A proton-coupled organic cation antiporter is involved in the blood-brain barrier transport of Aconitum alkaloids.

ETHNOPHARMACOLOGICAL RELEVANCE: The herbs of Aconitum are the essential Traditional Chinese medicine and have played an indispensable role in many Asian countries for thousands of years to treat critical illnesses, and chronic, stubborn diseases. However, Aconitum may induce severe neurotoxicity and even death. So far the mechanism of Aconitum penetrating the blood-brain barrier (BBB) is still unclear. AIM OF THE STUDY: To determine whether influx transporters contribute to the brain uptake of the highly toxic alkaloids in Aconitum including aconitine (AC), mesaconitine (MA) and hypaconitine (HA). MATERIALS AND METHODS: The uptake of AC, MA and HA was characterized using in vitro hCMEC/D3 model and in situ mouse brain perfusion. In hCMEC/D3 cells, the effect of incubation temperature, time, initial drug concentration, energy (NaN3), extracellular and intracellular pH (FCCP and NH4Cl), the prototypical substrates/inhibitors of known organic cation transporting carriers and trans-stimulation (pre-incubating with pyrilamine and diphenhydramine) on the cellular uptake were studied. In addition, the effect of silencing OCTN1, OCTN2 and PMAT by specific siRNA was investigated. In mice, the contribution of the proton-coupled antiporter on the brain uptake of Aconitum was investigated by chemical inhibition. RESULTS: In hCMEC/D3 cells, AC, MA and HA were each taken up in a temperature-, time- and concentration-dependent manner, which were reduced by NaN3 and FCCP. Regulation of extracellular and intracellular pH as well as trans-stimulation studies showed that AC, MA and HA were transported by a proton-coupled antiporter expressed at the plasma membrane that could also transport pyrilamine and diphenhydramine. Each uptake was markedly inhibited by various cationic drugs, but insensitive to the prototypical substrates/inhibitors of identified organic cation transporting carriers, such as OCTs, PMAT, MATEs and OCTNs. In addition, silence of OCTN1, OCTN2 and PMAT had no significant inhibitory effect on the uptake of AC, MA and HA. In mice, the brain uptake of each alkaloid measured by in situ brain perfusion was suppressed by diphenhydramine when the transport capacity of P-gp/Bcrp at the BBB was chemically inhibited. CONCLUSIONS: A novel proton-coupled organic cation antiporter plays a predominant role in the blood to brain influx of AC, MA and HA at the BBB, and thus affect the safety of Aconitum species.

ROS generated by CYP450, especially CYP2E1, mediate mitochondrial dysfunction induced by tetrandrine in rat hepatocytes.

AIM: Tetrandrine, an alkaloid with a remarkable pharmacological profile, induces oxidative stress and mitochondrial dysfunction in hepatocytes; however, mitochondria are not the direct target of tetrandrine, which prompts us to elucidate the role of oxidative stress in tetrandrine-induced mitochondrial dysfunction and the sources of oxidative stress. METHODS: Rat primary hepatocytes were isolated by two-step collagenase perfusion. Mitochondrial function was evaluated by analyzing ATP content, mitochondrial membrane potential (MMP) and the mitochondrial permeability transition. The oxidative stress was evaluated by examining changes in the levels of reactive oxygen species (ROS) and glutathione (GSH). RESULTS: ROS scavengers largely attenuated the cytotoxicity induced by tetrandrine in rat hepatocytes, indicating the important role of ROS in the hepatotoxicity of tetrandrine. Of the multiple ROS inhibitors that were tested, only inhibitors of CYP450 (SKF-525A and others) reduced the ROS levels and ameliorated the depletion of GSH. Mitochondrial function assays showed that the mitochondrial permeability transition (MPT) induced by tetrandrine was inhibited by SKF-525A and vitamin C (VC), both of which also rescued the depletion of ATP levels and the mitochondrial membrane potential. Upon inhibiting specific CYP450 isoforms, we observed that the inhibitors of CYP2D, CYP2C, and CYP2E1 attenuated the ATP depletion that occurred following tetrandrine exposure, whereas the inhibitors of CYP2D and CYP2E1 reduced the ROS induced by tetrandrine. Overexpression of CYP2E1 enhanced the tetrandrine-induced cytotoxicity. CONCLUSION: We demonstrated that CYP450 plays an important role in the mitochondrial dysfunction induced by the administration of tetrandrine. ROS generated by CYP450, especially CYP2E1, may contribute to the mitochondrial dysfunction induced by tetrandrine.

Comparative 28-day repeated oral toxicity of Longdan Xieganwan, Akebia trifoliate (Thunb.) koidz., Akebia quinata (Thunb.) Decne. and Caulis aristolochiae manshuriensis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Longdan Xieganwan, which contains Aristolochia species, is a traditional Chinese prescription. It has been used for thousands of years to "enhance liver". However, many cases of Longdan Xieganwan induced nephropathy were reported recently. AIM OF THE STUDY: This study was designed to compare the possible toxic effects of Longdan Xieganwan and three different Aristolochia species, i.e. Akebia trifoliate (Thunb.) koid (Akebia trifoliate), Akebia quinata (Thunb.) Decne. (Akebia quinata) and Caulis aristolochiae manshuriensis (Aristolochia manshuriensis). MATERIALS AND METHODS: Mice were orally administered these drugs for 28 days. Clinical signs, body weights, serum biochemistry, organ weights and histopathology were examined. RESULTS: Significantly decreased body weights and obvious nephropathy were noticed in the Aristolochia manshuriensis groups at doses higher than 0.24 g/kg/d. A few endothelial cell degenerations in renal glomerulus were observed in the Akebia trifoliate group at a high-dose of 2.00 g/kg/d. No significant changes were observed in the other groups. CONCLUSIONS: The no-observed-adverse-effect levels (NOAELs) for Aristolochia manshuriensis, Akebia trifoliate, Akebia quinata and Longdan Xieganwan in this study for mice were 0.06 g/kg/d, 0.40 g/kg/d, higher than 3.00 g/kg/d and higher than 10.00 g/kg/d, which were equivalent to 0.25 times, 5 times, 25 times and 10 times of normal human dose in clinical prescription, respectively.

Roles of reactive oxygen species and MAP kinases in the primary rat hepatocytes death induced by toosendanin.

Toosendanin (Tsn), a triterpenoid extracted from Melia toosendan Sieb et Zucc, possesses different pharmacological effects in human and important values in agriculture. However, liver injury has been reported when toosendanin or Melia-family plants, which contain toosendanin are applied. The mechanism by which toosendanin induces liver injury remains largely unknown. Here we reported that toosendanin induced primary rat hepatocytes death by mitochondrial dysfunction and caspase activation. Toosendanin led to decrease of mitochondrial membrane potential, fall in intracellular ATP level, release of cytochrome c to cytoplasm, activation of caspase-8, 9, and 3 and ultimately cell death. Level of reactive oxygen species (ROS) was also increased in hepatocytes after incubation with toosendanin. Catalase, the H2O2-decomposing enzyme, can prevent the reduction in ATP level and protect hepatocytes from toosendanin-induced death. The ERK1/2 (p44/42 MAP kinases) and JNK (c-Jun N-terminal kinase) were activated, but p38 MAPK was not activated by toosendanin. Inhibition of ERK1/2 activation sensitized hepatocytes to death and increased activity of caspase-9 and 3 in response to toosendanin. Inhibition of JNK attenuated toosendanin-induced cell death. These results suggested that toosendanin causes death of primary rat hepatocytes by mitochondrial dysfunction and caspase activation. Generation of ROS and MAP kinases activation might be involved in this process.

Effect of Feitai on bleomycin-induced pulmonary fibrosis in rats.

The current therapeutic approaches for pulmonary fibrosis, which is characterized by fibroblast proliferation and extracellular matrix remodeling, are unsatisfactory. Feitai, consisting of several herbs, is a folk formula for pulmonary tuberculosis therapy in China. To investigate the effects of Feitai on pulmonary fibrosis, Feitai was administered orally to bleomycin (BLM)-treated rats, and the lung toxicity effects were evaluated according to inflammatory cell count, protein concentration, and lactate dehydrogenase (LDH) activity in the bronchoalveolar lavage fluid (BALF), malondialdehyde level and hydroxyproline content in lung tissue 28 days post-BLM. Serial sections of the lung were stained with hematoxylin and eosin (HE) and Masson trichrome, respectively. The degree of fibrosis was assessed quantitatively using LEICA QWin image analyzer. Results showed that Feitai inhibited BLM-induced lung fibrotic lesions in a dose-dependent manner as reflected by decreased the lung hydroxyproline content and lung fibrosis fraction 28 days after BLM instillation. Treatment with Feitai also significantly ameliorated the BLM-induced lung toxicity effects detected in BALF and lung tissue. The effects in vitro on WI-38 human lung fibroblast cell line showed that Feitai significantly reduced the cell proliferation and transforming growth factor (TGF)-beta stimulated type I collagen synthesis. These results strongly demonstrate that Feitai may be useful in the treatment of pulmonary fibrosis.

Feitai attenuates bleomycin-induced pulmonary fibrosis in rats.

Pulmonary fibrosis is a common consequence of numerous pulmonary diseases. The current therapeutic approaches for this condition are unsatisfactory. Feitai, a composite formula consisting of several herbs, is used in China as a folk remedy for treating patients with pulmonary tuberculosis. In this study, we extensively investigate the effects and mechanisms of Feitai on bleomycin (BLM)-induced pulmonary fibrosis in rats. One hundred and twenty male Sprague-Dawley rats were randomly divided into four groups, referred to as the saline-water, saline-Feitai, BLM-water, and BLM-Feitai groups. Following a single instillation of BLM (5 mg/kg) or saline, rats were orally administered Feitai at a dose of 3 g/kg body weight or sterilized distilled water once daily. Rats were killed at 7, 14, or 28 d post-BLM. Inflammatory cell count, protein concentration, and lactate dehydrogenase activity in bronchoalveolar lavage fluid were measured, and myeloperoxidase activity and lipid peroxide content in lung homogenates were analyzed. Treatment with Feitai inhibited lung fibrotic progression induced by BLM, as indicated by the decrease in lung hydroproline content and lung fibrosis score at 28 d post-BLM. This was accompanied by significant amelioration of BLM-induced body weight loss, lung edema, and inflammatory response during the development of lung injury in the acute phase. The results strongly indicate the beneficial effects of Feitai in protecting against BLM-induced pulmonary fibrosis. Furthermore, the inflammatory response and lipid peroxidation were inhibited by Feitai, suggesting that the effect of this formula on BLM-induced lung injury and fibrosis is associated with antiinflammatory and antioxidant properties.