Neolignans with anti-inflammatory activity from Piper kadsura (Choisy) Ohwi.

Two novel neolignans, piperkadsurenin A (1) and kadsurenin N (2), along with six known neolignans (3-8) and two lignans (9-10) were isolated from the stems of Piper kadsura (Choisy) Ohwi. Extensive spectroscopic data interpretation and ECD calculations were used to identify the structures of the new compounds 1 and 2. Especially, compound 1 represents the first example of neolignan with cyclopenta[b]pyran framework. The anti-inflammatory efficacy of compounds 1-10 in vitro was systematically assessed through NO production inhibitory assay. Compounds 3 and 7 significantly inhibited LPS-induced NO generation in RAW 264.7 cells, with IC50 values of 34.29 ± 0.82 and 47.5 ± 5.81 µM, respectively.

Alkaloids from Piper longum Exhibit Anti-inflammatory Activity and Synergistic Effects with Chemotherapeutic Agents against Cervical Cancer Cells.

Piper longum L. is widely cultivated for food, medicine, and other purposes in tropical and subtropical regions. Sixteen compounds including nine new amide alkaloids were isolated from the roots of P. longum. The structures of these compounds were determined by spectroscopic data. All compounds showed better anti-inflammatory activities (IC50 = 1.90 ± 0.68-40.22 ± 0.45 µM) compared to indomethacin (IC50 = 52.88 ± 3.56 µM). Among the isolated compounds, five dimeric amide alkaloids exhibited synergistic effects with three chemotherapeutic drugs (paclitaxel, adriamycin, or vincristine) against cervical cancer cells. Moreover, these dimeric amide alkaloids also enhanced the efficacy of paclitaxel in paclitaxel-resistant cervical cancer cells. The combination treatment of one of these dimeric amide alkaloids and paclitaxel promoted cancer cell apoptosis, which is related to the Src/ERK/STAT3 signaling pathway.

The bioactive amide alkaloids from the stems of Piper nigrum.

Piper nigrum is an important aromatic plant, and its fruits (black and white pepper) are commonly used as food additives and spices. However, its stems were disposed as wastes. This research comprehensively investigated bioactive alkaloids of the stems, eight new dimeric amide alkaloids and eight known compounds were obtained. All obtained compounds showed excellent anti-inflammatory activity. Additionally, the dimeric amide alkaloids enhanced the anticancer effect of paclitaxel against cervical cancer cells. These results demonstrate that the stems of P. nigrum could be the sustainable source of bioactive alkaloids for development and utilization in the food and health fields.

Production and characterization of polyclonal and monoclonal antibodies of lamprey pore-forming protein.

In the most primitive jawless vertebrate lamprey, the complement-dependent cytotoxicity regulated by variable lymphocyte receptors (VLRs) plays an important role in the adaptive immunity. Our previous studies have shown that the lamprey pore-forming protein (LPFP) acted as the terminal effector of VLR to lyse and kill the target cells. Here, the recombinant GST-LPFP protein was expressed and purified in prokaryotic expression system, and then used as the immunogen to produce mouse monoclonal antibody and rabbit polyclonal antibody. With these antibodies, we proved that LPFP existed as homodimers in the lamprey serum, and could be recruited to the membrane of target cells after stimulation. In conclusion, the antibodies we produced could specifically recognize the LPFP protein, which could be the useful tools to further study the pore-forming mechanism of LPFP.

Extracts of Poria cocos improve functional dyspepsia via regulating brain-gut peptides, immunity and repairing of gastrointestinal mucosa.

BACKGROUND: Poria cocos (Schw.) Wolf (PC), a fungus, has been used for more than 2000 years as a food and medicine in China. It has a very good therapeutic effect for functional dyspepsia (FD). However, the material basis and mechanism of PC on FD were not reported. PURPOSE: To investigate the function and potential mechanisms of PC including its three extracts (triterpenoid, PCT; water-soluble polysaccharide, PCWP; acidic polysaccharide, PCAP) on FD. STUDY DESIGN: The study explored the therapeutic effect of PC and its three extracts on FD in rats for the first time and discussed its mechanisms based on brain-gut peptides, immunity and repair of the gastrointestinal mucosa. METHODS: The chemical components of PC extracts were analyzed and quantified using ultra high performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS) and gel permeation chromatography coupled with size exclusion chromatography (GPC/SEC). The FD rat models were established using weight-loaded forced swimming and alternate-day fasting for 42 days. After 14 days of treatment, the effect and mechanisms were investigated using ELISA, histopathology, immunohistochemistry as well as Western blot. RESULTS: Seventy-seven triterpenoids in PCT were identified. PCWP was primarily composed of component A (Mw: 3.831 × 107 Da), component B (Mw: 5.650 × 106 Da) and component C (Mw: 113,117 Da). PCAP was a homogeneous composition with an average Mw of 74,320 Da. PCT, PCWP and PCAP alleviated the symptoms of FD. These extracts promoted the repair of gastrointestinal mucosa and regulated the balance between the T helper cell (Th)1/Th2 axis and the Th17/Treg axis. PCT and PCWP regulated brain-gut peptides more effectively, PCWP and PCAP enhanced immunity more effectively. Further study demonstrated that these extracts may have enhanced immunity via the Toll-like receptor (TLR) and c-Jun N-terminal kinase (JNK) signaling pathways. CONCLUSIONS: PC extracts showed therapeutic effects on FD rats, and the mechanism of action involved multiple pathways. PCAP, which is often discarded in traditional applications, was effective. Our study provides new ideas for the application and development of PC extracts.

MicroRNA-483-5p Predicts Poor Prognosis and Promotes Cancer Metastasis by Targeting EGR3 in Nasopharyngeal Carcinoma.

BACKGROUND: MicroRNAs, as small non-coding RNAs, play an important role in tumorigenesis. MiR-483-5p was found to have a significant increase as a diagnostic biomarker of nasopharyngeal carcinoma (NPC), not only in plasma from NPC patients but also in tumor cell lines and biopsy tissues in our previous study. However, its function and mechanism in NPC are still unclear. METHODS: Tissue microarray including 178 primary NPC and 35 adjacent non-cancerous nasopharyngeal mucosal tissues was used to further validate the overexpression of miR-483-5p. Wound healing and invasion assays were conducted to verify its biological function. RNA sequencing (RNA-seq) and dual-luciferase reporter assay was performed to explore its target, and it was verified in fresh biopsy tissues from 23 NPC patients and 9 patients with chronic nasopharyngitis. RESULTS: MiR-483-5p was highly expressed in NPC tissues than in adjacent non-cancerous tissues. It was found to have a significant correlation with poor overall survival (OS) [hazard ratio (HR) = 2.89, 95% confidence interval (CI) = 1.00-8.35, p = 0.041] and progression-free survival (PFS) (HR = 1.95, 95%CI = 1.06-3.60, p = 0.029) of NPC patients. Silencing of its expression inhibited the migratory and invasive capacities of NPC cells in vitro. EGR3 (early growth response 3) was identified as a direct target, and inhibiting miR-483-5p expression markedly enhanced the expression of EGR3 at both the mRNA and protein levels. Besides, a significant decrease of EGR3 expression was found in fresh biopsy tissues from NPC patients, in contrast to miR-483-5p expression. Furthermore, directly decreasing the expression of EGR3 could enhance the migration and invasion of NPC cells. CONCLUSION: The newly identified miR-483-5p/EGR3 pathway provides further insights into the development and metastasis of NPC and may provide a potential therapeutic target for NPC treatment in order to improve survival of NPC patients.

Huganbuzure Granule Attenuates Concanavalin-A-Induced Immune Liver Injury in Mice via Regulating the Balance of Th1/Th2/Th17/Treg Cells and Inhibiting Apoptosis.

In Uygur medicine, Huganbuzure granule (HBG) is one of the classical prescriptions for liver protection. However, its role in immune liver injury remains unknown. This study evaluates the effect of HBG on concanavalin-A- (ConA-) induced immune liver injury and investigates its protective underlying mechanism. BALB/c mice were randomly divided into five groups (n = 24 mice per group): control, ConA, 1.6 g/kg HBG + ConA, 3.2 g/kg HBG + ConA, and 6 mg/kg prednisolone + ConA. HBG was intragastrically administrated once daily for ten consecutive days, prior to ConA (20 mg/kg) injection. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), superoxide dismutase (SOD), and malondialdehyde (MDA) in mouse serum were measured after ConA injection. Moreover, liver-related mRNA levels were evaluated by qPCR. The detection of liver-related proteins was assessed by immunohistochemistry and western blot analysis. Compared with the ConA group, HBG reduced the mRNA expression of IL-17A and IFN-γ and the protein expression of T-bet and ROR-γt. In addition, HBG increased the mRNA expression of IL-4 and TGF-ß and protein expression of GATA3 and Foxp3, indicating that HBG regulated the balance of Th1/Th2 and Th17/Treg. Furthermore, HBG alleviated immune liver injury by reducing oxidative stress, inhibiting apoptosis, and decreasing the expression of p-JNK, p-ERK, p-p38, p-JAK1, p-STAT1, p-STAT3, and IRF1. Our data suggested that HBG attenuated ConA-induced immune liver injury by regulating the immune balance and inhibiting JAK1/STATs/IRF1 signaling, thereby reducing apoptosis induced by JNK activation. The findings indicate that HBG may be a promising drug for immune liver injury.

Bioactive sesquiterpenes from Inula helenium.

Twenty-one eudesmane-type sesquiterpenes, including five new compounds, were isolated from the roots of Inula helenium. The structures of the new compounds (1-5) were determined by extensive spectroscopic data interpretation, single-crystal X-ray diffraction analysis and ECD calculations. Six compounds can synergistically enhance cisplatin effect against ovarian cancer cells, the structure - activity relationship for the synergistic effect of these compounds with cisplatin was revealed for the first time, which provides useful clues to develop novel sensitizers to overcome drug resistance in cancer. In addition, fifteen sesquiterpenes exhibited significant anti-inflammatory activity, which provided promising candidates for development of anti-inflammatory agent.

Yu Gan Long Ameliorates Hepatic Fibrosis by Inhibiting PI3K/AKT, Ras/ERK and JAK1/STAT3 Signaling Pathways in CCl4-induced Liver Fibrosis Rats.

Yu Gan Long (YGL) is a Chinese traditional herbal formula which has been reported to attenuate liver fibrosis for many years and we have explored its anti-fibrotic mechanism through blocking transforming growth factor (TGF-ß) in the previous study. But the mechanisms associated with platelet-derived growth factor (PDGF)-BB remain obscure. In this study, we further investigated the mechanism of YGL reducing carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Our results showed that YGL suppressed CCl4-induced upregulation of collagen IV (Col IV), type HI precollagen (PCHI), hyaluronuc acid (HA) and laminin (LN), which are implicated in liver fibrosis. Also, YGL reduced the α-smooth muscle actin (α-SMA) expression, which acts as the indicator of liver fibrosis. Furthermore, YGL decreased the serum levels of hepatic stellate cell (HSC) mitogen PDGF-BB and inflammation cytokines, including TNF-α, IL-1ß, IL-6. Markers involved in liver fibrosis, such as Ras, p-Raf-1, p-ERK1/2, p-JNK, p-P38, p-PI3K, p-AKT, p-JAKl, p-STAT3 were downregulated significantly after treatment with YGL. Our results indicated that YGL ameliorated CCl4-induced liver fibrosis by reducing inflammation cytokines production, and suppressing Ras/ERK, PI3K/AKT, and JAK1/STAT3 signaling pathways, which provided further evidence towards elucidation of the anti-fibrotic mechanism of YGL.

A Potential Anti-cancer Compound Separated from the Chloroform Extract of the Chinese Medicine Formula Shenqi San.

This study examined anti-cancer compounds present in the chloroform extract of the Chinese medicine formula Shenqi San (CE-SS). Silica gel column chromatography, Sephadex LH-20, octadecylsilyl (ODS) column chromatography, and high performance liquid chromatography (HPLC) were used to separate the compounds from CE-SS. The structural formulas of the separated compounds were determined using 1D 1H and 13C experiments as well as high resolution electrospray ionization mass spectroscopy (HRESIMS). The corresponding results were compared with the reported literature data. A total of six compounds were separated and their structures were identified on the basis of corresponding spectroscopic and physico-chemical properties. They were Saikogenin F (I), Prosaikogenin D (II), Prosaikogenin F (III), ß-sitosterol (IV), 3ß,16ß,23-trihydroxy-13,28-epoxyurs-11-ene-3-O-ß-D-glucopyranoside (V), and methyl ursolic acid (VI). The separated compounds were evaluated in vitro for their inhibitory ability against the proliferation of A549 cells via MTT assay. Apoptosis was investigated using Annexin V-FITC/propidium iodide (PI) by flow cytometry. Apoptosis-associated proteins were examined by Western blotting. All the compounds were observed to have inhibitory activities against the proliferation of A549 cells to different degrees. Flow cytometry showed that compound V increased the proportion of apoptotic A549 cells in a dose-dependent manner. Western blotting showed that compound V increased the expression of Bax, cleaved-caspase-3, cleaved-caspase-9 and cleaved-poly ADP-ribose polymerase (PARP), and decreased the expression of Bcl-2. These results indicated that compound V featured a significant inhibitory effect on A549 cells when compared with other compounds, and it may be considered a potential drug against cancers.

14-Deoxycoleon U-induced endoplasmic reticulum stress-mediated apoptosis, autophagy, and cell cycle arrest in lung adenocarcinoma.

OBJECTIVE: 14-Deoxycoleon U is a natural abietane-type diterpene and exerts an inhibitory effect on tumor cells proliferation, which suggests that 14-Deoxycoleon U may be a potent anti-cancerous lead compound for lung cancer treatment. This study was to evaluate potential of 14-Deoxycoleon U to treat lung adenocarcinoma in vitro and in vivo. METHODS: In the present study, the cell viability and apoptosis morphology of 14-Deoxycoleon U-treated A549 and LLC cells were explored using cell counting kit-8 assay and Hoechst 33258 staining. Then, the protein expressions about apoptosis, endoplasmic reticulum (ER) stress, autophagy and cell cycle were measured using Western blot. The autophagosome formation of 14-Deoxycoleon U-treated A549 cells was visualized using a confocal microscopy. LLC lung adenocarcinoma model was established. RESULTS: The results indicated that 14-Deoxycoleon U significantly inhibited A549 and LLC cell proliferation in a dose-dependent manner via caspase-dependent apoptosis. Furthermore, apoptosis of both cells was mediated by 14-Deoxycoleon U-induced ER stress. In addition, 14-Deoxycoleon U-induced A549 and LLC cell autophagy, thus promoting their death. Moreover, 14-Deoxycoleon U-induced cell cycle arrest in both cells via inhibition of cyclin D3, cyclin-dependent kinase 6, CDC2 and up-regulation of p21. In vivo results showed that administration of 14-Deoxycoleon U significantly suppressed LLC growth and adverse effects of 14-Deoxycoleon U on organs might be lower than of adriamycin. CONCLUSION: Overall, our results demonstrated that 14-Deoxycoleon U represses in vitro and in vivo growth of lung adenocarcinoma through ER stress-mediated apoptosis accompanied by autophagy and cell cycle arrest.

TEEG Induced A549 Cell Autophagy by Regulating the PI3K/AKT/mTOR Signaling Pathway.

TEEG (3ß,16ß,23-trihydroxy-13,28-epoxyurs-11-ene-3-O-ß-D-glucopyranoside) is derived from the chloroform extract of the Chinese medicine formula Shenqi San (CE-SS). In the present study, we aimed to elucidate the anticancer effect and possible molecular mechanism underlying the action of TEEG against the human non-small cell lung cancer (NSCLC) cell line A549 in vitro. A549 cells were incubated with different concentrations of TEEG. Cell proliferation was assessed by MTT assay. Autophagy was evaluated by immunofluorescence staining. Autophagy-associated proteins were examined by Western blot analysis. TEEG markedly inhibited A549 cell proliferation in a concentration-dependent manner. Immunofluorescence staining showed that TEEG induced autophagy in A549 cells. The LC3-II : LC3-I conversion ratio and the expression of Beclin-1, Atg5, Atg7, and Atg12 increased with the concentration of TEEG. In addition, increased TEEG concentration enhanced the expression of Class III p-PI3K and reduced the expression of Class I p-PI3K, p-AKT, p-mTOR, and p-P70S6K. These results indicate that TEEG induces autophagy of A549 cells through regulation of the PI3K/AKT/mTOR signaling pathway.

Extracts of Coleus forskohlii relieves cough and asthma symptoms via modulating inflammation and the extracellular matrix.

Asthma is characterized by airway inflammatory infiltration, which leads to airway remodeling and airway hyperreactivity. Coleus forskohlii (CFK) has been used to treat asthma, however, the mechanism involved is not clear. To explore the antiasthma mechanism of extracts of Coleus forskohlii (ECFK), guinea pigs were administered with a spray of phosphoric acid histamine, and rats were sensitized with ovalbumin (OVA). Hematoxylin and eosin staining (H&E) were used to evaluate pathological changes in lung tissue. Enzyme-linked immunosorbent assay (ELISA) was used to determine cytokine levels in serum and bronchoalveolar lavage fluid (BALF). Immunohistochemistry and Western blot analysis were used to assess the expression of intercellular cell adhesion molecule-1 (ICAM-1), phosphorylation of p65 (p-p65), matrix metallopeptidase 9 (MMP-9), and tissue inhibitor of metalloproteinase 1 (TIMP-1). After ECFK treatment, the asthma incubation period of guinea pigs was significantly prolonged. The H&E results showed that the number of eosinophils in the 12.8 g/kg ECFK group was significantly lower when compared with the control group. Moreover, ELISA results demonstrated that interleukin (IL)-4, IL-5, and IL-17 in serum and BALF were significantly decreased, and interferon-γ (IFN-γ) and IL-10 were increased after ECFK treatment. In addition, ECFK treatment resulted in downregulation of ICAM-1, p-p65, MMP-9, and TIMP-1 in lung tissue after being sensitized by OVA. In conclusion, our findings indicated that ECFK significantly alleviated OVA-induced inflammatory infiltration and airway remodeling in asthma. This study laid a theoretical foundation for the clinical use of ECFK.

Yu Gan Long reduces rat liver fibrosis by blocking TGF-ß1/Smad pathway and modulating the immunity.

Yu Gan Long (YGL) is a Chinese traditional herbal medicine that has been used in the treatment of liver fibrosis for many years in clinical practice. However, its anti-hepatofibrotic mechanism has not been studied yet. In this study, the effect and mechanism of YGL in reducing liver fibrosis was demonstrated in vivo. Our results showed that liver fibrosis biomarkers collagen IV (Col IV), type III precollagen (PCIII), hyaluronuc acid (HA) and laminin (LN), were increased after CCl4 treatment and decreased by YGL. Among the liver fibrosis indicators, α-smooth muscle actin (α-SMA) was decreased by YGL in the CCl4-treated rats, while MMP2 and MMP9 was upregulated followed by TIMP1 downregulation. Proteins involved in liver fibrosis such as p-Smad2, p-Smad3 and Smad4 were down-regulated, while Smad7 protein was up-regulated by YGL after CCl4-induced liver damage. YGL also suppressed the increase of TGF-ß1, TNF-α, IL-1ß, IL-6, IL-4 and IL-17 A induced by CCl4 treatment, while promoted IFN-γ expression. Finally, the transcription factors ROR-γt and GATA3 were decreased, while T-bet was increased after YGL treatment. These results suggested that YGL attenuated CCl4-induced hepatic fibrosis by accelerating the extracellular matrix degradation, blocking the TGF-ß1/Smad signaling pathway and modulating the balance among IL-4, IL-17 A and IFN-γ, demonstrating YGL protective effect and its potential mechanisms in treating liver fibrosis.

Activation of protein kinase B/Akt signaling pathway contributes to mechanical hypersensitivity induced by capsaicin.

We investigated the involvement of the protein kinase B/Akt (PKB/Akt) signaling pathway in the mechanical hypersensitivity induced in rats by capsaicin. Intradermal injection of capsaicin results in activation of PKB/Akt in the lumbar spinal cord, most prominently in the dorsal horn, starting by 5 min after capsaicin injection and lasting at least 1h. The activated PKB/Akt in the spinal cord is in neurons, since phospho-PKB/Akt (p-PKB/Akt) colocalizes with the neuronal marker, neuronal-specific nuclear protein (NeuN). The mechanical hypersensitivity is shown by the enhanced paw withdrawal frequency to applications of von Frey filaments with different bending forces (30, 100, 200 mN) on the rat paw. Pre-treatment with several different PKB/Akt inhibitors, including SH-6, Akt inhibitor IV, and Akt inhibitor V, blocked the mechanical hypersensitivity induced by intradermal injection of capsaicin, a measure of spinal cord central sensitization. Two structurally unrelated phosphoinositide 3-Kinase (PI3K, upstream of PKB/Akt) inhibitors, Wortmannin and LY294002, also prevented the mechanical hypersensitivity induced by intradermal injection of capsaicin. Furthermore, post-treatment with the PI3K inhibitor, Wortmannin, or PKB/Akt inhibitors, such as NL-71-101, SH-6, Akt inhibitor IV, and inhibitor V significantly reduced the established mechanical hypersensitivity induced by capsaicin. The PKB/Akt signaling pathway in the spinal cord is therefore involved in pain hypersensitivity.

Calcitonin gene-related peptide receptor activation produces PKA- and PKC-dependent mechanical hyperalgesia and central sensitization.

Calcitonin gene-related peptide (CGRP), acting through CGRP receptors, produces behavioral signs of mechanical hyperalgesia in rats and sensitization of wide dynamic range (WDR) neurons in the spinal cord dorsal horn. Although involvement of CGRP receptors in central sensitization has been confirmed, the second-messenger systems activated by CGRP receptor stimulation and involved in pain transmission are not clear. This study tested whether the hyperalgesia and sensitizing effects of CGRP receptor activation on WDR neurons are mediated by protein kinase A or C (PKA or PKC) signaling. Intrathecal injection of CGRP in rats produced mechanical hyperalgesia, as shown by paw withdrawal threshold tests. CGRP-induced hyperalgesia was attenuated significantly by the CGRP1 receptor antagonist, CGRP8-37. The effect was also attenuated significantly by a PKA inhibitor (H89) or a PKC inhibitor (chelerythrine chloride). Electrophysiological experiments demonstrated that superfusion of the spinal cord with CGRP-induced sensitization of spinal dorsal horn neurons. The CGRP effect could be blocked by CGRP8-37. Either a PKA or PKC inhibitor (H89 or chelerythrine) also attenuated this effect of CGRP. These results are consistent with the hypothesis that CGRP produces hyperalgesia by a direct action on CGRP1 receptors in the spinal cord dorsal horn and suggest that the effects of CGRP are mediated by both PKA and PKC second-messenger pathways.

Effects of intrathecal injections of melatonin analogs on capsaicin-induced secondary mechanical allodynia and hyperalgesia in rats.

Melatonin, its agonists/antagonists were administered intrathecally (i.t.) before/after intradermal injection of capsaicin. Capsaicin produced an increase in the paw withdrawal frequency (PWF) in the presumed area of secondary mechanical allodynia and hyperalgesia. Melatonin agonists in the absence of a capsaicin injection decreased the PWF significantly, whereas melatonin antagonists given intrathecally alone were ineffective in the absence of a capsaicin injection. Pre-treatment with a melatonin agonist i.t. caused a reduction in the PWF after capsaicin. In contrast, the PWF increased after capsaicin with pre-administration of a melatonin antagonist i.t. Combined pre-treatment with melatonin and a melatonin antagonist i.t. prevented the change in PWF induced by melatonin alone after capsaicin. Intrathecal post-treatment with a melatonin agonist reduced the enhanced PWF that followed an injection of capsaicin, but treatment with a combination of a melatonin agonist and its antagonist did not alter the responses. The PWF was unaffected when melatonin analogs were applied i.t. at the T6 level or were injected intramuscularly adjacent to the L4 vertebra. In spinal rats, the data showed comparable effects of melatonin analogs on capsaicin-induced secondary mechanical hyperalgesia. Animal motor function tested by 'activity box' showed that motion activity was not affected by i.t. melatonin or its antagonist. These results suggest that activation of the endogenous melatonin system in the spinal cord can reduce the generation, development and maintenance of central sensitization, with a resultant inhibition of capsaicin-induced secondary mechanical allodynia and hyperalgesia.