Bofutsushosan, a Japanese herbal (Kampo) medicine, attenuates progression of nonalcoholic steatohepatitis in mice

BACKGROUND: Obesity-induced liver disease (nonalcoholic fatty liver disease, NAFLD) is now the commonest cause of chronic liver disease in affluent nations. There are presently no proven treatments for NAFLD or its more severe stage, nonalcoholic steatohepatitis (NASH). Bofutsushosan (BTS), a Japanese herbal (Kampo) medicine, long used as an anti-obesity medicine in Japan and other Asian countries, has been shown to reduce body weight and improve insulin resistance (IR) and hepatic steatosis. The precise mechanism of action of BTS, however, remains unclear. To evaluate the ability of BTS to prevent the development of NASH, and determine the mediators and pathways involved. METHODS: C57BL/6 mice were injected intra-peritoneally with gold-thioglucose and fed a high-fat diet (HF) or HF diet admixed with either 2 or 5 % BTS for 12 weeks. The effectiveness of BTS in attenuating features of NASH and the mechanisms through which BTS attenuated NASH were then assayed through an assessment of the anthropometric, radiological, biochemical and histological parameters. RESULTS: BTS attenuated the progression of NASH through induction of adiponectin and its receptors along with an induction of PPAR-α and PPAR-γ, decreased expression of SREBP-1c, increased hepatic fatty acid oxidation and increased hepatic export of triglycerides. BTS moreover, reduced IR through phosphorylation of the protein kinase, Akt. CONCLUSIONS: BTS through induction of adiponectin signaling and Akt attenuated development of NASH. Identification of the active entity in BTS should allow development of novel treatments for NASH. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00535-013-0852-8) contains supplementary material, which is available to authorized users

The changes in PTTH titers in the hemolymph.

<p>PTTH titers in the hemolymph of post-diapause pupae after warming (A) and nondiapause pupae after pupal ecdysis (B) were determined by TR-FIA. The values are the means ±SEM (n = 8). Different letters above the bars indicate a significant difference (ANOVA followed by Tukey-Kramer multiple-comparison test, p <0.05).</p

The role of the Br-CC-CA in post-diapause development.

<p>(A) The effects of brain removal and implantation on adult development of post-diapause pupae. Diapausing pupae were chilled for 8 weeks and the Br-CC-CA was removed immediately after warming. A Br-CC-CA collected from another previously chilled pupa was implanted into a Br-CC-CA-deficient pupa. As a positive control, pupae were only injured (sham-operated). For comparison, a Br-CC-CA collected from an unchilled pupa 5 weeks after pupation (*) was implanted into a Br-CC-CA-deficient pupa. Percentage values were determined with 6–12 pupae per operation. The values shown are the means (±SEM) of three independent determinations. (B) Time-dependent effects of brain removal on the development of post-diapause pupae. The Br-CC-CA was removed from post-diapause pupae at various times after warming (n = 12). (C) The effect of CC-CA removal (allatectomy) on adult development of post-diapause pupae. The CC-CA was removed immediately after warming. Percentage values were determined with 6–12 pupae per operation. The values shown are the means (±SEM) of three independent determinations.</p

The role of PTTH in the initiation of adult development.

<p>(A) The effect of brain extract (Br-ex) injection. The Br-CC-CA or brain alone (Br) was removed from post-diapause pupae 12 h after warming and 12 h later, brain extract (2 units of PTTH equivalents) was injected. As a negative control, saline was injected into the brain-deficient pupae. (B) The effect of PTTH removal from the brain extract. The brains were removed from post-diapause pupae 12 h after warming, and the debrained pupae were injected with the brain extract pre-treated with an anti-<i>Mab</i>PTTH or control antibody. As a negative and a positive controls, saline and the same dose (3 units of PTTH equivalents) of the brain extract were injected, respectively (n = 6). The values shown are the means (± SEM) of three independent determinations. (C) The Effect of the implantation of PTTH-containing gel into the Br-CC-CA-deficient pupae. The Br-CC-CA was removed from post-diapause pupae immediately after warming, and the PTTH-containing gel (+PTTH) was implanted into the Br-CC-CA-deficient pupae (n = 8). Control pupae received the gel without PTTH (-PTTH).</p

Developmental changes in <i>PTTH</i> gene expression and PTTH content in the brain.

<p>(A) <i>PTTH</i> gene expression in the brains of day-0 nondiapause pupae and post-diapause pupae at various times after warming was analyzed by qRT-PCR. The values shown are the means (± SEM) of three independent determinations. (B) PTTH content in the brains of day-0 nondiapause pupae and post-diapause pupae at various times after warming was determined by TR-FIA. The values are the means ±SEM (n = 6). Different letters above the bars indicate a significant difference (ANOVA followed by Tukey-Kramer multiple-comparison test, p <0.05).</p

qRT-PCR analysis of <i>MabTorso</i> expression in the PGs.

<p>The PGs were dissected from nondiapause and diapause pupae 0 to 3 days after pupal ecdysis (A) or from post-diapause pupae at various times after warming (B), and relative expression levels of the <i>MabTorso</i> gene were determined by qRT-PCR, with the level in day-0 nondiapause pupae being 1. The values shown are the means (± SEM) of three independent determinations.</p

The responsiveness of the PGs to PTTH in post-diapause pupae.

<p>(A) The PGs of post-diapause pupae were dissected at various times after warming and incubated <i>in vitro</i> with or without PTTH (0.2 units/ml) for 2 h. The amount of ecdysteroid secreted into the medium was determined by ELISA. The values are the means ± SEM (n = 6). (B) Activation ratios were calculated from the data in A. (C, D) The PGs of post-diapause pupae 12 h after warming (C) and of day-1 nondiapause pupae (D) were dissected and incubated <i>in vitro</i> with or without PTTH at various concentrations (0.01 to 2 units/ml) for 2 h, and the amount of ecdysteroid secreted into the medium was determined by ELISA. The values are the means ± SEM (n = 8). (E) The responsiveness to PTTH of the PGs of post-diapause pupae and nondiapause pupae was expressed as activation ratio, which was calculated from the data in C and D. *, P < 0.05, Student t-test.</p

Duration of chilling period required for diapause termination.

<p>Diapausing pupae 6 weeks after pupal ecdysis were chilled at 4°C and a fraction (n = 10) of the chilled pupae transferred to an incubator set at 25°C every week. Adult development was judged by observing the wing vein apolysis from the epidermis within 10 days after warming, and the percentage of the animals that initiated adult development was calculated. The values shown are the means (±SEM) of three independent determinations.</p

Prothoracicotropic hormone acts as a neuroendocrine switch between pupal diapause and adult development.

Diapause is a programmed developmental arrest that has evolved in a wide variety of organisms and allows them survive unfavorable seasons. This developmental state is particularly common in insects. Based on circumstantial evidence, pupal diapause has been hypothesized to result from a cessation of prothoracicotropic hormone (PTTH) secretion from the brain. Here, we provide direct evidence for this classical hypothesis by determining both the PTTH titer in the hemolymph and the PTTH content in the brain of diapause pupae in the cabbage army moth Mamestra brassicae. For this purpose, we cloned the PTTH gene, produced PTTH-specific antibodies, and developed a highly sensitive immunoassay for PTTH. While the hemolymph PTTH titer in non-diapause pupae was maintained at high levels after pupation, the titer in diapause pupae dropped to an undetectable level. In contrast, the PTTH content of the post-pupation brain was higher in diapause animals than in non-diapause animals. These results clearly demonstrate that diapause pupae have sufficient PTTH in their brain, but they do not release it into the hemolymph. Injecting PTTH into diapause pupae immediately after pupation induced adult development, showing that a lack of PTTH is a necessary and sufficient condition for inducing pupal diapause. Most interestingly, in diapause-destined larvae, lower hemolymph titers of PTTH and reduced PTTH gene expression were observed for 4 and 2 days, respectively, prior to pupation. This discovery demonstrates that the diapause program is already manifested in the PTTH neurons as early as the mid final instar stage