New horizons for the role of RNA N6-methyladenosine modification in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignancy, presenting a formidable challenge to the medical community owing to its intricate pathogenic mechanisms. Although current prevention, surveillance, early detection, diagnosis, and treatment have achieved some success in preventing HCC and controlling overall disease mortality, the imperative to explore novel treatment modalities for HCC remains increasingly urgent. Epigenetic modification has emerged as pivotal factors in the etiology of cancer. Among these, RNA N6-methyladenosine (m6A) modification stands out as one of the most prevalent, abundant, and evolutionarily conserved post-transcriptional alterations in eukaryotes. The literature underscores that the dynamic and reversible nature of m6A modifications orchestrates the intricate regulation of gene expression, thereby exerting a profound influence on cell destinies. Increasing evidence has substantiated conspicuous fluctuations in m6A modification levels throughout the progression of HCC. The deliberate modulation of m6A modification levels through molecular biology and pharmacological interventions has been demonstrated to exert a discernible impact on the pathogenesis of HCC. In this review, we elucidate the multifaceted biological functions of m6A modifications in HCC, and concurrently advancing novel therapeutic strategies for the management of this malignancy.

A new species of the genus Synanthedon Hbner [1819] (Lepidoptera Sesiidae) from Taiwan.

A new species of clearwing moth, Synanthedon suhua sp. nov., is described from Taiwan in this article. Adults and genitalia of both sexes are illustrated, DNA barcodes provided, and potential damage to Quercus longinux (Fagaceae) discussed.

Endoplasmic reticulum stress and protein degradation in chronic liver disease.

Endoplasmic reticulum (ER) stress is easily observed in chronic liver disease, which often causes accumulation of unfolded or misfolded proteins in the ER, leading to unfolded protein response (UPR). Regulating protein degradation is an integral part of UPR to relieve ER stress. The major protein degradation system includes the ubiquitin-proteasome system (UPS) and autophagy. All three arms of UPR triggered in response to ER stress can regulate UPS and autophagy. Accumulated misfolded proteins could activate these arms, and then generate various transcription factors to regulate the expression of UPS-related and autophagy-related genes. The protein degradation process regulated by UPR has great significance in many chronic liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), viral hepatitis, liver fibrosis, and hepatocellular carcinoma(HCC). In most instances, the degradation of excessive proteins protects cells with ER stress survival from apoptosis. According to the specific functions of protein degradation in chronic liver disease, choosing to promote or inhibit this process is promising as a potential method for treating chronic liver disease.

LncRNA-H19 induces hepatic stellate cell activation via upregulating alcohol dehydrogenase III-mediated retinoic acid signals.

Activation of hepatic stellate cells (HSCs) is a pivotal event in liver fibrosis, characterized by enhanced retinoic acid signals. Although up-regulated retinoic acid signal responds further to maintain HSC activation, the underlying molecular mechanisms are largely unknown. In this study, we sought to investigate the role of lncRNA-H19 in regulation of retinoic acid signals, and to further examine the underlying mechanism in this molecular context. We found that lncRNA-H19 upregulation could enhance retinoic acid signals to induce HSC activation, whereas lncRNA-H19 knockdown completely disturbed retinoic acid signals. Moreover, the activation of retinoic acid signals impaired the lncRNA-H19 knockdown mediated HSC inactivation. Interestingly, we also found that enhanced retinoic acid signals by lncRNA-H19 was associated with a coordinate increase in retinol metabolism during HSC activation. Increased retinol metabolism contributed to obvious lipid droplet consumption. Importantly, we identified that alcohol dehydrogenase III (ADH3) was essential for lncRNA-H19 to enhance retinoic acid signals. The inhibition of ADH3 completely abrogated the lncRNA-H19 mediated retinoic acid signals and HSC activation. Of note, we identified dihydroartemisinin (DHA) as a natural inhibitor for lncRNA-H19. Treatment with DHA significantly decreased the expression of lncRNA-H19, reduced the expression of ADH3, blocked retinoic acid signals, and in turn, inhibited HSC activation. Overall, these results provided novel implications to reveal the molecular mechanism of increased retinoic acid signals during HSC activation, and identify lncRNA-H19/ADH3 pathway as a potential target for the treatment of liver fibrosis.

Development of a Novel Spawn (Block Spawn) of an Edible Mushroom, Pleurotus ostreatus, in Liquid Culture and its Cultivation Evaluation.

Mushroom cultivation has gained increased attention in recent years. Currently, only four types of spawn, including sawdust spawn, grain spawn, liquid spawn, and stick spawn, are commonly available for mushroom cultivation. This limited spawn diversity has led to difficulty in selecting suitable inoculum materials in some cultivation. In this study, three small blocks of lignocellulosic agro-wastes and one block of a synthetic matrix were prepared as support for growing Pleurotus ostreatus in liquid medium. Mycelium-adsorbed blocks were then evaluated for their potential as block spawn for fructification. Our results indicated that the edible fungus was adsorbed and abundantly grew internally and externally on loofah sponge and synthetic polyurethane foam (PUF) supports and also has the ability to attach and grow on the surface of sugarcane bagasse and corncob supports. The mycelia of P. ostreatus adhered on corncob exhibited the highest metabolic activity, while those on the PUF showed the least activity. Mycelial extension rates of block spawns made of agro-waste materials were comparable to that of sawdust spawn, but the block spawn of PUF showed a significantly lower rate. No significant differences in cropping time and yield were observed among cultivations between experimental block spawns and sawdust spawns. Moreover, the corncob block spawn maintained its fruiting potential during an examined period of 6-month storage. The developed block spawn could be practically applied in mushroom cultivation.

Diallyl trisulfide attenuates ethanol-induced hepatic steatosis by inhibiting oxidative stress and apoptosis.

Inhibiting the major characteristics of alcoholic fatty liver (AFL) such as lipid accumulation, oxidative stress and apoptosis is a promising strategy of treating AFL. Diallyl trisulfide (DATS) is the major constituent isolated from garlic, which shows promise in the treatment of chronic liver disease. However, the effects of DATS on ethanol-induced liver injury and the related mechanisms remain unclear. The aim of this study was to evaluate the potential protective effects of DATS on AFL and the potential mechanisms. A single intragastric dose of ethanol was given to rats in vivo, while ethanol-stimulated LO2 cells were used as an in vitro model. Our results demonstrated that DATS prevented ethanol-induced injury, as indicated by the reduced activities of aspartate transaminase (AST) and alanine aminotransferase (ALT) in the serum and culture medium, and inhibition of cell apoptosis. Furthermore, DATS reduced hepatic steatosis by up-regulating the expression of peroxisome proliferator-activated receptor-alpha (PPAR-α) and down-regulating the expression of sterolregulatory element binding protein 1c(SREBP-1c). In addition, DATS alleviated ethanol-induced oxidative stress by enhancing non-enzymatic antioxidant and enzymatic antioxidants contents and by reducing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA). These data collectively revealed that DATS protected ethanol-induced liver injury by inhibiting lipid accumulation and oxidative stress.

Diallyl trisulfide protects against ethanol-induced oxidative stress and apoptosis via a hydrogen sulfide-mediated mechanism.

Garlic is one natural source of organic sulfur containing compounds and has shown promise in the treatment of chronic liver disease. Dietary garlic consumption is inversely correlated with the progression of alcoholic fatty liver (AFL), although the exact underlying mechanisms are not clear. Our previous studies also have shown that diallyl trisulfide (DATS), the primary organosulfur compound from Allium sativum L, displayed anti-lipid deposition and antioxidant properties in AFL. The aim of the present study was to clarify the underlying mechanisms. In the present study, we used the intragastric infusion model of alcohol administration and human normal liver cell line LO2 cultured with suitable ethanol to mimic the pathological condition of AFL. We showed that accumulation of intracellular reactive oxygen species (ROS) was lowered significantly by the administration of DATS, but antioxidant capacity was increased by DATS. Additionally, DATS inhibited hepatocyte apoptosis via down-regulating Bax expression and up-regulating Bcl-2 expression, and attenuated alcohol-induced caspase-dependent apoptosis. More importantly, using iodoacetamide (IAM) to block hydrogen sulfide (H2S) production from DATS, we noted that IAM abolished all the above effects of DATS in ethanol-treated LO2 cells. Lastly, we found DATS could increase the expressions of cystathionine gamma-lyase (CSE) and cystathionine beta-synthase (CBS), the major H2S-producing enzymes. These results demonstrate that DATS protect against alcohol-induced fatty liver via a H2S-mediated mechanism. Therefore, targeting H2S may play a therapeutic role for AFL.

[Effect of emodin in attenuating endoplasmic reticulum stress of pancreatic acinar AR42J cells].

OBJECTIVE: To explore the effect of emodin on endoplasmic reticulum (ER) stress of pancreatic acinar AR42J cells. METHOD: Rat pancreatic acinar AR42J cells were cultured in 6-well plates, and divided into the normal control group, the model group (with the final concentration at 1 x 10(-7) mol · L(-1) for cerulean and lipopolysaccharide at 10 mg · L(-1)) and the emodin group (10, 20, 40 µmol · L(-1)). Cells in each group were cultured in three multiple pores for 24 h, and their supernate was removed after cell attachment. The normal control group was added with haploids, the model group was added with the modeling liquid for haploids, and the treatment groups were added with different concentrations of emodin at 15-20 min before the modeling liquid. The cells were continuously cultured for 3 h under 37 °C and 5% CO2. Their intracellular protease and lipase expressions were detected with kits. The cellular morphology was observed under optical microscope. The level of calcium in endoplasmic reticulum was measured under laser confocal microscopy. Western blot assay were used to determine the protein expression of ER-related signaling molecules. RESULT: Emodin could significantly inhibit levels of amylase, lipase and intracellular calcium and ER. CONCLUSION: Emodin could reduce pancreatic acinar cell injury induced by the combination of cerulean and lipopolysaccharide. Its action mechanism is correlated with the inhibition of intracellular calcium overload and ER stress.

Effect of aqueous extracts of several kinds of herbs on human platelet aggregation and expression of P-selectin in vitro.

OBJECTIVE: To study the effect of aqueous extract of several kinds of herbs on human platelet aggregation and expression of P-selectin in vitro. METHODS: Blood was collected from volunteers. Effects of the prepared water extracts of herbs on platelet aggregation were monitored on a Packs-4 aggregometer. The fluorescence intensity of water extracts of Caulis Spatholobi, Flos Carthami and Rhizoma Curcumae on the expression of P-selectin in human platelets of healthy persons was measured with flow cytometry. RESULTS: Out of several herbs investigated, Flos Carthami and Rhizoma Curcumae potently inhibited platelet aggregation after incubation with platelet-rich plasma (PRP) for 15 min. Caulis Spatholobi Flos Carthami and Rhizoma Curcumae inhibited adenosine-5'-diphosphate (ADP) or platelet activating factor (PAF)-induced platelet aggregation in PRP in a dose-dependent manner. In contrast to Flos Carthami and Rhizoma Curcumae, Caulis Spatholobi could not inhibit thrombin-induced platelet aggregation. Despite its inability to inhibit thrombin-induced platelet aggregation in PRP, Caulis Spatholobi had a greater anti-aggregating activity in PRP induced by ADP or PAF. Caulis Spatholobi and Flos Carthami showed significant inhibitory effects on the expression of P-selectin. CONCLUSIONS: Caulis Spatholobi, Flos Carthami and Rhizoma Curcumae have potent anti-platelet properties, and their inhibitory actions are mediated via different mechanisms. Caulis Spatholobi inhibited ADP-induced platelet aggregation but not by thrombin, indicating that its mechanism of action might be independent of the thromboxane pathway. The effect of Caulis Spatholobi and Flos Carthami were associated with suppressing the expression of P-selectin.

PKM2 regulates hepatocellular carcinoma cell epithelial-mesenchymal transition and migration upon EGFR activation.

Pyruvate kinase isozyme type M2(PKM2) was first found in hepatocellular carcinoma(HCC), and its expression has been thought to correlate with prognosis. A large number of studies have demonstrated that epithelial-mesenchymal transition (EMT) is a crucial event in hepatocellular carcinoma (HCC) and associated metastasis, resulting in enhanced malignancy of HCC. However, the roles of PKM2 in HCC EMT and metastasis remain largely unknown. The present study aimed to determine the effects of PKM2 in EGF-induced HCC EMT and elucidate the molecular mechanisms in vitro. Our results showed that EGF promoted EMT in HCC cell lines as evidenced by altered morphology, expression of EMT-associated markers, and enhanced invasion capacity. Furthermore, the present study also revealed that nuclear translocation of PKM2, which is regulated by ERK pathway, regulated ß-catenin-TCF/LEF-1 transcriptional activity and associated EMT in HCC cell lines. These discoveries provide evidence of novel roles of PKM2 in the progression of HCC and potential therapeutic target for advanced cases.

Da-Huang-Fu-Zi-Tang attenuates liver injury in rats with severe acute pancreatitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Da-Huang-Fu-Zi-Tang (DHFZT) is a famous traditional Chinese prescription with strong anti-inflammatory effects. Our previous work found that DHFZT could act against pancreatic injury in rats with severe acute pancreatitis (SAP) via inhibiting the Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) signaling pathway in pancreatic tissues. AIM OF THE STUDY: To investigate the therapeutic effects of DHFZT on liver injury in SAP rats, and the effects on JAK2/STAT3 signaling in liver tissue and Kupffer cells (KCs). MATERIALS AND METHODS: Fifty SD male rats were randomly divided into five groups: sham operation group (SO), SAP model group, DHFZT treatment groups (12, 24, and 48 mg/kg body weight). The model of SAP was constructed by injecting sodium taurocholate (3.5%) into pancreatic and biliary ducts. One hour before constructing the model, DHFZT was perfused into the stomach. All rats were sacrificed after 24h following the operation; livers were examined with hematoxylin and eosin staining. The protein expression of pJAK2 and pSTAT3 in liver tissue was detected by immunohistochemical staining. The activity of ALT, IL-6 and TNF-α in serum was detected. KCs of each group were isolated. After culture for 4h, the protein expression of JAK2, pJAK2, STAT3 and pSTAT3, the mRNA expression of IL-6 and TNF-α in KCs were examined. RESULTS: Sodium taurocholate induced liver injury concomitant with increased expression of pJAK2 and pSTAT3 in liver tissue and KCs. Pretreatment with DHFZT significantly attenuated liver injury induced by SAP, and concurrently, effectively lowered the serum ALT level. Furthermore, our studies showed that DHFZT obviously decreased the expression of pJAK2 and pSTAT3 in liver tissue and KCs. CONCLUSIONS: DHFZT could ameliorate liver injury in rats with SAP.

[Prevention and treatment of age-related macular degeneration by extract of Fructus lycii and its constituents lutein/zeaxanthin: an in vive and in vitro experimental research].

OBJECTIVE: To investigate the in vivo inhibition of extract of Fructus lycii (FL) on the expressions of cathepsin B (Cat B) and cystatin C (Cys C) in high-fat diet and hydroquinone (HQ) induced model mice with age-related macular degeneration (AMD), and to explore the in vitro effects of lutein and zeaxanthin on hydrogen peroxide (H2O2,) induced expressions of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) on ARPE-19 cells. METHODS: Fifty female 8-month-old C57BL/6 mice were recruited in this research. Ten mice fed with regular diet was taken as the age control group. The rest 40 mice were fed with high fat diet for 6 months, followed by adding HQ (0. 8%) in the drinking water for 3 consecutive months. Then the modeled mice were randomly divided into the model control group (n =10), the high (at the daily dose of 3.75 g/kg), middle (at the daily dose of 2.50 g/kg), and low dose (at the daily dose of 1.25 g/kg) FL groups, 10 in each group. The extract of FL at each dose was respectively administered to mice by gastrogavage for 3 successive months. By the end of the experiment, the mice were killed and their eyeballs were removed. The protein expressions of Cat B and Cys C were observed by immunohistochemical assay. The mRNA and protein expressions of Cat B and Cys C were detected by real-time PCR and Western blot respectively. The drug concentrations of H2O2, lutein, and zeaxanthin were screened and detected using the activity of cell proliferation. The protein expressions of MMP-2 and TIMP-2 were detected using Western blot. RESULTS: Compared with the age control group, the mRNA and protein expressions of Cat B and Cys C were significantly higher in the in vivo model control group (P <0.05, P <0.01). The mRNA expressions of Cat B and Cys C were weaker in the middle and high dose FL groups than in the model control group (P <0. 05, P <0. 01). In in vitro cells, lutein and zeaxanthin could down-regulate the protein expressions of MMP-2 and TIMP-2 in H202 induced ARPE-19 cells (P <0. 05, P <0. 01). CONCLUSIONS: Extract of FL could down-regulate the high protein expressions of Cat B and Cys C in high-fat diet and HQ induced model mice. Lutein and zeaxanthin could down-regulate the protein expressions of MMP-2 and TIMP-2 in H202 induced ARPE-19 cells.

Tetramethylpyrazine induces G0/G1 cell cycle arrest and stimulates mitochondrial-mediated and caspase-dependent apoptosis through modulating ERK/p53 signaling in hepatic stellate cells in vitro.

Activation of hepatic stellate cells (HSCs) is a pivotal event in the pathogenesis of liver fibrosis. Pharmacological induction of HSC apoptosis could be a promising strategy for fibrosis regression. Natural product tetramethylpyrazine (TMP) exhibits potent antifibrotic activities in vivo. However, the molecular mechanisms remain to be defined. The present study aimed at investigating the anti-proliferative and pro-apoptotic effects of TMP on HSCs and elucidating the underlying mechanisms. Our results demonstrated that TMP had no apparent cytotoxic effects on hepatocytes, but significantly inhibited HSC proliferation and induced cell cycle arrest at the G0/G1 checkpoint. These effects were associated with TMP regulation of cyclin D1, p21, p27 and p53. Furthermore, we found that TMP disrupted mitochondrial functions and led to activation of caspase cascades in HSCs. Mechanistic investigations revealed that TMP selectively blocked the extracellular signal-regulated kinase (ERK) signaling and activated p53, which was required for TMP induction of caspase-dependent mitochondrial apoptosis in HSCs. Autodock simulations predicted that TMP could directly bind to ERK2 with two hydrogen bonds and low energy score, indicating that ERK2 could be a direct target molecule for TMP within HSCs. Moreover, TMP altered expression of some marker proteins relevant to HSC activation. These data collectively revealed that TMP modulation of ERK/p53 signaling led to mitochondrial-mediated and caspase-dependent apoptosis in HSCs in vitro. These studies provided mechanistic insights into the antifibrotic properties of TMP that may be exploited as a potential option for hepatic fibrosis.

Luteolin reduces the invasive potential of malignant melanoma cells by targeting ß3 integrin and the epithelial-mesenchymal transition.

AIM: To investigate whether luteolin, a highly prevalent flavonoid, reverses the effects of epithelial-mesenchymal transition (EMT) in vitro and in vivo and to determine the mechanisms underlying this reversal. METHODS: Murine malignant melanoma B16F10 cells were exposed to 1% O(2) for 24 h. Cellular mobility and adhesion were assessed using Boyden chamber transwell assay and cell adhesion assay, respectively. EMT-related proteins, such as E-cadherin and N-cadherin, were examined using Western blotting. Female C57BL/6 mice (6 to 8 weeks old) were injected with B16F10 cells (1×10(6) cells in 0.2 mL per mouse) via the lateral tail vein. The mice were treated with luteolin (10 or 20 mg/kg, ip) daily for 23 d. On the 23rd day after tumor injection, the mice were sacrificed, and the lungs were collected, and metastatic foci in the lung surfaces were photographed. Tissue sections were analyzed with immunohistochemistry and HE staining. RESULTS: Hypoxia changed the morphology of B16F10 cells in vitro from the cobblestone-like to mesenchymal-like strips, which was accompanied by increased cellular adhesion and invasion. Luteolin (5-50 µmol/L) suppressed the hypoxia-induced changes in the cells in a dose-dependent manner. Hypoxia significantly decreased the expression of E-cadherin while increased the expression of N-cadherin in the cells (indicating the occurrence of EMT-like transformation), which was reversed by luteolin (5 µmol/L). In B16F10 cells, luteolin up-regulated E-cadherin at least partly via inhibiting the ß3 integrin/FAK signal pathway. In experimental metastasis model mice, treatment with luteolin (10 or 20 mg/kg) reduced metastatic colonization in the lungs by 50%. Furthermore, the treatment increased the expression of E-cadherin while reduced the expression of vimentin and ß3 integrin in the tumor tissues. CONCLUSION: Luteolin inhibits the hypoxia-induced EMT in malignant melanoma cells both in vitro and in vivo via the regulation of ß3 integrin, suggesting that luteolin may be applied as a potential anticancer chemopreventative and chemotherapeutic agent.

Acupuncture combined with curcumin disrupts platelet-derived growth factor ß receptor/extracellular signal-regulated kinase signalling and stimulates extracellular matrix degradation in carbon tetrachloride-induced hepatic fibrosis in rats.

BACKGROUND: Acupuncture treatment has been increasingly used to treat chronic liver diseases. We previously reported that acupuncture combined with curcumin, a natural antifibrotic compound, could remarkably attenuate liver fibrosis in chemically intoxicated rats, but the underlying molecular mechanisms are poorly understood. The present study was aimed at investigating the effects of acupuncture combined with curcumin on platelet-derived growth factor (PDGF) signalling and extracellular matrix (ECM) regulation in the fibrotic liver. METHODS: A total of 60 Sprague-Dawley male rats were randomly divided into control, model, sham, acupuncture, curcumin and combination treatment groups. During the establishment of fibrosis using carbon tetrachloride (CCl(4)), acupuncture at LR3, LR14, BL18 and ST36 and/or curcumin treatment by mouth were performed simultaneously. After treatment, serum PDGF levels were measured. Protein and mRNA expression of key effectors in PDGF pathway and fibrinolysis in the liver was determined. RESULTS: Acupuncture combined with curcumin potently reduced serum PDGF levels and selectively disrupted the PDGF-ßR/extracellular signal-regulated kinase (ERK) cascade. Combination treatment also significantly repressed expression of connective tissue growth factor and upregulated expression of matrix metalloproteinase-9, promoting fibrinolysis in the fibrotic liver. CONCLUSIONS: The beneficial effects of acupuncture and its combination with curcumin could be attributed to the disruption of PDGF-ßR/ERK pathway and stimulated ECM degradation in the fibrotic liver. Acupuncture treatment significantly enhanced curcumin effects at the molecular level. These findings may provide molecular insights into the potential of acupuncture combined with curcumin for prevention of hepatic fibrosis.

[Effects of acupuncture intervention on hepatic platelet-derived growth factor signaling pathway in CCl4-induced hepatic fibrosis rats].

OBJECTIVE: To observe the effect of acupuncture stimulation of "Taichong" (LR 3), "Qimen" (LR 14), etc. on hepatic platelet-derived growth factor (PDGF) signal pathway activity at the protein and mRNA levels in hepatic fibrosis rats. METHODS: Forty-six SD rats were randomly divided into control (10 rats), model (12 rats), acupuncture (12 rats) and non-acupoint (12 rats) groups. Hepatic fibrosis model was established by intraperitoneal injection of mixture solution of 50% CCl4 and olive oil [1:1, 3 times on the 1st week (W), twice/W thereafter for 5 more weeks]. During modeling, acupuncture stimulation of "Taichong" (LR 3), "Qimen" (LR 14), "Ganshu" (BL 18) and "Zusanli" (ST 36) was conducted simultaneously. At the end of the experiments, all the rats were sacrificed for collecting their liver and blood samples, followed by separation of the hepatic stellate cells (HSCs). ELISA, Western blot and Real-time quantitative PCR techniques were used to detect the content of serum PDGF and expression levels of PDGF-beta receptor (PDGF-beta R), extracellular signal-regulated kinase (ERK1/2), c-jun N-terminal kinase (JNK) and P 38 genes and proteins of HSCs, respectively. RESULTS: Compared to the control group, serum PDGF content, and expression levels of PDGF-beta R mRNA and protein, ERK mRNA and protein and P 38 protein of HSCs in the model group were upregulated significantly (P < 0.01, P < 0.05). In comparison with the model group, serum PDGF content, and the expression levels of PDGF-beta R mRNA and protein, ERK mRNA and protein of HSCs in the acupuncture group were down-regulated apparently (P < 0.05, P < 0.01). No significant differences were found between the acupuncture and non-acupoint groups in serum PDGF content and between the model group and non-acupoint group in the expression levels of PDGF-beta R mRNA and protein, ERK mRNA and protein, JNK protein and P 38 protein of HSCs, as well as between the model group and acupuncture group in the expression levels of JNK protein and P 38 protein of HSCs (P > 0.05). CONCLUSION: Acupuncture intervention can effectively down-regulate serum PDGF content, and expression levels of PDGF-beta R mRNA and protein, ERK mRNA and protein of HSCs in liver fibrosis rats, which may contribute to its effect in improving liver fibrosis through down-regulating PDGF signal pathway activity.

[Inhibitory effect of acupuncture on hepatic extracellular matrix production in carbon tetrachloride-induced liver fibrosis rats].

OBJECTIVE: To investigate the protective effect of acupuncture intervention on liver in carbon tetrachloride induced hepatic fibrosis rats and to reveal its impact on extracellular matrix production in the liver tissue. METHODS: A total of 46 SD rats were randomly divided into control group (n = 10), model group (n = 12), sham group (n = 12) and acupuncture group (n = 12). Hepatic fibrosis model was established by intraperitoneal injection of 50% olive oil containing CCl (1 mL/kg), 3 times in the 1st week and twice per week from the 2nd to the 6th week. During the fibrosis model establishment, acupuncture of "Taichong" (LR 3), "Qimen" (LR 14), "Ganshu" (BL 18) and "Zusanli" (ST 36) was carried out simultaneously. In the sham group, non-acupuncture points (0.5 cm left to the above-mentioned real points) were punctured. The treatment was conducted 3 times a week in the first three weeks and then twice a week for the last three weeks. Serum levels of hyaluronic acid (HA), liminin (LN) and precollagen (PC III) were determined by enzyme linked immunosorbent assay for assessing the hepatic fibrosis degree. Primary hepatic stellate cells (HSC) were separated. Western blot assay was used to detect the expression of alpha-smooth muscle actin (SMA), alpha 1 (l) collagen, fibronectin, matrix metalloproteinase (MMP)-9 (components of extracellular matrix, ECM) and tissue inhibitor of metalloprotei-nase-1 (TIMP-1, an inhibitor of MMP-9) proteins of HSC. Real-time quantitative polymerase chain reaction was used to detect the expression levels of alpha-SMA, alpha 1 (1) collagen and fibronectin genes of HSC. RESULTS: Compared with the control group, contents of serum HA, LN and PC III, expression levels of alpha-SMA, alpha 1 (I) collagen and fibronectin proteins and genes, and TIMP-1 protein of HSC were significantly increased in the model group (P < 0.01, P < 0.05), while MMP-9 protein (an enzyme for degradating ECM) expression level of HSC in the model group was down-regulated significantly (P < 0.01), suggesting a formation of hepatic fibrosis, impairment of the liver tissue fibrosis and imbalance of degradation of ECM. H.E. staining showed an ameliorated liver injury (disorder of hepatocyte arrangement, hepatocyte necrosis, formation of pseudolobule, etc.) in the acupuncture group in comparison with the model group. In comparison with the model group, serum HA and LN contents, expression levels of alpha-SMA, alpha 1(I) collagen and fibronectin proteins, and alpha-SMA mRNA and fibronectin mRNA of HSC were downregulated considerably in the acupuncture group (P < 0.05, P < 0.01). On the contrary, MMP-9 protein expression level of HSC was up-regulated remarkably in the acupuncture group compared with the model group (P < 0.05). No significant changes were found in all the aforementioned indexes in the sham group, and serum PC III content as well as alpha 1 (I) collagen mRNA and TIMP-1 protein expression levels of HSC in the acupuncture group compared with those in the model group (P > 0.05). CONCLUSION: Acupuncture treatment can significantly relieve CCl4-induced hepatic fibrosis in hepatic fibrosis rats probably by inhibiting the synthesis and deposite of HA and LN, down-regulating expression levels of alpha-SMA, alpha 1(1) collagen and fibronectin proteins, and alpha-SMA mRNA and fibronectin mRNA of HSC as well as up-regulating MMP-9 protein expression of HSC.

Acupuncture combined with curcumin attenuates carbon tetrachloride-induced hepatic fibrosis in rats.

BACKGROUND: Increasingly, studies demonstrate the effectiveness of acupuncture therapy against liver fibrosis. Curcumin is a natural product with antifibrotic effects, but has poor pharmacokinetic profiles. This study aimed to evaluate whether acupuncture combined with curcumin could more potently attenuate liver fibrosis in chemical intoxicated rats. METHODS: 60 Sprague-Dawley male rats were randomly divided into control, model, sham, acupuncture, curcumin and combination therapy groups. During the establishment of fibrosis using carbon tetrachloride (CCl(4)), acupuncture at LR3, LR14, BL18 and ST36 and/or curcumin treatment by mouth were performed simultaneously. After treatment, pathological indexes and histology for hepatic injury and fibrogenesis were detected. The expression of extracellular matrix (ECM) components was also determined. RESULTS: Acupuncture combined with curcumin potently protected the liver from CCl(4)-induced injury and fibrogenesis, as indicated by reduced levels of serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, hyaluronic acid, laminin and procollagen III. Combined use also led to significant liver histological improvements. Furthermore, combined use effectively inhibited ECM expression such as α-smooth muscle actin, fibronectin and α1(1) collagen. CONCLUSIONS: Acupuncture treatment could significantly enhance the antifibrotic efficacy of curcumin on CCl(4)-induced hepatic fibrosis in rats in vivo, suggesting that a combination of acupuncture with curcumin may be exploited for the prevention of hepatic fibrosis.

Injectable caltrop fruit saponin protects against ischemia-reperfusion injury in rat brain.

The present study aimed to investigate the protective effects of injectable caltrop fruit saponin preparation (ICFSP) on ischemia-reperfusion injury in rat brain. Rats were injected with ICFSP and then subjected to cerebral ischemia-reperfusion injury induced by middle cerebral artery occlusion. Then the neurological deficit score was evaluated by Bederson's method. The infarct size was assessed by TTC staining. The content of malondialdehyde (MDA) and nitric oxide (NO), and the activity of superoxide dismutase (SOD) in rat cerebrum were measured with kits, and the content of 6 K prostaglandin F1α (6-K-PGF 1α), thromboxane B2 (TXB2) and endothelin (ET) in blood plasma was measured by radioimmunoassay. The results demonstrated that ICFSP led to a decrease in infarct size (p < 0.01), neurological deficit score (p < 0.05) and plasma content of TXB2 and ET (p < 0.05), and an increase of the plasma level of 6-K-PGF 1α (p < 0.05) and SOD activity in cerebrum, where the MDA and NO content were decreased. The treatment improved forelimb function. ICFSP showed a similar potency compared to that of Ligustrazine hydrochloride parenteral solution (LHPS) and nimodipine (Nim). We concluded that ICFSP protects the brain damage caused by ischemia-reperfusion injury in rats, and this may be closely related to the regulation of reactive oxygen species (MDA and SOD activity) and NO levels in the rat cerebrum, as well as vasoactive factors in the plasma (6-K-PGF 1α, TXB2 and ET).

Cryptotanshinone has diverse effects on cell cycle events in melanoma cell lines with different metastatic capacity.

PURPOSE: Cryptotanshinone is a major active component of Salvia miltiorrhiza, which is often used as Chinese herbal medicine in cancer therapy. Here, we systematically assessed the anti-tumor effect of Cryptotanshinone on two melanoma cell lines with low/high-metastatic capacity (B16/B16BL6). METHODS: MTT and LDH assays were used to evaluate cell growth and cytotoxicity. We assessed the effect of Cryptotanshinone on cell apoptosis or proliferation by Annexin V, TUNEL or BrdU assay. Cell cycle distribution was detected by flow cytometry. The integrity of cell cycle checkpoints was determined by mutational analyses of B-RAF and N-RAS, and the expression of cell cycle-associated proteins by western blotting. RESULTS: Treatment with Cryptotanshinone had no obvious effect on cell apoptosis but significantly inhibited cell proliferation. Cryptotanshinone slightly increased the expression of p53, Chk1, and Chk2 in both B16 and B16BL6. Interestingly, Cryptotanshinone induced G1 arrest with a concomitant increase in p21 expression in B16BL6 cells. However, in B16 cells, Cryptotanshinone induced the G2/M arrest through its induction of Cdc25c. Regulation of Cyclin A1, Cyclin B1 and Cdk1/cdc2 expression might contribute to the different cell cycle patterns in B16 and B16BL6 after Cryptotanshinone treatment. CONCLUSIONS: Cryptotanshinone could have diverse effects on cell cycle events in melanoma cell lines with different metastatic capacity. This property might offer an opportunity to study underlying mechanisms for the different antitumor effects of administered Cryptotanshinone in B16 and B16BL6 cells.