then stored in a freezer at −30 °C. Thereafter, the leaf used for calculation of the dry matter ratio was oven-dried at 80 °C for 72 h, and then the dry weight was measured. g), and the supernatants were collected. Methanol (0.5 mL) was added to the residue, and sonication and centrifugation were performed as per methods described in the first step. The supernatants obtained from the execution of two steps were combined and centrifuged for 10 min , and then filtered using disposable syringe filter units . PA and RA contents were measured according to the method reported by Ogawa et al. . BrieflyThe PA and RA contents were measured using a UHPLC  equipped with a UPLC BEH C18 column . The temperature of the column oven was maintained at 50 °C. The detection wavelengths of PA and RA were set at 230 nm and 326 nm, respectively.t-test or the Tukey-Kramer test at p < 0.05.Data were statistically evaluated by performing one-way analysis of variance (ANOVA) using the SPSS program for Windows . To investigate significant differences among treatments, the mean value of measured data was compared using the


Tripterygium wilfordii and Paeonia lactiflora is often used to attenuate the toxicity of TP, but its compatibility mechanism has not been fully elucidated. The aim of this study was to investigate the pharmacokinetics and tissue distribution of a combined regimen of TP and paeoniflorin (PF) after transdermal administration in male and female Sprague Dawley (SD) rats via a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The results showed that after percutaneous administration of TP and PF, there was no significant difference in AUC t)(0- (area under the curve) of TP, the peak concentration decreased by 58.17%, and the peak time was delayed. The AUC (0-t) of PF increased significantly (P < 0.01), the peak-reaching concentration and AUC (0-∞) increased, and the half-life and average retention time were shortened, indicating that TP absorption in rats may be delayed. After percutaneous administration of TP and PF, the content of TP in the heart, liver, spleen, lungs, and kidneys of male rats significantly decreased at 2 h (P < 0.05) and the drug concentration in the liver tissues significantly decreased at 2 h, 4 h, and 8 h (P < 0.05). The TP content in the spleen of female rats significantly decreased at 2 h and 4 h (P < 0.05) and also decreased in other tissues, but not significantly. After percutaneous administration of TP and PF, the PF content in the heart, liver, spleen, lungs, and kidneys of male and female rats had no significant difference. However, after percutaneous administration of TP and PF, the TP concentration in the skin increased, suggesting that the amount of TP retained in the skin increased, thereby reducing its content in blood and tissues, producing a reduction in toxicity effect.Triptolide (TP) has shown potential in rheumatoid arthritis (RA) treatment, but the narrow therapeutic window limits its clinical application. In clinical practice, the compatibility of  Tripterygium wilfordii hook, TP, is responsible for anti-inflammatory, antitumor, antifertility, and immunomodulatory effects [Tripterygium wilfordii in the treatment of rheumatoid arthritis [Tripterygium wilfordii was often administered in combination with Paeonia lactiflora, and it was reported that the combination of the two could enhance efficacy and reduce toxicity [Tripterygium wilfordii combined with Paeonia lactiflora is unclear.Triptolide (TP) is an epoxy-xylene lactone compound. The common ingredient of  effects –3. Clini effects . Our resrthritis –7. It watoxicity –12; howeTripterygium wilfordii can maintain a stable blood drug concentration [Tripterygium wilfordii has been prepared into microemulsion gel, and pharmacodynamic experiments conducted on CIA model rats found that the topical preparation of Tripterygium wilfordii had a curative effect [The transdermal drug delivery system (TDDS) refers to a system in which drugs are administered on the surface of the skin, pass through each layer of the skin at a constant rate, enter systemic circulation, and produce systemic or local therapeutic effects . The prentration . Additioe effect , 16. HowPaeonia lactiflora) after transdermal administration in order to explain the mechanism of toxicity reduction caused by combining the two drugs.Therefore, in this study, modern techniques and analytical methods were used to further study the pharmacokinetics and tissue distribution of TP and PF (the main active component of μm) were used.Triptolide , paeoniflorin , AB Triple Quadrupole 4500 , AB Triple Quadrupole 5500 , and Agilent InfinityLab Poroshell 120 SB-C18  rats weighing 220–250 g were obtained from Hunan Slake Jingda Experimental Animal Co., Ltd., and acclimated in the laboratory for seven days. Prior to the experiments, rats were housed with free access to food and water on a 12 h light-dark cycle at ambient temperature (22–24°C) and roughly 50% relative humidity. All animal procedures were approved by the Ethics Committee of the Experimental Animal Science and Technology Center of Jiangxi University of Traditional Chinese Medicine (No. JZLLSC2019-0149).−1 for 2 min, stirred, and cooled in an ice-water bath to obtain TP cream.Oil phase contained isopropyl myristate (10%), glyceryl monostearate (14%), and Span-60 (2%); aqueous phase contained Tween-80 (2%), Garandam (0.2%), and water (68.8%); drug: 0.01% TP is dissolved in 3% anhydrous ethanol. First, the oil phase was preheated in a water bath at 80°C for 30 min. Next, the drug was poured into the oil phase and stirred evenly, after which the water phase was quickly poured into the oil phase. The mixture was sheared at high speed of 10,000 r·min−1 for 2 min, stirred, and cooled in an ice-water bath to obtain PF Cream.Oil phase contained isopropyl myristate (10%), glyceryl monostearate (14%), and Span-60 (2%); aqueous phase contained Tween-80 (2%), Garandam (0.2%), water (58.2%), and PF (10%). The oil phase was preheated in a water bath at 80°C for 30 min, after which the water phase was quickly poured into the oil phase. The mixture was sheared at high speed of 10,000 r·min−1 for 2 min, stirred, and cooled in an ice-water bath to obtain TP-PF cream.Oil phase contained isopropyl myristate (10%), glyceryl monostearate (14%), and Span-60 (2%); aqueous phase contained Tween-80 (2%), paeoniflorin (10%), and Galandan (0.2%), water (58.2%); drug: 0.01% TP is dissolved in 3% anhydrous ethanol. First, the oil phase was preheated in a water bath at 80°C for 30 min. Next, the drug was poured into the oil phase and stirred evenly, after which the water phase was quickly poured into the oil phase. The mixture was sheared at high speed of 10,000 r·min−1·D−1), PF (1 g·kg−1·D−1), and TP-PF creams were uniformly applied in the area from which the back hair was removed. The surface of the treatment area was covered with polyethylene and fixed with a medical adhesive cloth. Then, 0.25 mL blood samples were collected in heparinized Eppendorf tube via the posterior orbital venous plexus before dosing and subsequently at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, and 24 h. After centrifuging at 4000 r·min−1 for 10 min, the plasma samples were obtained and frozen at −20°C until analysis. For the tissue distribution study, three groups of rats  underwent drug administration in the same manner. Heart, liver, spleen, lungs, kidneys, skin, ovaries, and testes were removed at 2 h, 4 h, and 8 h after dosing. Tissue samples were weighed and rinsed with physiological saline solution; then, the samples were blotted using filter paper and stored at −20°C until analysis.For the pharmacokinetic study, rats were divided into three groups : the TP, PF, and TP-PF groups. Before the experiment, the rats were fasted for 16 h but allowed water. The back hair (5 cm × 4 cm) of the rats was removed. TP  (5 ng/mL) working solution, and 20 μL ammonia water were added in turn. All tissues were homogenized in triple normal saline. To a 100 μL of tissues homogenate sample, 10 μL mixed standard, 10 μL IS (5 ng/mL) working solution, and 20 μL ammonia water were added in turn. Plasma and tissue samples were then vortex-mixed for 30 s and extracted with 0.8 mL ethyl acetate by vortex mixing for 3 min. After centrifugation at 12,000 r·min−1 for 3 min, 0.78 mL upper organic layer was transferred. Next, 10 μL ammonia and 400 μL ethyl acetate were added to the lower layer and vortex-mixed for 2 min. After centrifugation at 12,000 r·min−1 for 3 min, 400 μL upper organic layer was transferred, and the two upper organic layers were combined. The combined upper organic layer was evaporated to dryness at 50°C under a gentle stream of nitrogen. The residue was reconstituted in 100 μL methanol, vortex-mixed for 1 min, and centrifuged at 16,000 r·min−1 for 10 min. Finally, a 50 μL aliquot was injected into the liquid chromatography-tandem mass spectrometry (LC-MS/MS) system for analysis.To a 100 The chromatographic conditions for TP were as follows: solvent A was 0.1% formic acid (V/V) in water, and solvent B was a gradient elution with acetonitrile. The elution conditions were as follows: 0–1 min, 65% A; 1–1.5 min, 65–52% A; 1.5–2.5 min, 52% A; 2.5–2.6 min, 52%–10% A; 2.6–3.5 min, 10% A; 3.5–4.1 min, 10%–65% A; 4.1–5 min, 65% A. The flow rate was 0.4 mL/min, and the column temperature was 40°C. The TP was detected in positive ion mode. An electrospray ion source was used, and the ion source temperature was 550°C, ionized voltage was 5500 V, curtain gas was at 35 psi, and impact gas was at 7 psi. Auxiliary gas 1 (GS1) was 55 psi, auxiliary gas 2 (GS2) was 50 psi, inlet voltage (EP) was 10 V, and outlet voltage (CXP) was 14 V. The quantification ion pairs of TP were 361.2/144.8. Additionally, the quantification ion pairs of internal carbamazepine were 236.8/194.0.The chromatographic conditions for PF were as follows: solvent A was 0.1% formic acid (V/V) in water, and solvent B was a gradient elution with acetonitrile. The elution conditions were as follows: 0–1 min, 90–65% A; 1–1.5 min, 65–44% A; 1.5–3 min, 44% A; 3–3.1 min, 44%–90% A; 3.1–5 min, 90% A. The flow rate was 0.35 mL/min, and the column temperature was 40°C. The TP was detected in positive ion mode. An electrospray ion source was used, and the ion source temperature was 550°C, ionized voltage was 5500 V, curtain gas was at 35 psi, and impact gas was at 9 psi. Auxiliary gas 1 (GS1) was 50 psi; auxiliary gas 2 (GS2) was 50 psi; inlet voltage (EP) was 10 V; outlet voltage (CXP) was 6 V. The quantification ion pairs of PF were 498.2/179.0. Also, the quantification ion pairs of internal carbamazepine were 236.8/194.0.P < 0.05 was considered to indicate statistical significance, and P < 0.01 was deemed to indicate a highly significant difference.All the data were expressed as mean ± standard deviation (SD), and statistical analyses were performed by SPSS 17.0. The pharmacokinetic data were fitted by DAS 2.0 software and then tested by SPSS 17.0. Pharmacokinetic parameters and analyte levels in the male and female rats were assessed by one-way ANOVA followed by a least-significant-difference test. Specificity was evaluated by comparing the chromatogram of blank plasma with that of blank plasma spiked with the mixed standard of TP (400 ng/mL) and PF , as previously described and that of plasma obtained from rats after administration of TP and PF after treatment for 30 min, respectively. For the pharmacokinetic study, the results showed that TP, PF, and IS were eluted, and no detectable interfering peaks were found . For theμg/mL. IS was used at concentrations of 5 ng/mL. Taking the ratio of the target component to the peak area of IS as the longitudinal coordinate and the concentration as the transverse coordinate, linear regression was performed. The standard curve was drawn with the reciprocal of the concentration as the weighted coefficient. For the pharmacokinetic study, the calibration curves constructed by plotting the peak area ratios of TP and PF to IS versus the nominal concentrations in the standard biological samples using linear regression analysis are listed in r2) of TP and PF were 0.9949 and 0.9928, respectively. For the tissue distribution study, the linear regression analysis is listed in Tables r2) of TP in the heart, liver, spleen, lungs, kidneys, skin, ovaries, and testes were 0.9979, 0.9941, 0.9991, 0.999, 0.9981, 0.9985, 0.9949, and 0.998, respectively. The correlation coefficients (r2) of PF in the heart, liver, spleen, lungs, kidneys, skin, ovaries, and testes were 0.998, 0.996, 0.997, 0.997, 0.993, 0.995, 0.998, and 0.993, respectively.Linearity of the developed analytical method was investigated by analyzing the matrix-matched construction via an internal standard approach, using mixed probe drugs at a series of concentrations. TP was used at the following concentrations: 50, 100, 250, 500, 1250, 2500, and 5000 ng/mL at 25°C. PF was used at concentrations of 1, 5, 10, 100, 250, 500, and 1000 μg/mL, 100 μg/mL, and 900 μg/mL) on the same day to ensure interday accuracy and precision. The present study estimated the intraday precision by analyzing six replicate QC samples on three consecutive days. The relative standard deviation (RSD) was used to assess precision, and accuracy was defined as the percent ratios of the calculated concentration to the nominal concentrations. For the pharmacokinetic study, the intra- and interday precision and accuracy for QC samples are listed in In total, six replicate analyses of the quality control (QC) samples  were prepared at three different concentrations  were used to assess the freeze-thaw cycle stabilities of the mixed TP, PF, and IS. All QC samples were stored at −80°C and subjected to three freeze-thaw cycles. Each cycle lasted for 20 h, and the concentrations were determined using LC-MS. For the pharmacokinetic study, the results of stability studies showed that the RSD was less than 15%, indicating that the sample has good stability under various conditions Tables  and S5. (0-∞) of TP in the combination group increased (P < 0.01), Cmax was decreased by 58.17%, the AUC t). The AUC t)(0- of PF in the compatibility group was increased (P < 0.01). Both C max and AUC (0-∞) were increased, and the half-life and average residence time were shortened (P > 0.05).The plasma concentration-time curves of TP and PF in each group of creams after transdermal administration are depicted in P < 0.05), and the drug concentration in liver tissue was decreased at 2 h, 4 h, and 8 h (P < 0.05) (P < 0.05) and was decreased in other tissues as well (P > 0.05) (P > 0.05)   . The con > 0.05) . There w Figures . After t < 0.05) .Cmax of PF increased by 2.38 times; AUCt) increased; and half-life and average retention time were shortened, suggesting that the combination of the two drugs can promote the transdermal absorption of PF after percutaneous administration and may enhance its efficacy [After the transdermal administration of the combination of TP and PF, there was no significant difference in the area under the blood concentration-time curve of TP, the peak concentration decreased, and the peak time was delayed, indicating that the absorption of TP in rats was slowed and delayed after the combined administration of the two drugs. The change of the drug-time curve also showed that TP had a more stable blood concentration change after compatibility. The above suggested that the compatibility of the two may weaken the toxicity of TP after percutaneous administration. efficacy . This coefficacy , reducinefficacy , which mAfter transdermal administration of TP and PF, the content of TP in the tissues of male rats decreased. This may be caused by the specific expression of CYP3A2 in male rats and the accelerated metabolism of TP . HoweverIn conclusion, this is the first report to evaluate the pharmacokinetics and tissue distribution of the combination of TP and PF in rats after transdermal administration. The absorption of TP in rats was slowed and delayed. Additionally, the change of plasma concentration was more stable, and the absorption of PF in rats was accelerated. Furthermore, the TP content in the heart, spleen, lungs, and kidneys of male rats decreased significantly at 2 h. The drug concentration in liver tissue decreased significantly at 2 h, 4 h, and 8 h. However, there was no significant difference in the PF content in the heart, liver, spleen, lungs, and kidneys. In this report, we studied the pharmacological effects and tissue distribution of the combination of TP and PF after transdermal administration in male and female rats for the first time. This work will provide useful information for the clinical application and further research of TP.