Interplay of neuropilin-1 and semaphorin 3A after partial hepatectomy in rats

AIM:To elucidate the role of neuropilin-1(Nrp-1) and semaphorin 3A(Sema3A) in sinusoidal remodeling during liver regeneration in rats.METHODS:Male Wistar/ST rats at 7 wk of age,weighing about 200 g,were used for all animal experiments.In vivo,at 24,48,72,96,144 and 192 h after twothirds partial hepatectomy(PHx),the remnant livers were removed.Liver tissues were immunohistochemically stained for Nrp-1,Sema3A and SE-1,a liver sinusoidal endothelial cell(SEC) marker.Total RNA of the liver tissue was extracted and reversely transcribed into cDNA.The mRNA expression of Sema3A was analyzed by quantitative real-time polymerase chain reaction and normalized to that of ribosomal protein S18.In vitro,SECs were isolated from rat liver and cultured in endothelial growth medium containing 20 ng/mL vascular endothelial cell growth factor.Migration of SECs in primary culture was assessed by cell transwell assay with or without recombinant Sema3A.Apoptotic cells were determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method.RESULTS:In vitro,immunohistochemistry study revealed that Sema3A and Nrp-1 were constitutively expressed in hepatocytes and SECs,respectively,in normal rat liver tissues.Nrp-1 expression in SECs was quantified by the percentage of immunostained area with antiNrp-1 antibody in relation to the area stained with SE-1.Between 24 h and 96 h following resection of liver,Nrp-1 expression in SECs was transiently increased.Compared with the baseline(5.2% ± 0.1%),Nrp-1 expression in SECs significantly increased at 24 h(17.3% ± 0.7%,P < 0.05),48 h(39.1% ± 0.6%,P < 0.01),72 h(46.9% ± 4.5%,P < 0.01) and 96 h(29.9% ± 3.8%,P < 0.01) after PHx,then returned to the basal level at termination of liver regeneration.Interestingly,the expression of Sema3A was inversely associated with that of Nrp-1 in liver after PHx.Sema3A mRNA expression was significantly reduced by about 75% over the period 24-144 h after PHx(P < 0.05),and returned to basal levels at 192 h after PHx.In vitro,SECs isolated from rats after PHx(PHx-SECs) were observed to migrate to the lower chamber of the cell transwell system after incubation for 24 h,but not cells from normal rats(CONT-SECs),indicating that mobility of PHx-SECs increases as compared with that of CONT-SECs.Moreover,recombinant Sema3A significantly attenuated migration in PHx-SECs in primary culture(vehicle-treated 100% ± 7.9% vs Sema3A-treated 42.6% ± 5.4%,P < 0.01),but not in CONT-SECs.Compared with CONTSECs,the apoptotic rate of PHx-SECs decreased by 78.3%(P < 0.05).There was no difference in apoptosis between CONT-SECs that were treated with vehicle and Sema3A.However,in PHx-SECs,apoptosis was induced by the presence of 5 nmol Sema3A for 24 h(vehicle-treated 21.7% ± 7.6% vs Sema3A-treated 104.3% ± 8.9%,P < 0.05).In addition,immunohistochemistry confirmed the increased expression of Nrp-1 in PHx-SECs,while it was noted to a lesser extent in CONT-SECs.CONCLUSION:The interplay of Nrp-1 and Sema3A shown in our results may lead to a better understanding of interaction between sinusoidal remodeling and SECs during liver regeneration.

Topical Application of Tenshino-Softgel^TMReduces Epidermal Nerve Fiber Density in a Chronic Dry Skin Model Mouse

Background: Dry skin induces antihistamine-resistant itch, as well as epidermal hyperinnervation, which is partly responsible for peripheral itch sensitization. In acute dry skin, topical application of emollients prevents the penetration of nerve fibers into the epidermis. However, the effects of emollients on itch and epidermal hyperinnervation in individuals with chronic dry skin are poorly understood. Objective: This study examined the effects of Tenshino-softgelTM (TSG) on itch-related behavior, epidermal hyperinnervation and skin barrier function in a chronic dry skin model mouse. Methods: Chronic dry skin was induced by application of acetone/ether (1:1) mixture and water (AEW) to the rostral parts of the back of hairless mice twice daily for six consecutive days. As treatment, TSG or, as control, Vaseline (V) was applied to the same areas twice daily. Skin barrier function was evaluated by measuring transepidermal water loss (TEWL) before each treatment. Scratching behavior was recorded and analyzed using a SCLABA&reg-real system, and skin samples were collected for immunohistochemical assays. Results: TEWL tended to be lower and scratching bouts fewer in AEW + TSG- than in AEW-treated mice. The numbers of protein gene product 9.5-immunoreactive fibers and substance P-immunoreactive fibers were each significantly lower in the epidermis of AEW + TSG- than of AEW-treated mice, but the expression of nerve growth factor in epidermis was similar in the three groups. Semaphorin 3A expression was significantly higher in the epidermis of AEW + TSG- than of AEW- and AEW + V-treated mice. Conclusion: Topical application of TSG may attenuate itch induced by chronic dry skin through a mechanism involving the inhibition of epidermal hyperinnervation.