Silibinin induced apoptosis of human epidermal cancer A431 cells by promoting mitochondrial NOS.

The antitumor effects of silibinin are of increasing interest, though its mechanism is not yet clear. The goal of this study was to clarify the mechanism of silibinin-induced cell death in the A431 human epidermoid carcinoma cell line. We used a cell viability assay, flow cytometry, nitric oxide (NO) assay, and western blotting to examine relationships between silibinin, NO generation and apoptosis in A431 cells. Silibinin inhibited A431 cell growth in a dose-dependent manner, inducing mitochondrial damage, and apoptosis at a high dose. At the same time, high dose silibinin increased NO levels in A431 cells and the endothelial nitric oxide synthase (eNOS) inhibitor NG-nitro-L-arginine methylester (L-NAME) attenuated silibinin-induced cell growth inhibition. By western blotting, silibinin caused increased eNOS phosphorylation in the mitochondria. The AMP-activated protein kinase inhibitor compound C significantly decreased p-eNOS expression, while blocking eNOS did not affect p-AMPK levels, suggested that AMPK acted upstream of eNOS. This study showed that silibinin increased NO levels in A431 cells by activating the AMPK-eNOS pathway, leading to mitochondrial dysfunction and apoptosis. In this mechanism of action, mitochondrial eNOS played an important role. The results provided new understanding of the functions of intracellular NO.

28-Day Oral Chronic Toxicity Study of Arctigenin in Rats.

Arctium lappa (burdock) is the most popular daily edible vegetable in China and Japan because of its general health tonic effects. Previous studies focused on the beneficial role of Arctigenin but neglected its potential side-effects and toxicities. In the present study, the sub-chronic toxicity profile of Arctigenin following 28 days of consecutive exposure was investigated in rats. The results showed that during the drug exposure period, Arctigenin-12 mg/kg administration resulted in focal necrosis and lymphocytes infiltration of heart ventricular septal muscle cells. In the kidney cortical zone, the renal tubular epithelial cells were swollen, mineralized, and lymphocyte infiltrated. In the liver, the partial hepatocyte cytoplasm showed vacuolation and fatty changes, focal necrosis, and interstitial lymphocyte infiltration. In the rats that underwent 36 mg/kg/day administration, there was bilateral testis and epididymis atrophy. In the lung and primary bronchus, erythrocytes and edema fluid were observed. Changes of proestrus or estrus were observed in the uterus, cervix, and vagina intimal epithelial cells. Lymphocytic focal infiltration occurred in the prostate mesenchyme. The high dosage of Arctigenin only decreased the body weight at day 4. At the end of the recovery period, histopathological changes were irreversible, even after withdrawal of the drug for 28 days. Focal necrosis still existed in the heart ventricular septal muscle cells and hepatocytes. Lymphocyte infiltrations were observed in the heart, renal cortex, hepatocyte, and pancreas exocrine gland. Meanwhile, atrophy occurred in the testicles and pancreas. In addition, in the Arctigenin-12 mg/kg group, creatinine (CREA) and brain weight were both significantly increased. The toxicokinetical study demonstrated that Arctigenin accumulated in the organs of rats. The food consumption, hematological, and biochemical parameters were not associated with the above results. These contradictory results might result from the lesions induced by Arctigenin, which were not sufficiently serious to change the parameters. These results suggest that Arctium lappa should be consumed daily with caution because of the potential toxicity induced by Arctigenin. According to all results, the lowest observed adverse effect level (LOAEL) was induced by 12 mg/kg daily exposure to Arctigenin, and the No-observed-adverse-effect-level (NOAEL) should be lower than 12 mg/kg.

[Bushen Huoxue Recipe Inhibited Vascular Calcification in Chronic Renal Failure Rats by Regulating BMP-2/Runx2/Osterix Signal Pathway].

OBJECTIVE: To observe the effect of Bushen Huoxue Recipe (BHR) on inhibiting vascular calcification (VC) in chronic renal failure (CRF) rats by regulating BMP-2/Runx2/Osterix signal pathway, and to explore its possible mechanism. METHODS: Thirty SD rats were randomly divided into the normal group, the model group, and the BHR group, 10 in each group. Rats in the model group and the BHR group were administered with 250 mg/kg adenine suspension by gastroagavage and fed with 1.8% high phosphorus forage, once per day in the first 4 weeks, and then gastric administration of adenine suspension was changed to once per two days in the following 5-8 weeks. Rats in the BHR group were administered with BHR at the daily dose of 55 g/kg by gastrogavage in the first 8 weeks, once per day. Equal volume of normal saline was given to rats in the normal group by gastrogavage for 8 weeks. Histological changes in renal tissue and aorta VC were observed by HE staining and alizarin red staining respectively. Levels of calcium (Ca), phosphorus (P), serum creatinine (Cr), blood urea nitrogen (BUN), and intact parathyroid hormone (iPTH) in serum were detected. Protein expression levels of bone morphogenetic protein (BMP-2), Runt related transcription factor (Runx2) , and Osterix were detected by Western blot. RESULTS: HE staining showed that compared with the normal group, disordered glomerular structure, tubular ectasia and dropsy, intracavitary inflammatory cell infiltration, dark brown crystal deposition in kidney tubules, renal interstitial fibrosis, and decreased number of renal blood vessels in the model group. Compared with the model group, normal glomerular numbers increased more, reduced degree of tubular ectasia, decreased number of inflammatory cells, and reduced adenine crystal deposition in the BHR group. Alizarin red staining showed that compared with the normal group, calcified nodes could be found in the model group, with extensive deposition of red particle in aorta. Compared with the model group, calcified nodes were reduced in the BHR group. Compared with normal group, serum levels of P, SCr, BUN, and iPTH significantly increased, serum Ca level significantly decreased, protein expressions of BMP-2, Runx2, Osterix also increased in the model group (P < 0.05, P < 0.01). Compared with the model group, serum levels of P, SCr, BUN, and iPTH levels significantly decreased, serum Ca level significantly increased, protein expressions of BMP-2, Runx2, Osterix also decreased in the BHD group (P < 0.05, P < 0.01). CONCLUSION: BHD could improve renal function, Ca-P metabolism, and renal histological changes in CHF rats, down-regulate the expression level of BMP-2/Runx2/Osterix signal pathway in vascular calcification of CRF, which might be one of the mechanisms for inhibiting VC in CHF.

Hydrogen sulfide reduces regional myocardial ischemia injury through protection of mitochondrial function.

Hydrogen sulfide (H2S) is a signaling gasotransmitter, involved in various physiological and pathological processes. H2S-donating drugs have been tested to conjugate the beneficial effects of H2S with other pharmaceutical properties. It has been shown that the endogenous cystathionine-γ-lyase (CSE)/H2S pathway participates in myocardial ischemia injury in isolated hearts in rats. The present study aimed to investigate the cytoprotective action of H2S against acute myocardial ischemia injury in rats. Isolated rat hearts were perfused and subjected to ischemic conditions for 4 h. The hearts were assigned to five groups: Sham, model, infarct plus low-dose (5 µmol/l) NaHS, infarct plus middle-dose (10 µmol/l) NaHS and infarct plus high-dose (20 µmol/l) NaHS. The administration of NaHS enhanced the activity of CSE, increased the content of H2S and reduced infarct volumes following myocardial ischemia injury. Furthermore, the administration of NaHS attenuated the injury to organelles (including the mitochondria, nucleus and myofilaments) by reducing lactate dehydrogenase activity, decreasing the level of mitochondrial malondialdehyde and increasing the activities of superoxide dismutase and glutathione peroxidase in the ischemic myocardial mitochondria. These protective effects of H2S against myocardial ischemia injury appeared to be mediated by its antioxidant activities and the preservation of mitochondrial function.

Reciprocal effects of Guizhi decoction to the Guizhi decoction syndrome by toll-like receptor mRNA expression and cytokines secretion.

OBJECTIVE: To explore the pathological mechanisms of Guizhi Decoction () syndrome and the therapeutic molecular mechanisms of the Guizhi Decoction, Mahuang Decoction (), Sangju Decoction ( ) and Yinqiao Powder (), as well as the potentially biological basis that Guizhi Decoction is most effective only for the patients with Guizhi Decoction syndrome in clinical practice. METHODS: We first got serum samples from the patients suffering from both upper respiratory tract infection and Guizhi Decoction syndrome identified by the doctors of Chinese medicine (CM) in the clinic. Four formulas with therapeutic actions of pungent warmth or pungent coolness for superficial syndromes were chosen and four kinds of rat serum samples each containing one of the above-mentioned herbal formulas were collected, then the effects of Guizhi Decoction syndromes' patient serum as well as the effects of sera containing the formulas after being stimulated by the patient serum samples on both the mRNA expression of certain toll-like receptor (TLR) subtypes and the release of some inflammatory cytokines in RAW264.7 cells were tested and analyzed in vitro. RESULTS: The expression of TLR-3, TLR-4 and TLR-9 mRNA among the 9 tested TLR subforms were up-regulated in the macrophages stimulated by the sera from untreated upper respiratory infection patients with the Guizhi Decoction syndrome (symptomcomplex). The products such as interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α and interferon (IFN)-ß from stimulated macrophages through TLR signaling pathways were also increased correspondingly. Interestingly, the changes induced by the Guizhi Decoction syndrome patients' sera were masked significantly after the macrophages were incubated with the sera from donors treated with Guizhi Decoction. Similarly, the three other exterior-releasing formulas were all effective in reversing the up-regulated changes of certain TLR subforms to different degrees, but both the number of targeted TLRs and efficacy of them seemed to be inferior to that of Guizhi Decoction. CONCLUSION: Evidence from these experiments might contribute to the scientific explanation of both the pharmacological mechanisms of Guizhi Decoction and also the CM theory that Guizhi Decoction is specifically prescribed for the treatment of Guizhi Decoction syndrome (The gearing formula to the symptom-complex).

[Effects of hydrogen sulfide on myocardial mitochondrial injury during acute myocardial ischemia in rats].

OBJECTIVE: To investigate the effect of hydrogen sulfide (H2S) on mitochondrial function in acute myocardial ischemia in rats. METHODS: Acute myocardial ischemia models were established by ligating the left anterior descending coronary artery (LADC) of rats. Fourty-eight male SD rats were randomly divided into 6 groups (n = 8): sham operation group, ischemia group, ischemia + sodium hydrosulfide (NaHS) low, middle and high dose groups and ischemia + DL-proparglycine(PPG) group. The ultrastructures of myocardial mitochondria were observed with electron microscope. The content of H2S in plasma and the activity of cystathionine-gamma-lyase (CSE) in myocardial tissue of rats were respectively detected. The swelling and activity of myocardial mitochondria were determined. The activities of ATPase, GSH-Px, SOD and the content of malondial-dehyde (MDA) in myocardial mitochondria of rats were also measured. RESULTS: Compared with those of the sham operation group, the content of H2S in plasma, the activity of CSE in myocardial tissue and the activity of myocardium mitochondria were significantly decreased. The activities of ATPase, SOD, GSH-Px in myocardial mitochondria were significantly decreased, The content of malondial dehyde(MDA) in myocardial mitochondria and the swelling of mitochondria were distinctly increased in the ischemia group (P < 0.01). Compared with those of the ischemia group, the content of H2S in plasma and the activity of CSE in myocardial tissue were increased, and the activities of mitochondria, ATPase, SOD, and GSH-Px in myocardial mitochondria were significantly increased in ischemia + NaHS low, middle and high-dose groups; the swelling of mitochondria and the content of MDA in myocardial mitochondria were significantly decreased in ischemia + NaHS middle and high-dose groups (P < 0.05 or P < 0.01). The administration of PPG could partially reduce the myocardial protection of hydrogen sulfide (P < 0.05 or P < 0.01). CONCLUSION: It could be concluded that the administration of hydrogen sulfide could enhance the activities of mitochondrial ATPase, SOD, GSH-Px, decrease the level of mitochondrial lipid peroxidation, and play a protective effect against acute myocardial ischemia.

Emodin down-regulates expression of TRPV1 mRNA and its function in DRG neurons in vitro.

Emodin is a principle ingredient isolated from rhubarb rhizome, which is commonly used for constipation or pain-related diseases in traditional Chinese medicine (TCM) practice. The transient receptor potential vanilloid 1 ion channel proteins (TRPV1) are abundantly expressed in the peripheral sensory neurons and are assumed to act as a kind of nociceptor involved in the perception of pain and development of hyperalgesia. The aim of this study was to further unravel the analgesic mechanisms of rhubarb through investigating the effects of its main constitutive ingredient emodin on the expression of TRPV1 mRNA as well as on its calcium- mediating functions in vitro. The primary DRG neurons with a high purity and viability were obtained, and the TRPV1 mRNA expression levels were examined by using real-time RT-PCR and the elevated amplitudes of intracellular [Ca(2+)]i in the DRG neurons evoked by TRPV1 agonist capsaicin were examined by confocal microscopy. The results showed that emodin could significantly down-regulate both the mRNA expression of TRPV1 and the capsaicin-evoked intracellular fluorescent intensity in the DRG neurons under both 37 degrees C and 39 degrees C in vitro. Concomitantly, all of the changes induced by emodin could not be blocked by pretreatment of the primary neurons with capsazepine, an antagonist of TRPV1. In conclusion, we established that the mRNA expression level of TRPV1 and its calcium-mediating function in naive DRG neurons could be down-regulated by emodin through perhaps the non-TRPV1 channel pathways, and this might be the molecular mechanisms for rhubarb to inhibit hyperalgesia induced by inflammatory stimuli.

Anti-nociceptive mechanism of baicalin involved in intervention of TRPV1 in DRG neurons in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellaria baicalensis Georgi (Lamiaceae) is often included as an ingredient in traditional Chinese compound prescriptions for the treatment of fever-related or inflammatory conditions. AIM OF THE STUDY: The present work was to further uncover the analgesic mechanisms of baicalin (a known principal constituent of Scutellaria baicalensis) by investigating its effects on the expression of TRPV1 mRNA as well as on its functions as mediators of calcium entrance into the cytoplasm of dorsal root ganglion (DRG) neurons in vitro. MATERIALS AND METHODS: By using CPT as an agent to eliminate the non-neuronal cells and using serum-free neurobasal as culture medium, primary cultures of rat DRG neurons with high purity and viability were established. On this basis, effects of baicalin on both the expression of TRPV1 mRNA and on the function of TRPV1 in vitro under two various temperature conditions were studied. The TRPV1 mRNA expression levels were examined by using qRT-PCR and analyzed by the method of 2(-DeltaDeltaCT). The elevation amplitudes of intracellular [Ca(2+)]i evoked by TRPV1 agonist capsaicin in DRG neurons were examined by the calcium fluorescence imaging method under confocal microscopy. RESULTS: Baicalin was shown to down-regulate the mRNA expression levels of TRPV1 at both 37 and 39 degrees C, and under the latter temperature, the intracellular fluorescent intensity evoked by capsaicin was significantly decreased following incubation with baicalin in vitro. We also demonstrated that the actions of baicalin to TRPV1 were not achieved through pathways of TRPA1 or TRPV subfamily members. CONCLUSIONS: Collectively, these results provide compelling evidence that the down-regulated actions of baicalin to TRPV1 in DRG neurons might account for part of the anti-nociceptive mechanism of baicalin.

Sini tang prevents depression-like behavior in rats exposed to chronic unpredictable stress.

Sini Tang, a Chinese traditional prescription containing three herbs, has been widely used for Yang-deficiency. Recent clinical studies have shown that Sini Tang could treat and improve depression symptoms, but the mechanisms underlying the antidepressant effect of Sini Tang remains unknown. In rats with chronic unpredictable stress (CUS), we examined the effects of Sini Tang on sucrose preference and open field exploratory behavior. The levels of corticosterone level in plasma and corticotropin-releasing hormone (CRH) mRNA expression in hypothalamus were also measured by enzyme-linked immunosorbent assays (ELISA) and real-time reverse transcription PCR (RT-PCR), respectively. Rats subjected to CUS exhibited decreases in sucrose preference and ambulation in the open field test. These were all attenuated by Sini Tang in a dose-dependent manner. Biochemically, Sini Tang also reversed CUS-induced increases in corticosterone in plasma and CRH mRNA in the hypothalamus. The behavioral effects of the Sini Tang were correlated to the biochemical actions. These results suggest that Sini Tang produces an antidepressant-like effect, which appears to involve CRH in the brain.

Effects of cinnamaldehyde on PGE2 release and TRPV4 expression in mouse cerebral microvascular endothelial cells induced by interleukin-1beta.

Cinnamaldehyde is a principle compound isolated from Guizhi-Tang (GZT), which is a famous traditional Chinese medical formula used to treat influenza, common cold and other pyretic conditions. Transient receptor potential vanilloid subtype 4 (TRPV4) is expressed in the anterior hypothalamus and may act as thermosensor. The purpose of the present study was to investigate the effects of cinnamaldehyde on the production of prostaglandin E2 (PGE2) and the expression of TRPV4 in mouse cerebral microvascular endothelial cell strain (b.End3). In the research work, the b.End3 cells were cultured in DMEM medium containing interleukin-1beta (IL-1beta) in the presence or absence of ruthenium red (RR), a kind of known TRPV4 inhibitor, or different concentrations of cinnamaldehyde. The results suggested that IL-1beta significantly increase production of PGE2 and cinnamaldehyde evidently decrease IL-1beta-induced PGE2 production, while RR showed no inhibitory effect on PGE2 production. Moreover, it was identified that TRPV4 was expressed at the mRNA and protein levels in b.End3 cells. IL-1beta could up-regulate the expression of TRPV4, RR and cinnamaldehyde could down-regulate the high expression of mRNA and protein of TRPV4 by IL-1beta induced in b.End3 cells. In conclusion, cinnamaldehyde decreased the production of PGE2 and the expression of TRPV4 in b.End3 cells induced by IL-1beta.