A pH-Adjustable Tissue Clearing Solution That Preserves Lipid Ultrastructures: Suitable Tissue Clearing Method for DDS Evaluation

Visualizing biological events and states to resolve biological questions is challenging. Tissue clearing permits three-dimensional multicolor imaging. Here, we describe a pH-adjustable tissue clearing solution, Seebest (SEE Biological Events and States in Tissues), which preserves lipid ultrastructures at an electron microscopy level. Adoption of polyethylenimine was required for a wide pH range adjustment of the tissue clearing solution.The combination of polyethylenimine and urea had a good tissue clearing ability for multiple tissues within several hours. Blood vessels stained with lipophilic carbocyanine dyes were deeply visible using the solution. Adjusting the pH of the solution was important to maximize the fluorescent intensity and suppress dye leakage during tissue clearing. The spatial distribution of doxorubicin and oxidative stress were observable using the solution. Moreover, spatial distribution of liposomes in the liver was visualized. Hence, the Seebest solution provides pH-adjustable, rapid, sufficient tissue clearing, while preserving lipid ultrastructures, which is suitable for drug delivery system evaluations

Saireito (TJ-114), a Japanese traditional herbal medicine, reduces 5-fluorouracil-induced intestinal mucositis in mice by inhibiting cytokine-mediated apoptosis in intestinal crypt cells.

Clinical chemotherapy frequently causes intestinal mucositis as a side effect, which is accompanied by severe diarrhea. We recently showed that the cytokine-mediated apoptotic pathway might be important for the development of intestinal mucositis induced by 5-fluorouracil (5-FU). Saireito, the traditional Japanese herbal (Kampo) medicine, is widely used to treat diarrhea and various inflammatory diseases in Japan. In the present study, we investigated the effect of saireito on 5-FU-induced intestinal mucositis in mice, especially in relation to apoptosis in the intestinal crypt. Male C57BL/6 mice were given 5-FU (50 mg/kg), i.p. once daily for 6 days. Intestinal mucositis was evaluated histochemically. Saireito (100-1000 mg/kg) was administered p.o. twice daily for 6 days. Repeated 5-FU treatment caused severe intestinal mucositis including morphological damage, which was accompanied by body weight loss and diarrhea. Daily administration of saireito reduced the severity of intestinal mucositis in a dose-dependent manner. Body weight loss and diarrhea during 5-FU treatment were also significantly attenuated by saireito administration. The number of apoptotic and caspase-3-activated cells in the intestinal crypt was increased, and was accompanied by up-regulated tumor necrosis factor (TNF)-α and interleukin (IL)-1β mRNA within 24 h of the first 5-FU injection. However, all of these measures were significantly lower after saireito administration. These results suggest that saireito attenuates 5-FU-induced intestinal mucositis. This action may come from the reduction of apoptosis in the intestinal crypt via suppression of the up-regulation of inflammatory cytokines. Therefore, saireito may be clinically useful for the prevention of intestinal mucositis during cancer chemotherapy

Effect of saireito on apoptosis and caspase-3 activation in the intestinal crypt induced by 5-fluorouracil (5-FU).

<p>5-FU (50 mg/kg) was injected i.p. while saireito (1000 mg/kg) was administered p.o. twice, 30 min before and 8 h after 5-FU injection. The jejunum was excised 24 h after 5-FU injection, sectioned, and TUNEL assay (A, 400×) and cleaved-caspases-3 immunostaining (B, 400×) were performed. The number of apoptotic (C) and caspase-3-activated cells (D) were counted. Data are presented as the mean ± SEM of 6 mice. *<i>P < 0.05</i>, versus control (vehicle alone); ^#<i>P < 0.05</i>, versus normal (5-FU-untreated).</p

Effect of saireito on the anti-tumor action, body weight loss, and diarrhea induced during 5-fluorouracil (5-FU) treatment in Colon 38 tumor-implanted mice.

<p>5-FU (20 mg/kg) was injected i.p. once daily for 6 days (days 7–12), while saireito (1000 mg/kg) was administered p.o. twice daily for 14 days (days 7–21), starting 7 day after tumor implantation. The volume (mm³) of solid tumor (A), body weight (B), and diarrhea score (C) were determined every 2 or 3 days, starting 7 days after the implantation. Data are presented as the mean ± SEM of 6–8 mice. *<i>P < 0.05</i>, versus control (vehicle alone); ^#<i>P < 0.05</i>, versus normal (5-FU-untreated).</p

Effect of saireito and daikenchuto on changes in body weight and diarrhea during 5-fluorouracil (5-FU) treatment.

<p>5-FU (50 mg/kg) was injected i.p. once daily while saireito (100–1000 mg/kg) and daikenchuto (2700 mg/kg) were administered p.o. twice daily for 6 days (days 0–5). Body weight is shown as a percentage of initial body weight (A), whereas the severity of diarrhea is scored using the four-grade scale (0 to 3) (B). Data are presented as the mean ± SEM of 6–8 mice. *<i>P < 0.05</i> versus control (vehicle alone), ^#<i>P < 0.05</i> versus normal (5-FU-untreated).</p

Three-dimensional high-performance liquid chromatograph pattern of saireito (TJ-114).

<p>Three-dimensional high-performance liquid chromatograph pattern of saireito (TJ-114).</p