Anti-Hyperglycemic Properties of Crude Extract and Triterpenes from Poria cocos

Poria cocos, Bai Fu Ling in Chinese, is used in traditional Chinese medicine to treat diabetes. However, its claimed benefits and mechanism are not fully understood. This study aimed to investigate the effect and action of P. cocos on type 2 diabetes. We first performed phytochemical analysis on the crude extract and factions of P. cocos. P. cocos crude extract at 50 mg/kg body weight or more significantly decreased blood glucose levels in db/db mice. Based on a bioactivity-directed fractionation and isolation (BDFI) strategy, chloroform fraction and subfractions 4 and 6 of the P. cocos crude extract possessed a blood glucose-lowering effect. Dehydrotumulosic acid, dehydrotrametenolic acid, and pachymic acid were identified from the chloroform sub-fractions 4, 3, and 2, respectively. Dehydrotumulosic acid had anti-hyperglycemic effect to a greater extent than dehydrotrametenolic acid and pachymic acid. Mechanistic study on streptozocin- (STZ-) treated mice showed that the crude extract, dehydrotumulosic acid, dehydrotrametenolic acid, and pachymic acid of P. cocos exhibited different levels of insulin sensitizer activity. However, the P. cocos crude extract and triterpenes appeared not to activate PPAR-γ pathway. Overall, the data suggest that the P. cocos extract and its triterpenes reduce postprandial blood glucose levels in db/db mice via enhanced insulin sensitivity irrespective of PPAR-γ

Antidiabetic effect and mode of action of cytopiloyne

Cytopiloyne was identified as a novel polyacetylenic compound. However, its antidiabetic properties are poorly understood. The aim of the present study was to investigate the anti-diabetic effect and mode of action of cytopiloyne on type 2 diabetes (T2D). We first evaluated the therapeutic effect of cytopiloyne on T2D in db/db mice. We found that one dose of cytopiloyne reduced postprandial glucose levels while increasing blood insulin levels. Accordingly, long-term treatment with cytopiloyne reduced postprandial blood glucose levels, increased blood insulin, improved glucose tolerance, suppressed the level of glycosylated hemoglobin A1c (HbA1c), and protected pancreatic islets in db/db mice. Next, we studied the anti-diabetic mechanism of action of cytopiloyne. We showed that cytopiloyne failed to decrease blood glucose in streptozocin- (STZ-)treated mice whose β cells were already destroyed. Additionally, cytopiloyne dose dependently increased insulin secretion and expression in β cells. The increase of insulin secretion/expression of cytopiloyne was regulated by protein kinase C α (PKC α ) and its activators, calcium, and diacylglycerol (DAG). Overall, our data suggest that cytopiloyne treats T2D via regulation of insulin production involving the calcium/DAG/PKC α cascade in β cells. These data thus identify the molecular mechanism of action of cytopiloyne and prove its therapeutic potential in T2D

Cell replacement and visual restoration by retinal sheet transplants

Retinal diseases such as age-related macular degeneration (ARMD) and retinitis pigmentosa (RP) affect millions of people. Replacing lost cells with new cells that connect with the still functional part of the host retina might repair a degenerating retina and restore eyesight to an unknown extent. A unique model, subretinal transplantation of freshly dissected sheets of fetal-derived retinal progenitor cells, combined with its retinal pigment epithelium (RPE), has demonstrated successful results in both animals and humans. Most other approaches are restricted to rescue endogenous retinal cells of the recipient in earlier disease stages by a ‘nursing’ role of the implanted cells and are not aimed at neural retinal cell replacement. Sheet transplants restore lost visual responses in several retinal degeneration models in the superior colliculus (SC) corresponding to the location of the transplant in the retina. They do not simply preserve visual performance – they increase visual responsiveness to light. Restoration of visual responses in the SC can be directly traced to neural cells in the transplant, demonstrating that synaptic connections between transplant and host contribute to the visual improvement. Transplant processes invade the inner plexiform layer of the host retina and form synapses with presumable host cells. In a Phase II trial of RP and ARMD patients, transplants of retina together with its RPE improved visual acuity. In summary, retinal progenitor sheet transplantation provides an excellent model to answer questions about how to repair and restore function of a degenerating retina. Supply of fetal donor tissue will always be limited but the model can set a standard and provide an informative base for optimal cell replacement therapies such as embryonic stem cell (ESC)-derived therapy

The Study of Ketoconazole on Testicular Steroidogenesis in Male Animal

摘 要 Ketoconazole (KCZ)為imidazole類的抗黴菌劑，為瞭解KCZ抑制類固醇內泌素生合成的作用，本實驗利用成年公犬及小鼠睪丸間質細胞瘤MA-10細胞研究KCZ減少雄性素生合成的可能機制。以每日每公斤30 mg KCZ單一口服劑量連續投予公犬七日及以每公斤30 mg KCZ單一口服劑量投予公犬，並分別檢測公犬於服用KCZ前後血清類固醇內泌素生成的變化及服藥後24小時內的血清睪固酮分泌狀況；另外以每公斤30 mg及10 mg KCZ單一口服劑量分別間隔投予對人類絨毛膜性腺激素刺激正常反應之公犬，以檢測公犬於不同劑量KCZ投予前後，以及在人類絨毛膜性腺激素刺激後的血清睪固酮濃度，結果顯示KCZ抑制公犬的助孕素轉換成睪固酮及氫基皮質酮，且呈現對公犬睪固酮分泌之暫時性抑制作用，而對公犬睪固酮分泌之作用具有顯著的劑量差異性 (P<0.05)，而此顯著差異性未顯現於KCZ於公犬對人類絨毛膜性腺激素刺激反應下血清睪固酮生合成作用。此外亦利用小鼠睪丸間質細胞瘤MA-10細胞，研究KCZ在in vitro下對睪丸分泌助孕素及環單磷酸核腺之影響，結果指出KCZ抑制間質細胞之adenylate cyclase holoenzyme催化部分之作用。由以上的結果顯示KCZ除了經由選擇性抑制cytochrome P-450相關性酵素及競爭性佔據雄性素受納器外，亦可能藉阻斷adenylate cyclase的活性，來達到抑制睪丸分泌睪固酮的作用。Summary Ketoconazole (KCZ) is an imidazole antifungal agent. According to the inhibitory effect of KCZ on steroid hormones biosynthesis, our laboratory utilized the adult male dogs and MA-10 mouse Leydig tumor cells for investigating the possible mechanism of causing androgen deficiency by KCZ. The male dogs were orally administrated with 30 mg KCZ/ kg of body weight/day for a week for detecting the changes of serum steroid hormones production. A single oral dose of 30 mg KCZ/ kg of body weight was also administered to other male dogs and followed by 24-hour observation. Another male dogs with normal response to human chorionic gonadotropin (hCG) were orally administered with 10 mg KCZ/ kg of body weight and (30 mg KCZ /kg of body weight, respectively. Thereafter, the effects of different-dose KCZ on male dog's testosterone production and its response to hCG stimulation were also investigated. The results indicated that KCZ inhibited the conversion from progesterone to testosterone and cortisol in male dogs. Meanwhile, KCZ exerted temporary inhibition on testosterone biosynthesis within 24 hours in male dogs. In addition, the significant difference between dosages on the effects of KCZ treatment on testosterone secretion in male dogs was observed. However, the effect of KCZ treatment on testosterone response to hCG stimulation between dosages was not significant. Moreover, the effects of KCZ on secretion of progesterone and cAMP in MA-10 cells were investigated in vitro. These data indicated that KCZ induced the inhibition of a catalytic component of adenylate cyclase holoenzyme in MA-10 cells. Excepting the selective inhibition of cytochrome P-450 enzymes and competitive binding of androgen receptors by KCZ, we concluded the block of adenylate cyclase activity by KCZ was another possible mechanism to inhibit testicular testosterone secretion.目 次 中文摘要……………………………………………………………………1 英文摘………………………………………………………………………2 縮寫表………………………………………………………………………3 第一章 文獻探討………………………………………………………6 第一節 Ketoconazole的作用機制…………………………………6 第二節 Ketoconazole的藥理作用………………………………10 第三節 Ketoconazole抑制雄性素的生成………………………13 第四節 Ketoconazole抑制氫基皮質酮的生成…………………16 第五節 Ketoconazole的其他作用………………………………19 第六節 小鼠睪丸間質細胞瘤MA-10細胞…………………………22 第二章 材料與方法……………………………………………………23 第一節 實驗犬資料及小鼠間質細胞瘤MA-10細胞培養方法……23 第二節 Ketoconazole對公犬睪丸類固醇生合成作用之研究…24 實驗一 高劑量Ketoconazole對睪固酮及氫基皮質酮生合成 之研究……………………………………………24 實驗二 Ketoconazole對人類絨毛膜性腺激素刺激睪固酮 生合成之研究……………………………………26 實驗三 高劑量Ketoconazole對睪固酮分泌之影響……26 第三節 Ketoconazole對小鼠間質MA-10細胞助孕素及cAMP 生合成之研究……………………………………………27 第四節 統計分析…………………………………………………29 第三章 結果…………………………………………………………30 第一節 高劑量Ketoconazole對公犬睪固酮及氫基皮質酮生合成 之影響……………………………………………………30 第二節 Ketoconazole在公犬對人類絨毛膜性腺激素刺激睪固酮 生合成之影響……………………………………………31 第三節 高劑量Ketoconazole對公犬睪固酮分泌之影響………31 第四節 Ketoconazole對小鼠間質MA-10細胞助孕素及cAMP 生合成之影響……………………………………………32 第四章 討論……………………………………………………………48 附錄 附錄一 MA-10細胞生成助孕素相關的促進式化學冷光檢測圖…57 附錄二 發表文獻…………………………………………………58 參考文獻…………………………………………………………………81 圖 表 目 錄 表 次 Table 1. Serum Levels of Cholesterol before, during and after Cessation of High-dose Ketoconazole Administration…35 Table 2. Serum Levels of Cortisol, Testosterone and Progesterone before, during and after Cessation of High-dose Ketoconazole Administration…………………36 Table 3. Spermatozoal Numbers before, during and after Cessation of High-dose Ketoconazole Administration…37 Table 4. Basal and Post-hCG Stimulation Serum Testosterone Levels in Male Dogs Given Different Doses of Ketoconazole……………………………………………………38 Table 5. Effect of Ketoconazole and db-cAMP on Progesterone Release in MA-10 Cells3……………………………………39 Table 6. Effect of Ketoconazole on Forskolin and Sodium Fluoride Stimulated Progesterone and cAMP Release in MA-10 Cells……………………………………………………40 圖 次 Fig. 1 Structure of Ketoconazole…………………………………41 Fig. 2 Impact of Ketoconazole of Steroidogenesis Pathways…42 Fig. 3 Changes of Plasma Testosterone Concentrations before and after the Administration of Ketoconazole in Six Intact Male Adult Dogs…………………………………………………43 Fig. 4 The Inhibitory Effect of Ketoconazole on Progesterone Release in MA-10 Cells………………………………………44 Fig. 5 The Inhibitory Effect of Ketoconazole on Progesterone Release in MA-10 Cells under LH Stimulation……………45 Fig. 6 The Inhibitory Effect of Ketoconazole on cAMP and Progesterone Release in MA-10 Cells in the presence of IBMX and LH………………………………………………………46 Fig. 7 Model of the Action of Ketoconazole on the Adenylate Cyclase in MA-10 Cells………………………………………4

Field trial of medicinal plant, Bidens pilosa, against eimeriosis in broilers

Eimeriosis is a severe protozoan disease in poultry. Because of increasing concern about drug residue and drug resistance with the use of anticoccidial drugs, natural products are emerging as an alternative and complementary approach to control avian eimeriosis. Our previous publication showed that feed supplemented with B. pilosa (BP) was effective at combating chicken eimeriosis in experimental settings. However, its efficacy against chicken eimeriosis under field conditions is not known. Here, we investigated the efficacy of BP against eimeriosis on an organic chicken farm. We found that feed supplemented with BP, at the dose of 0.025% of feed or more, significantly reduced Eimeria infection. This treatment increased body weight gain and reduced feed conversion ratio, leading to superior growth performance. It lowered morbidity/mortality rate, decreased oocysts per gram of feces and gut pathology and augmented the anticoccidial index. Collectively, these data demonstrated the potential of BP to control chicken eimeriosis on chicken farms. BP can, therefore, be used as an effective means to control eimeriosis

Bidens pilosa and its active compound inhibit adipogenesis and lipid accumulation via down-modulation of the C/EBP and PPARγ pathways

Obesity and its complications are a major global health problem. In this study, we investigated the anti-obesity effect and mechanism of an edible plant, Bidens pilosa, and its active constituent. We first assessed the long-term effect of B. pilosa on body composition, body weight, blood parameters in ICR mice. We observed that it significantly decreased fat content and increased protein content in ICR mice. Next, we verified the anti-obesity effect of B. pilosa in ob/ob mice. It effectively and dose-dependently reduced fat content, adipocyte size and/or body weight in mice. Moreover, mechanistic studies showed that B. pilosa inhibited the expression of peroxisome proliferator activated receptor γ (PPARγ), CCAAT/enhancer binding proteins (C/EBPs) and Egr2 in adipose tissue. Finally, we examined the effect of 2-β-D-glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne (GHT) on adipogenesis in adipocytes. We found that B. pilosa significantly decreased the adipogenesis and lipid accumulation. This decrease was associated with the down-regulation of expression of Egr2, C/EBPs, PPARγ, adipocyte Protein 2 (aP2) and adiponectin. In summary, this work demonstrated that B. pilosa and GHT suppressed adipogenesis and lipid content in adipocytes and/or animals via the down-regulation of the Egr2, C/EBPs and PPARγ pathways, suggesting a novel application of B. pilosa and GHT against obesity

Exendin-4 improves resistance to Listeria monocytogenes infection in diabetic db/db mice

The incidence of diabetes mellitus is increasing among companion animals. This disease has similar characteristics in both humans and animals. Diabetes is frequently identified as an independent risk factor for infections associated with increased mortality. In the present study, homozygous diabetic (db/db) mice were infected with Listeria (L.) monocytogenes and then treated with the anti-diabetic drug exendin-4, a glucagon-like peptide 1 analogue. In aged db/db mice, decreased CD11b+ macrophage populations with higher lipid content and lower phagocytic activity were observed. Exendin-4 lowered high lipid levels and enhanced phagocytosis in macrophages from db/db mice infected with L. monocytogenes. Exendin-4 also ameliorated obesity and hyperglycemia, and improved ex vivo bacteria clearance by macrophages in the animals. Liver histology examined during L. monocytogenes infection indicated that abscess formation was much milder in exendin-4-treated db/db mice than in the control animals. Moreover, mechanistic studies demonstrated that expression of ATP binding cassette transporter 1, a sterol transporter, was higher in macrophages isolated from the exendin-4-treated db/db mice. Overall, our results suggest that exendin-4 decreases the risk of infection in diabetic animals by modifying the interaction between intracellular lipids and phagocytic macrophages

Data on the effect of Cytopiloyne against Listeria monocytogenes infection in mice

Cytopiloyne (CP), a novel polyacetylene compound extracted from B. pilosa, shows a multi-bioactivity, including immunomodulatory and antidiabetes. Here, we investigated the anti-Listeria effect of cytopiloyne in mice by assessing mortality, clearance of L. monocytogenes, and pathology examination. The data presented herein are supplemental to our research article entitled “Cytopiloyne, a polyacetylenic glucoside from Bidens pilosa, acts as a novel anticandidial agent via regulation of macrophages” [1]