Gynura procumbens and selected metabolites: Amelioration of depressive-like behaviors in mice and risperidone-induced hyperprolactinemia in rats.

Gynura procumbens (Lour.) Merr., utilized in traditional Chinese medicine, is known for its liver-protective, liver-soothing, and depression-alleviating properties. This research examines the antidepressant and anti-hyperprolactinemia potentials of an ethanol extract from G. procumbens stems (EEGS) and specific metabolites. To model depression and hyperprolactinemia, chronic unpredictable mild stress (CUMS) was induced in mice and risperidone was administered to rats, respectively. Treatments involved administering low (5 mg/kg), medium (25 mg/kg), and high (125 mg/kg) doses of EEGS and certain metabolites to both models. Behavioral assessments were conducted in the CUMS-induced mice, while the CA3 neuronal damage in mice and histopathological alterations in rat mammary glands were evaluated using Nissl and Hematoxylin & Eosin staining techniques, respectively. EEGS decreased immobility times in the forced swimming and tail suspension tests in mice, enhancing their exploration of the central zone. It elevated the serum levels of 5-hydroxytryptamine, norepinephrine, estradiol, luteinizing hormone (LH), and testosterone in mice. Moreover, EEGS restored the neuronal cell arrangement in the CA3 area, reduced interleukin-1beta mRNA production, and increased the expression of interleukin-10 and beta-catenin mRNA. In the context of risperidone-induced hyperprolactinemia, EEGS lowered blood prolactin levels, reduced the dimensions of rat nipples, and enhanced LH, progesterone, and dopamine levels, alongside mitigating mammary hyperplasia. Among the EEGS selected metabolites, the combined effect of chlorogenic acid and trans-p-coumaric acid was found to be more effective than the action of each compound in isolation. Collectively, the findings indicate that EEGS and its selected metabolites offer promising antidepressant benefits while counteracting hyperprolactinemia.

Promotion Effect of Coexposure to a High-Fat Diet and Nano-Diethylnitrosamine on the Progression of Fatty Liver Malignant Transformation into Liver Cancer.

Overconsumption of high-fat foods increases the risk of fatty liver disease (FLD) and liver cancer with long pathogenic cycles. It is also known that the intake of the chemical poison nitrosamine and its nanopreparations can promote the development of liver injuries, such as FLD, and hepatic fibrosis, and significantly shorten the formation time of the liver cancer cycle. The present work confirmed that the coexposure of a high-fat diet (HFD) and nano-diethylnitrosamine (nano-DEN) altered the tumor microenvironment and studied the effect of this coexposure on the progression of fatty liver malignant transformation into liver cancer. Gene transcriptomics and immunostaining were used to evaluate the tumor promotion effect of the coexposure in mice. After coexposure treatment, tumor nodules were obviously increased, and inflammation levels were elevated. The liver transcriptomics analysis showed that the expression levels of inflammatory, fatty, and fibrosis-related factors in the coexposed group were increased in comparison with the nano-DEN- and high-fat-alone groups. The Kyoto Encyclopedia of Genes and Genomes (KEGG) results showed that coexposure aggravated the high expression of genes related to the carcinomatous pathway and accelerated the formation of the tumor microenvironment. The immunohistochemical staining results showed that the coexposure significantly increased the abnormal changes in proteins related to inflammation, proliferation, aging, and hypoxia in mouse liver tissues. The coexposure of high fat and nano-DEN aggravated the process of steatosis and carcinogenesis. In conclusion, the habitual consumption of pickled foods containing nitrosamines in a daily HFD significantly increases the risk of liver pathology lesions progressing from FLD to liver cancer.

Gradual deterioration of fatty liver disease to liver cancer via inhibition of AMPK signaling pathways involved in energy-dependent disorders, cellular aging, and chronic inflammation.

Introduction: Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer kind. According to recent research, a fatty liver increases the risk of hepatocellular cancer. Nevertheless, the AMPK signaling pathway is crucial. In addition, 5'-AMP-activated protein kinase (AMPK) is strongly linked to alterations in the tumor microenvironment, such as inflammation, hypoxia, and aging. The objective of this study is to evaluate the impact of the AMPK signaling pathway on the progression of fatty liver to HCC. Methods: In this study, we established a mouse liver cancer model using high-fat diets and nano-nitrosamines (nano-DEN). In addition, we employed a transcriptomic technique to identify all mRNAs detected in liver samples at the 25th weekexpression of proteins linked with the LKB1-AMPK-mTOR signaling pathway, inflammation, aging, and hypoxia was studied in microarrays of liver cancer tissues from mice and humans. These proteins included p-AMPK, LKB1, mTOR, COX-2, ß-catenin, HMGB1, p16, and HIF-1α. Results: Data were collected at different times in the liver as well as in cancerous and paracancerous regions and analyzed by a multispectral imaging system. The results showed that most of the genes in the AMPK signaling pathway were downregulated. Prakk1 expression was upregulated compared to control group but downregulated in the cancerous regions compared to the paracancerous regions. Stk11 expression was downregulated in the cancerous regions. Mtor expression was upregulated in the cancerous regions. During liver cancer formation, deletion of LKB1 in the LKB1-AMPK-mTOR signaling pathway reduces phosphorylation of AMPK. It contributed to the upregulation of mTOR, which further led to the upregulation of HIF1α. In addition, the expression of ß-catenin, COX-2, and HMGB1 were upregulated, as well as the expression of p16 was downregulated. Discussion: These findings suggest that changes in the AMPK signaling pathway exacerbate the deterioration of disrupted energy metabolism, chronic inflammation, hypoxia, and cellular aging in the tumor microenvironment, promoting the development of fatty liver into liver cancer.

Enhanced healing of oral chemical burn by inhibiting inflammatory factors with an oral administration of shengFu oil.

ShengFu oil is a compounded Chinese medicinal prescription, and provides antibacterial, anti-inflammatory, and analgesic effects, favoring burn wound repair. In this study, we aimed at investigating the effects of topical applications of ShengFu oil and its active ingredients in oral chemical burns and elucidating its regulatory effects on ß-catenin, COX-2, and MMP-9 expression caused by exposure to acid or alkaline agents. ShengFu oil contains 16 components, such as Frankincense, Radix Scutellariae and Radix Rehmanniae, and the main active ingredients from Frankincense are α-pinene, linalool, and n-octanol. Mouse models of oral chemical burns were induced by using glacial acetic acid or sodium hydroxide. Hematoxylin and eosin staining and immunohistochemical staining were used to detect the protein expressions of ß-catenin, COX-2, and MMP-9 in wound tissues. They were further quantified by multispectral imaging analysis to clarify the effective mechanism of ShengFu oil for intervening inflammatory factors and active components. Our results indicated that the application of ShengFu oil on oral chemical burns effectively stopped the oral burn bleeding and reduced the inflammatory reaction in the damaged tissues, demonstrating that ShengFu oil can promote wound tissue repair in burns caused by heat, acids, and alkalis. The immunohistochemical staining results illustrated that ShengFu oil and its active ingredients significantly reversed the abnormal changes in inflammation-related proteins in mouse tongue tissues that were caused by chemical burns. Regarding long-term toxic effects of ShengFu oil on the gastrointestinal tract, liver, and kidney system, the results of hematoxylin and eosin staining experiments depicted that ShengFu oil was safe and effective for liver, kidney, intestine, esophagus, and tongue. All of these demonstrated that ShengFu oil and its active ingredients are effective and safe in preventing and treating oral chemical burns by interfering with the inflammatory microenvironment.

Synergistic effects of trans-p-coumaric acid isolated from the ethanol extract of Gynura procumbens in promoting intestinal absorption of chlorogenic acid and reversing alcoholic fatty liver disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Our previous studies found that the ethanol extract of Gynura procumbens (EEGS) reduced hepatic steatosis in alcoholic fatty liver disease (AFLD). AIM OF THE STUDY: To explore the active ingredients from EEGS and their relevant mechanism of action in alleviating alcoholic liver injuries. AIM OF THE STUDY: To explore the active ingredients from EEGS and their intestinal absorption characteristics as an approach for understanding mechanism of action in alleviating alcoholic liver injuries. MATERIALS AND METHODS: Monitored by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), chemical constituents from the prepared EEGS were isolated by means of solvent extraction, repeated column chromatography, preparative HPLC and other methods, and their structures were identified based on spectroscopic methods. The in vivo intestinal absorption rate of chlorogenic acid (CA), the active component of the EEGS, both in a single form and in the EEGS were monitored by the single-pass intestinal perfusion (SPIP) method in rats. The protective effect of EEGS and its active components on alcoholic liver injuries was evaluated in the alcoholic liver injury model of C57BL/6J male mice induced by Lieber-DeCarli alcohol liquid feed. RESULTS: Three noncaffeoyl quinic acid components were isolated and identified from the EEGS, namely, 3-trans-p-coumaroyl quinic acid (0.9%), 3-cis-p-coumaroyl quinic acid (2.7%), and trans-p-coumaric acid (0.6%). In vivo intestinal absorption of CA decreased with the increase of pH value of perfusion solution in the range of 5.5-7.8. The maximum absorption percentage of CA alone was 6.7 ± 2.4%, while the maximum absorption percentage of CA in the EEGS was 16.0 ± 2.2%, which was 2.4 times higher than that of CA alone. The results of animal experiments showed that the degree of fatty liver of mice treated with EEGS was significantly lower than that of the CA, trans-p-coumaric acid, and the combination group of CA and trans-p-coumaric acid alone. CONCLUSION: The above results indicated that trans-p-coumaric acid isolated from the dried stems of Gynura procumbens assisted CA being absorbed into the body and worked together with CA to improve the function of liver lipid metabolism, reduce hepatic lipid accumulation in a mouse model of AFLD and effectively counteract alcohol-induced fatty liver disease.

Ethanol supernatant extracts of Gynura procumbens could treat nanodiethylnitrosamine-induced mouse liver cancer by interfering with inflammatory factors for the tumor microenvironment.

ETHNOPHARMACOLOGICAL RELEVANCE: Gynura procumbens (Lour.) Merr, (Family Asteraceae), which serves as both medicine and food in traditional ethnic medicine, has the effects of diminishing inflammation, relieving cough, reducing blood glucose and lipids levels, mitigating hepatotoxicity, and can be used for liver cancer prevention and treatment. AIM OF THE STUDY: To explore how the ethanol extract of Gynura procumbens stems (EEGS) can effectively intervene in the tumor microenvironment, it is necessary to study the mechanism of EEGS on the chemical toxicant nanodiethylnitrosamine (nanoDEN) that induces liver cancer. MATERIALS AND METHODS: EEGS contains large quantities of caffeoylquinic acid (CAC) and non-caffeoylquinic acid (n-CAC), which can be separated by high-performance liquid chromatography. The liver cancer model that was induced by the chemical toxin, nanoDEN, was used to clarify the effective mechanism for tumor intervention of the EEGS and its active ingredients. RESULTS: (1) after interventions with the four drugs on liver cancer, the tumor nodules were obviously reduced and inflammation levels improved. (2) The immunohistochemical staining results showed that both the EEGS and its active ingredients could significantly reverse the abnormal changes in inflammation, proliferation, aging and hypoxia-related proteins in mouse liver tissues that were caused by nanoDEN. (3) Real-time PCR results showed that compared with the nanoDEN group, the expression levels of inflammatory, fatty, and fibrosis-related factors in each group after drug intervention were decreased. (4) The transmission electron microscopy measurements showed that the EEGS significantly reversed the nanostructure changes in hepatocytes that were induced by nanoDEN. CONCLUSION: The EEGS component of Gynura procumbens is effective in preventing and treating liver cancer by interfering with the inflammatory microenvironment during oncogenesis induced by nanoDEN.

Kirenol, darutoside and hesperidin contribute to the anti-inflammatory and analgesic activities of Siegesbeckia pubescens makino by inhibiting COX-2 expression and inflammatory cell infiltration.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese traditional medicine of Siegesbeckia pubescens Makino (SM), which has the effect of healing rheumatism and promoting joint health, is often used to treat rheumatoid arthritis and ischemic stroke. AIM OF THE STUDY: To clarify the mechanisms underlying the anti-inflammatory and analgesic influence of active components in the ethanol extract of Siegesbeckia pubescens Makino (ESM). MATERIALS AND METHODS: The active ingredients in the ESM were identified practicing high-performance liquid chromatography-diode array detection (HPLC-DAD). Four models including xylene-induced ear oedema, complete Freund's adjuvant (CFA)-induced hind paw oedema, acetic acid-induced pain writhing and lipopolysaccharide (LPS)-induced RAW264.7 cell migration, were used to clarify the anti-inflammatory and analgesic mechanisms of the active ingredients in the ESM. RESULTS: (1) Three active ingredients of kirenol, darutoside and hesperidin were identified in the ESM, with relative proportion of 0.6%, 0.2% and 0.01%, respectively; hesperidin was reported for the first time in the ESM. (2) Both the ESM and its active ingredients could effectively alleviate the degree of swelling of the auricle and toes, increase the threshold of heat pain, decrease the overexpression of inflammatory protein cyclooxygenase-2 (COX-2) in the skin tissue of the tested parts of the toes, and reduce the number of writhes induced by acetic acid in mice. (3) ESM and its active ingredients also dose-dependently inhibited the migration of RAW264.7 cells. CONCLUSIONS: ESM and its active ingredients can effectively attenuate the expression of inflammatory factors induced by chemical inflammation, prevent the infiltration of inflammatory cells, and exert good anti-inflammatory and antinociceptive activities.

Nanoparticle-Encapsulated Liushenwan Could Treat Nanodiethylnitrosamine-Induced Liver Cancer in Mice by Interfering With Multiple Critical Factors for the Tumor Microenvironment.

We previously isolated an ethanol fraction of LSW (Liushenwan pill, a traditional Chinese medicine) which has been shown to prevent and treat liver cancer induced by nanodiethylnitrosamine (nanoDEN) in mice. In the present study, we utilized a high-pressure microfluidics technique to generate LSW lipid nanoparticles (nano-LSW) to reduce its toxicity, and enhance its inhibitory effect on tumor growth, and further evaluate its therapeutic effect using a nanoDEN-induced mouse model of liver cancer. Our in vitro results indicated that nano-LSW-low could induce apoptosis in HepG2 cells, but exhibited low toxicity in L02 cells. Furthermore, the in vivo results indicated that nano-LSW-low exerted minimal or no damage to normal hepatocytes, kidney, and small intestine tissues. In addition, our results showed that at the 20th week, the inflammatory infiltration in the mice in the model group increased severely, and partial pimelosis and fibrosis occurred. In contrast, the liver tissues in the mice treated with nano-LSW exhibited only slight inflammatory infiltration, without pimelosis and fibrosis. At the 30th week, 4 out of 5 liver tissues in the model group showed hyperplastic nodules by hematoxylin and eosin (H&E) staining. However, the liver tissues in the nano-LSW treatment group did not showed hyperplastic nodules. Immunohistochemical staining showed that, in contrast to the model group, the levels of COX-2, PCNA, ß-catenin, and HMGB1 protein expressions were significantly lower in the nano-LSW-low group at the 20th and 30th week. Compared to model group, the COX-2, TNF-α, Smad-2, and TGF-ß1 mRNA levels obviously decreased in the liver tissue after the nano-LSW-low treatment. Taken together, nano-LSW-low may serve as a potent therapeutic agent for preventing liver cancer by interfering with multiple critical factors for the tumor microenvironment during oncogenesis.

Effect of electroacupuncture on relieving central post-stroke pain by inhibiting autophagy in the hippocampus.

INTRODUCTION: To explore the underlying mechanism of electroacupuncture (EA) treatment on central post-stroke pain (CPSP), and provide basic evidence for the EA treatment on CPSP. METHODS: Firstly, 40 male SD rats were successfully established with a model of CPSP, under the intervention of different EA frequencies (2 Hz and 15 Hz) and fluoxetine (5 ml/kg and 0.4 mg/ml), whose brain tissue was then removed for paraffin-embedded sectioning; secondly, LPS induced the primary brain cells in the hippocampus to cause inflammation model which were added NS398 (inhibitor of COX-2) and DKK-1 (inhibitor of ß-catenin) later. The lesion sites of brain tissue were observed by Nissl staining and Transmission Electron Microscope (TEM) and autophagy-related proteins (LC3B, p62, LAMP-1), COX-2 and ß-catenin were detected by Western Blot and immunohistochemical staining. Finally, the correlation between LC3B, COX-2, and ß-catenin was calculated by multispectral quantification. RESULTS: (1) In the EA group (15 Hz), the number of Nissl bodies increased, autophagy-related protein LC3B-Ⅱ/Ⅰ, LAMP-1, COX-2, and ß-catenin was lowly expressed, p62 was highly expressed; (2) COX-2, ß-catenin and LC3B are positively correlated with each other (COX-2 & ß-catenin: r = 0.923; COX-2 & LC3B: r = 0.818; ß-catenin & LC3B: r = 0.801); (3) Nissl bodies of primary brain cells of the hippocampus under LPS were like animal experiments; after addition of DKK-1, high expression of ß-catenin and COX-2 induced by LPS was significantly down-regulated, and LC3B-II/I was significantly down-regulated, and p62 protein only had up-regulation trend; after addition of NS398, COX-2 and LC3B-II/I was significantly down-regulated. CONCLUSION: EA may inhibit autophagy in the hippocampus by reducing ß-catenin/COX-2 protein expression and effectively alleviating CPSP. SIGNIFICANCE STATEMENT: Previous studies have found that EA can reduce the expression of NK-1R in damaged rats by inhibition of COX-2 and ß-catenin loops, which controls the activation of glial cells in the damaged area and the apoptosis of neuronal cells, and alleviated pain. In the male SD rat model, we evaluated this effect that EA inhibits autophagy in the hippocampus by reducing ß-catenin/COX-2 protein expression in the brain tissue. In addition, we assessed expression levels of autophagy-related proteins and genes on the inflammatory primary brain cells model. From the experiment, we found EA may inhibit autophagy in the hippocampus by reducing ß-catenin/COX-2 protein expression. These findings provide a foundation for the interpretation of the mechanism of EA on relieving CPSP in clinical practice.

AMPK-ERK/CARM1 Signaling Pathways Affect Autophagy of Hepatic Cells in Samples of Liver Cancer Patients.

Liver cancer is one of the most common malignant tumors, with the death rate ranking fourth among all types of cancer. Over the past few decades, several studies have reported that liver tumorigenesis is associated with dysfunction in autophagy. However, the detailed mechanism remains unclear. In this paper, we used tissue micro-array (TMA) of liver cancer to detect proteins associated with the regulation of autophagic signaling in non-cancerous and cancerous regions by immunohistochemical staining. Those proteins contained 4-HNE, p-AMPK, Erk1/2, p-Erk1/2, CARM1, TFEB, LAMP1, and p62. According to the degrees of tumor differentiation in patients (well differentiated group vs. moderately and poorly differentiated group), we analyzed each protein's expression in the ratio of the "cancerous region/non-cancerous region" in two groups. Current data showed that there were AMPK-ERK/CARM1 autophagic signaling pathways during the formation of liver cancer. The above-mentioned changes in signals indicated an upregulation of autophagy in cancerous regions, which means overactivated autophagy plays an important role in liver cancer.

Solid lipid nanoparticle induced apoptosis of macrophages via a mitochondrial-dependent pathway in vitro and in vivo.

Background and aims: Lipid nanoparticles (LNs) are widely applied in drug delivery systems because they can incorporate and stabilize lipophilic and hydrophilic molecules. LNs are generally considered quite safe and convenient for in vivo applications. However, we previously observed that certain types of LNs could cause a loss of Kupffer cells, a kind of resident macrophage in the liver. As a result, we investigated the details of this phenomenon. Methods: MTT assay, Annexin-V-FITC/PI double staining, JC-1 staining, flow cytometry, Western blot and transmission electron microscopy were used in cell-based experiments. Additionally, serum biochemical analyses, H&E staining and immunofluorescence staining were performed to detect the acute and chronic changes of tissue structure and the number of Kupffer cells in mouse liver tissue samples. Results: Application of LN depolarized and swelled the mitochondria of Raw264.7 cells, and disrupted the balance of Bax/Bcl-2. This led to cleavage and activation of caspase-3 and PARP, and then induced apoptosis of Raw264.7 cells. In addition, either acute or chronic applications of LN were sufficient to disrupt the structure of the hepatic portal vein and reduce the number of Kupffer cells in mice. Conclusions: LNs could induce apoptosis of macrophages through a mitochondrial-dependent pathway.

Anti-inflammatory and antinociceptive effects of active ingredients in the essential oils from Gynura procumbens, a traditional medicine and a new and popular food material.

ETHNOPHARMACOLOGICAL RELEVANCE: Gynura procumbens is applied topically for the treatment of traumatic injuries in South China. MATERIALS AND METHODS: This study was conducted to identify the active ingredients in the essential oils from Gynura procumbens (GPEO) by Gas Chromatography-Mass Spectrometer (GC-MS) and to elucidate the mechanisms underlying the anti-inflammatory and antinociceptive effects in vivo and in vitro. RESULTS: A reduction in dswelling and pain were observed in mice treated with GPEO or its active ingredients (α-pinene, 3-carene, and limonene) compared with mice treated with a solvent control. GPEO or its three active ingredients had potent pharmacological effects on COX-2 overexpression and LPS-induced migration of Raw264.7 macrophages. All three ingredients inhibited nociceptive stimulus-induced inflammatory infiltrates and COX-2 overexpression, which could be responsible for the anti-inflammatory effect of GPEO. However, only 3-carene produced an antinociceptive effect. CONCLUSION: Consistent with the traditional usage in Southern China, GPEO may serve as a promising potent external therapeutic agent for the treatment of chronic pain.

The Ethanol Supernatant Extracts of Liushenwan Could Alleviate Nanodiethylnitrosamine-Induced Liver Cancer in Mice.

Liver cancer is one of the leading causes of cancerous deaths worldwide. At present, the treatment of hepatocellular carcinoma (HCC) remains to be a problem globally. Liushenwan (LSW), an ancient Chinese medicine previously used to treat localized infections, was recently reported to possess anticancer activity. Here in this study, we aim to examine the effect of LSW-ET (LSW-ET is the supernatant fraction of LSW from ultrasound assisted ethanol extraction) in prevention and treatment on nanodiethylnitrosamine- (nanoDEN-) induced HCC in mice. In nanoDEN-induced HCC mice treated with LSW-ET by oral (po) or intragastric gavage (ig), we observed an alleviation of serum ALT and AST levels, amelioration in histopathological stainings, and an inhibition in liver tumor growth. In addition, compared with the nanoDEN group, downregulation of multiple pivotal factors (COX-2, ß-catenin, PCNA, and HMGB-1) was observed in LSW-ET-po and LSW-ET-ig groups. Taken together, the delivery of LSW-ET by oral could be a potential prevention and treatment of liver cancer.

Strategic Combination of Isocratic and Gradient Elution for Simultaneous Separation of Polar Compounds in Traditional Chinese Medicines by HPLC.

A simple high-performance liquid chromatography (HPLC) method for the simultaneous separation of the highly polar and weakly polar components of traditional Chinese medicines was developed via a strategic combination of isocratic and gradient elution methods. Liu-Shen-Wan and Liu-Wei-Di-Huang-Wan were used as representative examples of traditional Chinese medicines. This is the first time that 6 components of varying degrees of polarity in Liu-Shen-Wan had been successfully resolved in a single chromatographic run using an ultraviolet-visible detector with a fixed wavelength of 296 nm. In contrast to conventional gradient separation methods, this novel method offered a viable route for separation of the highly and weakly polar fractions simultaneously, thus greatly reducing the time and cost of analysis. This method therefore provides a more efficient way to determine the polar components present in traditional Chinese medicines. It would find potential application in drug screening, drug authentication, and product quality control.

Lipopolysaccharide Stimulated the Migration of NIH3T3 Cells Through a Positive Feedback Between ß-Catenin and COX-2.

How ß-catenin/COX-2 contribute to inflammation-induced fibroblasts migration remains poorly understood. Therefore, in this study, lipopolysaccharide (LPS) was used as a stimulus to accelerate the migration of NIH3T3 cells, which mimicked the tissue repair process. LPS treatment increased the cell migration in concentration-and time-dependent manner. And NS398, a COX-2 inhibitor, inhibited LPS-induced NIH3T3 cells migration. DKK-1, an antagonist of the Wnt/ß-catenin signaling, also inhibited that migration. However, TWS119, an inducer of ß-catenin via GSK-3ß, increased the cell migration. LPS or TWS119 treatment increased COX-2, ß-catenin, TGF-ß1, and HMGB-1 expressions, and that could be attenuated by NS398 or DKK-1 addition. LPS induced the PGE2 production, and PGE2 increased the expression and nuclear translocation of ß-catenin, while EP2 blocker, AH6809, alleviated those effects. TWS119 increased the luciferase activity in the COX-2 promoter. In conclusion, LPS stimulated the NIH3T3 fibroblasts migration through a positive feedback between ß-catenin and COX-2, in which PGE2, EP2, TGF-ß1, and HMGB-1 played as signal molecules.

Shengfu Oil Enhances the Healing of Full-Thickness Scalded Skin Accompanying the Differential Regulation of ß-Catenin, Dlk1, and COX-2.

Shengfu oil is a traditional Chinese medicine formula containing 16 ingredients, including Scutellariae radix, Olibanum, and Rehmanniae radix. In this study, we aimed to enhance the wound healing of rabbit full-thickness scalded skin by Shengfu oil and to elucidate its regulatory effects on ß-catenin, Dlk1, and COX-2. We found that Shengfu oil exhibited significant anti-inflammatory, analgesic, and antimicrobial activities. The structure of wound tissues in Shengfu oil group was intact, including regenerated cutaneous appendages, indicating better healing capability of Shengfu oil compared to the controls. The protein expression of ß-catenin, Dlk1, and COX-2 in wound tissues were investigated by immunohistochemistry staining and were further quantitated with the use of multispectral imaging analysis. The protein expression of ß-catenin and Dlk1 in the Shengfu oil group was higher than that in the sesame oil group in early wound repair, accompanied by the lower expression of COX-2; the protein expression of ß-catenin decreased in the middle of wound healing; the protein expression of ß-catenin and Dlk1 increased at the end of wound healing. These results strongly suggest that Shengfu oil can enhance wound healing by regulating the expression of ß-catenin, Dlk1, and COX-2 due to its excellent anti-inflammatory, analgesic, and antimicrobial activities.

The Involvement of ß-Catenin/COX-2/VEGF Axis in NMDA-Caused Retinopathy.

NMDA, a molecule that is capable of producing the loss of retinal ganglia cells (RGCs), has been widely studied; however, the detailed mechanism is not yet clarified. Previously, Wnt/ß-catenin signaling has been suggested to be involved in the NMDA-induced retinopathy. In addition, previous investigations in our group demonstrated the presence of a Wnt/ß-catenin/COX-2 axis in dorsal root ganglions (DRGs). Therefore, here in this paper, we tested whether there is an association of such axis with NMDA-induced RGC loss. Rat retinal damage models generated by intravitreal injection of NMDA were used to measure the expression levels of ß-catenin, COX-2, and VEGF in retinas, and the neuron numbers of the retinal GCL of rats were counted. Then, pharmacological tools (MK801, a NMDA receptor inhibitor; Dickkopf homolog 1, a specific inhibitor of the Wnt pathway; NS-398, a COX-2 inhibitor; and bevacizumab, IVB, a VEGF inhibitor) were introduced to evaluate the detailed roles of Wnt/ß-catenin, COX-2, and VEGF in retinopathy of rats. Results demonstrated that all three factors in sequence are positively regulated neuronal loss induced by NMDA. These observations indicated that the Wnt pathway/COX-2/VEGF axis plays a pathogenic role in retinopathy and represented novel therapeutic targets.

Pharmacokinetic Analysis of Four Bioactive Iridoid and Secoiridoid Glycoside Components of Radix Gentianae Macrophyllae and Their Synergistic Excretion by HPLC-DAD Combined with Second-Order Calibration.

An HPLC-DAD method combined with second-order calibration based on the alternating trilinear decomposition (ATLD) algorithm with the aid of region selection was developed to simultaneously and quantitatively characterize the synergistic relationships and cumulative excretion of the four bioactive ingredients of Radix Gentianae Macrophyllae in vivo. Although the analytes spectra substantially overlapped with that of the biological matrix, the overlapping profiles between analytes and co-eluting interferences can be successfully separated and accurately quantified by the ATLD method on the basis of the strength of region selection. The proposed approach not only determined the content change but also revealed the synergistic relationships and the cumulative excretion in vivo of the four ingredients in urine and feces samples collected at different excretion time intervals. In addition, several statistical parameters were employed to evaluate the accuracy and precision of the method. Quantitative results were confirmed by HPLC-mass spectrometry. Satisfactory results indicated that the proposed approach can be utilized to investigate the pharmacokinetics of Radix Gentianae Macrophyllae excretion in vivo.

The dark side of "the force" - lipid nanoparticles enhance the oncogenesis of diethylnitrosamine and result in liver cancer in mice.

Nano-carriers, especially lipid nanoparticles have been used widely in "a good manner", for instance in the treatment of cancer, by enhancing the targetability and reducing required dose. Here in the contrary, we presented a new possibility: nanoDEN, a nanoparticle-packed "bad guy", which is more effective and efficient in generating liver tumor in mice. We have shown that nanoDEN, same as diethylnitrosamine (DEN), induced overexpression of multiple pivotal factors (including COX-2, ß-catenin and PCNA) during oncogenesis. Moreover, nanoDEN increased the apoptosis of liver cells compared with DEN alone. This apoptotic effect of nanoDEN is more efficient on normal cells than on cancer cells. Taken into consideration the fact that there are endogenous nanoparticles naturally formed inside our body, our research enlarged our views of all the aspects of oncogenic chemicals, while also established a better method of producing animal model of liver cancer, which has future investigational and therapeutical potential.

Chronic high-dosage fish oil exacerbates gut-liver axis injury in alcoholic steatohepatitis in mice: the roles of endotoxin and IL-4 in Kupffer cell polarization imbalance.

In the present study, intestinal tight junctions (TJs) and Kupffer cell polarization were investigated in an alcoholic steatohepatitis (ASH) mouse model to uncover the potential side effects of overexposure to fish oil or omega-3 fatty acids. The mice were fed ad libitum with a liquid diet containing ethanol and fish oil. In the meantime, ethanol was given every 5-7 days by gavage to simulate binge drinking. After the 7th binge, steatosis, necrosis, inflammatory infiltration, and bridging fibrosis were observed in the liver by histological staining. After the 13th binge, the inducers, markers and other downstream genes/proteins of the Kupffer cell M1/M2 phenotype in the liver, serum, and small intestine were analysed. The results suggested that a chronic high dosage of fish oil alone reduced the mRNA levels of most genes tested and showed a tendency to damage the intestinal zonula occludens-1 localization and reduce the number of M2 Kupffer cells. Meanwhile, the combination of fish oil and ethanol damaged the intestinal TJs, resulting in an increased endotoxin level in the liver. Gut-derived endotoxin polarized Kupffer cells to the M1 phenotype, whereas the number of cells with the M2 phenotype (markers: CD163 and CD206) was decreased. Interleukin-4 (IL-4), an M2 Kupffer cell inducer, was also decreased. Moreover, in vitro experiments showed that IL-4 reversed eicosapentaenoic acid-induced CD163 and CD206 mRNA suppression in RAW 264.7 cells. Overall, our results showed that a chronic high dosage of fish oil exacerbated gut-liver axis injury in alcoholic liver disease in mice, and endotoxin/IL-4-induced Kupffer cell polarization imbalance might play an important role in that process.