The American Journal of Chinese Medicine

Electroacupuncture (EA) has been shown to induce potent analgesic effects on neuropathic pain in both patients and rodents. Cell therapy to release antinociceptive agents near the pain processing centers of the spinal cord is a promising next step in the development of treatment modalities. This study investigated the effects of the combination of EA and cell therapy by glial cell line-derived neurotrophic factor (GDNF) on neuropathic pain in rats. The hyperalgesic state was induced by chronic constriction injury (CCI) of the sciatic nerve and fibroblasts genetically modified to secrete bioactive GDNF (FBs-GDNF) were used for cell therapy. Fifty-eight rats with neuropathic pain were randomly divided into five groups (CCI+PBS, n = 11; CCI+FBs-GDNF, n = 12; CCI+EA+PBS, n = 11; CCI+EA+FBs-pLNCX2, n = 12; CCI+EA+FBs-GDNF, n = 12). On the 7th day after CCI, the rats received intrathecal transplantation of FBs-GDNF or control fibroblasts (FBs-pLNCX2). In the meantime, EA was administered once every other day from the 7th day after CCI surgery for 21 days. The paw withdrawal latency (PWL) to radiant heat was measured every other day. The results showed that the ipsilateral PWL of the rats from all three EA treatment groups significantly increased starting on the 12th day compared with the PBS control group. Strikingly, the group which received EA treatment and FBs-GDNF transplantation (CCI+EA+FBs-GDNF) showed a significantly decreased thermal hyperalgesia after 2 weeks post CCI surgery compared with the groups which received EA treatment and FBs-pLNCX2 transplantation (CCI+EA+FBs-pLNCX2) or PBS (CCI+EA+PBS) as well as the FBs-GDNF transplantation group without EA treatment (CCI+FBs-GDNF). Our data suggest that EA and cell therapy can synergistically attenuate hyperalgesia in neuropathic pain rats.

Reducing the expression of endothelial cell adhesion molecules (ECAMs) is known to decrease inflammation-induced vascular complications. In this study, we explored whether rhein can reduce the inflammation-induced expression of ECAMs in human umbilical vein endothelial cells (HUVECs) with or without lipopolysaccharide (LPS) stimulation. HUVECs were treated with different concentrations of rhein with or without 2.5 μg/ml LPS stimulation. Cell viability was assayed using the MTT method. Real-time PCR and Western blot analysis were used to measure the transcription and expression levels of ECAMs, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-SELECTIN and related signaling proteins. The results indicated that rhein (0–20 μmol/L) and LPS (0–10 μg/ml) had no effect on the viability of HUVECs. LPS could promote the expression of VCAM-1, ICAM-1 and E-SELECTIN. Rhein appeared to target VCAM-1, ICAM-1 and E-SELECTIN, with the transcription and expression of all three factors being reduced by the rhein treatment (10 and 20 μmol/L). The transcription and expression of VCAM-1 were also reduced by treatment with rhein (10 and 20 μmol/L) in the presence of LPS stimulation. In conclusion, rhein treatment reduced the expression of VCAM-1 in HUVECs via a p38-dependent pathway.

Non-alcoholic fatty liver disease (NAFLD) has become a major challenge to the healthcare system. This study was designed to evaluate the effect of the triterpenoid-rich fraction (TF) from Ilex hainanensis Merr. on NAFLD. Male Sprague-Dawley (SD) rats were fed a normal diet (control) or high fat diet (NAFLD model). After four weeks, the high fat diet group was orally administrated TF (250 mg/kg) for another two weeks. High fat diet fed rats displayed hyperlipidemia and a decline in liver function compared with control. However, administration with TF could effectively improve these symptoms, as demonstrated by decreasing the plasma levels of triglyceride (p <0.05), total cholesterol (p < 0.01), low-density lipoprotein cholesterol (p < 0.05), alanine transaminase (p < 0.05), aspartate aminotransferase (p < 0.01), liver index (p < 0.05) and insulin resistance index (p < 0.05) while increasing the high-density lipoprotein cholesterol (p < 0.05). Meanwhile, histopathological examination of livers also showed that TF could reduce the incidence of liver lesions induced by high fat diet. Furthermore, TF could alleviate oxidative stress and inflammation status indicated by the decline malondialdehyde and superoxide dismutase levels (p < 0.01, both) and levels of interleukin 6 and tumor necrosis factor-α (p < 0.05). In addition, immunohistochemistry showed TF evidently elevated the peroxisome proliferator-activated receptor (PPARα) expression (p < 0.01), while it diminished the Cytochrome P450 2E1 (CYP2E1) expression (p < 0.01) in liver. These results demonstrate that TF has potential ability to protect liver against NAFLD by regulating lipids metabolism and alleviating insulin resistance, inflammation and oxidative stress. This effect might be associated with regulating PPARα and CYP2E1 expression.

In Ayurveda,Withania somnifera (WS) is used as a medicine to maintain mental and physical health as well as to enhance memory. In this study, the methanolic extract of WS(mWS) was tested for its electrical influence on hippocampal CA1 pyramidal neurons using a patch clamp technique. In current clamp mode under a high chloride pipette solution, mWS (400 ng/μl) induced remarkable membrane depolarization (9.75 ± 2.54 mV, n = 6) of CA1 neurons. The mWS-induced depolarization was dose-dependent, reproducible, and persistent in the presence of 0.5 μM tetrodotoxin (TTX, 10.17 ± 0.04 mV, n = 6). In voltage clamp mode (holding potential = -60 mV), mWS induced a dose-dependent non-desensitizing inward current that persisted in the presence of TTX (0.5 μM), suggesting that the response induced by mWS was purely a postsynaptic event. Interestingly, these inward currents were partially blocked by strychnine, a glycine receptor blocker. Further, mWS potentiated the NMDA response in hippocampal CA1 neurons at low concentrations. Overall, these results suggest that there are compounds in WS with possible glycine mimetic activities, which may be potential targets for inducing memory consolidation in hippocampal CA1 neurons.

Toki-shakuyaku-san (TSS), an herbal formula based on traditional Chinese medicine, is commonly used in obstetrics. To examine the effects of TSS on the normal mouse fetus and placenta, TSS was administered to normal pregnant mice and their placentas and fetuses were studied. First, the effects of maternal TSS treatment on implantation were investigated. Administration of TSS from gestation day 0.5 (G0.5) to G6.5 showed that litter size was not altered at embryonic day 11.5 (E11.5), but the number of resorbed fetuses was slightly decreased. Then, to investigate effects on fetal and placental growths after implantation, TSS was administered from G5.5. At E14.5, the body weight of fetuses from TSS-treated dams was significantly increased. Gene expression of insulin-like growth factor 2 (Igf2), one of the most important modulators of fetal growth, was significantly increased in the placentas and fetuses of TSS-treated dams. In addition, the expression of particular placental developmental genes and nutrient transporter genes was significantly increased in TSS-treated placentas. At E18.5, after longer-term administration of TSS, fetal and placental weights were not altered, but the expression of the placental developmental and nutrient transporter genes remained elevated compared with controls. These results suggest that maternal TSS treatment in normal mice enhances the expression of Igf2, placental developmental genes and nutrient transporter genes, resulting in increased fetal weight. No obvious changes were observed in the expression of these genes after longer-term maternal TSS treatment.

This study was designed to compare the anti-inflammatory potential of a Magnolia officinalis L. bark extract solely or in combination with extracts prepared from either Polygonum aviculare L., Sambucus nigra L., or Isodon japonicus L. in bacterial lipopolysaccharide (LPS) stimulated human gingival fibroblasts (HGF-1) and human U-937 monocytes, as cell models of periodontal disease. HGF-1 and U-937 cells were incubated with LPS from either Porphyromonas gingivalis or Escherichia coli together with the four plant extracts alone or in combination. Secretion of anti-inflammatory cytokines from HGF-1 and U-937 cells was measured by means of a multiplexed bead assay system. Magnolia officinalis L. bark extract, at concentrations of 1 μg/mL and 10 μg/mL, reduced interleukin 6 (IL-6) and interleukin-8 (IL-8) secretion from HGF-1 cells to 72.5 ± 28.6% and reduced matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) secretion from U-937 cells to 8.87 ± 7.97% compared to LPS-treated cells (100%). The other three extracts also reduced secretion of these inflammatory markers but were not as effective. Combination of 9 μg/mL Magnolia officinalis L. extract with 1 μg/mL of each of the other extracts maintained the anti-inflammatory effect of Magnolia officinalis L. extract. Combination of 5 μg/mL Magnolia officinalis L. extract with 5 μg/mL Isodon japonicus L. extract also maintained the anti-inflammatory potential of the Magnolia officinalis L. extract, whereas increasing concentrations of any of the other plant extracts in the combination experiments reduced the Magnolia officinalis L. extract efficacy in U-937 cells.

The increased life expectancy of the population has led to increasing incidences of cancer, chronic inflammatory and autoimmune diseases. Thus the continuous search for new drugs is necessary because ineffectiveness and adverse effects have been described for standard drugs. Essential oils are important sources of bioactive metabolites and several clinical trials have been developed using them. The Pterodon genus has been used in traditional medicine to treat rheumatic disorders, thus this work investigated the properties of essential oil from Pterodon polygalaeflorus fruits (EsOPpg) on acute inflammation and lymphocyte activation. The essential oil was obtained by hydrodistillation and its components were identified by GC/MS. The anti-inflammatory response was assessed using the air pouch model. Antinociceptive potential was evaluated using the writhing model. Lymphocyte phenotyping, cell cycle and apoptosis were analyzed by flow cytometry. EsOPpg promoted a reduction in leukocyte counts and protein concentration in the exudate, and reduced vasodilatation and inflammatory cell infiltrate in air pouch tissue. No antinociceptive effect was demonstrated for the doses tested. EsOPpg inhibited lymphocyte proliferation, arresting the cell cycle in G₁ phase, and induced apoptosis in these cells. EsOPpg downregulated both the total number of CD8⁺ T cells and the activated subpopulation (CD8⁺CD69⁺), while promoting upregulation of the total number of CD19⁺ and CD19⁺CD69⁺ B cells. In conclusion, Pterodon polygalaeflorus essential oil diminished the acute inflammatory response and inhibited lymphocyte proliferation, reducing neutrophil recruitment into the cavity and air pouch tissue and promoting distinct modulations of the activation level of each lymphocyte subpopulation.

Sargassum fulvellum (Turner) C. Agardh has been used to treat various inflammatory diseases, including lump, dropsy, swollen and painful scrotum, and urination problems for several centuries with no side effects. This study aims to investigate the anti-inflammatory effect of the hexane fraction of S. fulvellum (HFS) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and phorbol 12-myristate 13-acetate (PMA)-induced mouse-ear edema. The anti-inflammatory activity of HFS in LPS-stimulated RAW 264.7 cells was investigated by assessing the inhibition of nitric oxide (NO) and pro-inflammatory cytokine production during Griess reaction and enzyme-linked immunosorbent assay (ELISA), respectively. The molecular mechanisms that underlie the anti-inflammatory action of HFS were investigated by analyzing the activation of transcription factor and its upstream signaling proteins. Additionally, an in vivo study of the anti-inflammatory effect of HFS was carried out using PMA-induced mouse-ear edema. HFS inhibited LPS-induced NO production in a dose-dependent manner and suppressed the expression of inducible NO synthase (iNOS) in the RAW 264.7 cells. Further, HFS reduced the production of pro-inflammatory cytokines in the LPS-stimulated RAW 264.7 cells. HFS significantly inhibited LPS-induced nuclear factor kappa B (NF-κB) transcriptional activity and NF-κB translocation into the nucleus by preventing degradation of inhibitor κB-α. Moreover, HFS inhibited the activation of Akt and mitogen-activated protein kinases (MAPKs) in the LPS-stimulated RAW 264.7 cells. Furthermore, HFS suppressed PMA-induced mouse-ear edema. The above data indicate that the anti-inflammatory effects of HFS on LPS-stimulated cells are associated with the suppression of NF-κB through the inhibition of MAPKs and Akt phosphorylation.

Paeonia lactiflora Pallas (P. lactiflora, Ranunculaceae) is a common ingredient of Sheng-Ma-Ge-Gen-Tang (SMGGT; Shoma-kakkon-to) and Ge-Gen-Tang (GGT; kakkon-to). SMGGT and GGT are different prescriptions of traditional Chinese medicine with different ingredients designed for airway symptoms. Both SMGGT and GGT have anti-viral activity against human respiratory syncytial virus (HRSV). Therefore, P. lactiflora was hypothesized to be the effective ingredient of both SMGGT and GGT against HRSV. However, P. lactiflora does not have any proven antiviral activity. This study used both human upper (Human larynx epidermoid carcinoma cell line, HEp-2) and lower (human lung carcinoma cell line, A549) respiratory tract cells to test the hypothesis that a hot water extract of P. lactiflora could effectively inhibit plaque formation induced by HRSV infection. The ability of P. lactiflora to stimulate anti-viral cytokines was evaluated by enzyme-linked immunosorbent assay (ELISA). The results showed that P. lactiflora was time-dependently and dose-dependently effective against HRSV in HEp-2 and A549 cells, particularly supplemented before viral inoculation (p < 0.0001). 10 μg/ml P. lactiflora had a comparable anti-HRSV activity with 10 μg/ml ribavirin, a broad-spectrum antiviral agent. P. lactiflora was dose-dependently effective against viral attachment (p < 0.0001), with a better effect on A549 cells (p < 0.0001). P. lactiflora was time-dependently (p < 0.0001) and dose-dependently (p < 0.0001) effective against viral penetration. Moreover, P. lactiflora stimulated IFN-β secretion without any effect on TNF-α secretion. Therefore, P. lactiflora could be beneficial at preventing HRSV infection by inhibiting viral attachment, internalization, and stimulating IFN secretion.

This work was designed to identify the effect of 5,7,3′-triacetyl hesperetin (TAHP) on rat adjuvant arthritis (AA) and further clarify the possible role of TAHP on modulating Janus kinase signal transducers and activators (JAK/STAT in this process. Freund's complete adjuvant was used to induce AA in rats. TAHP (33, 66, 132 mg/kg) was administered intragastrically. Secondary paw swelling, polyarthritis index, index of immune organs and histopathological assessment were used to evaluate the effects of TAHP on AA in rats. IL-6 in serum and in synovial tissues was examined with ELISA and RT-PCR. In addition, JAK2/STAT3 pathway-related key molecules mRNA expression in synovial tissues of AA rats were detected by RT-PCR and western blot respectively. It was found that TAHP (66, 132 mg/kg) could significantly inhibit secondary paw swelling, restore the index of immune organs and reduce polyarthritis index. Results of histopathological assessment showed that TAHP clearly ameliorated the pathological changes in AA rats. TAHP could downregulate the level of IL-6 in serum and in synovial tissues of AA rats. Besides, treatment with TAHP could decrease mRNA expressions of STAT3 and JAK2, as well as the ratio of p-JAK2/JAK2 protein and p-STAT3/STAT3 protein from synovial tissues. Thus, the paper demonstrated that TAHP had a therapeutic effect on AA in rats and the mechanisms were partly associated with modulating proinflammatory cytokine IL-6 production in serum and in synovial tissues and inhibiting excessive activation of JAK2/STAT3 signaling pathway which might play a crucial role in the pathogenesis of AA.

Celastrol is one of the principal active ingredients of Tripterygium wilfordii Hook.f., a toxic Chinese medical herb traditionally prescribed for controlling pain and inhibiting inflammation in various chronic inflammatory diseases, including rheumatoid arthritis (RA). Resistance to apoptosis of fibroblast-like synoviocytes is considered a major characteristic of RA. In this study, we test celastrol's cytotoxic effect and potential mechanisms in human rheumatoid synovial fibroblasts (RA-FLS). In the cytotoxic assay, we found that celastrol dose-dependently decreased RA-FLS viability and increased LDH release. The apoptotic nuclear morphology was observed after celastrol treatment as determined by DAPI fluorescence staining. Flow cytometry analysis with PI and Annexin V revealed that celastrol induced RA-FLS cell cycle arrest in the G2/M phase and apoptosis. Furthermore, celastrol dramatically increased expression of Bax/Bcl-2, proteolytic cleavage of Caspase-3, -9, PARP, and decreased expression of FasR. In addition, celastrol treatment resulted in DNA damage. Collectively, we concluded that celastrol inhibits RA-FLS proliferation by inducing DNA damage, cell cycle arrest, and apoptosis in vitro, which might provide data for its application in RA treatment.

PI3K/AKT/mTOR signaling promotes cell survival, proliferation and progression in cancer cells. Targeting this pathway may lead to the development of novel therapeutic approaches for human cancers. Here, we examined the effects of (-)-epigallocatechin-3-gallate (EGCG) on the PI3K/AKT/mTOR pathway in pancreatic cancer cells, and assessed its therapeutic potential. In this study, the proliferation and apoptosis of PANC-1 cells were examined by MTT assay and flow cytometry, respectively. The expression of genes and proteins involved in the PI3K/AKT/mTOR pathway were measured by RT-PCR and western blotting, respectively. Our results revealed that EGCG dramatically inhibited the proliferation of PANC-1 cells and induced apoptosis simultaneously. Furthermore, it upregulated PTEN mRNA and protein expression levels, as well as downregulating the expression of phospho-AKT and phospho-mTOR. In conclusion, these results suggest that EGCG can suppress proliferation and induce apoptosis of PANC-1 cells in a time- and dose-dependent manner; moreover, EGCG also can upregulate PTEN expression and downregulate the expression of pAKT and p-mTOR to modulate the PI3K/AKT/mTOR signaling pathway.

The cytotoxin family of cobra venom proteins, also called cardiotoxins, can activate both necrotic and apoptotic cell death pathways in cancer cells. Cytotoxin 1 (CTX1)from Naja atra Cantor venom is a 60 amino acid, 6698 Da protein with as yet untested anticancer efficacy and cell selectivity. We tested the toxicity of CTX1 on a number of cancer cell lines (MCF-7, P388, K562, and H22) and on one normal human cell line (16HBE). The rank order of cytotoxicity was MCF-7 > P388 ≈ K562 >H22 ≈ 16HBE, indicating that the effect of CTX1 on certain cancer cell types was relatively selective.Treatment with CTX1 greatly prolonged the survival of P388 ascites tumors bearing KM mice compared to cyclophosphamide treatment. Cell viability, apoptosis, and lysosomal permeability assays all demonstrated that CTX1 induced dose- and time-dependent cell death, with most cells exhibiting the morphological and biochemical features of late apoptosis and necrosis. Mitochondrial membrane potential was lost in CTX1-treated P388 cells. In addition, CTX1 induced an increase in both lysosomal membrane permeability and cathepsin B protease activity. These analyses reveal that CTX1 possesses significant and selective anticancer activity, likely by inducing programmed cell death through mitochondrial and/or lysosomal pathways.

Norcantharidin (NCTD) is currently used for anticancer therapy but the exact mechanism of action remains unknown. Pre-replicative complexes (pre-RCs) are essential for cell DNA replication and highly related to malignant proliferation. Here, we examined the inhibitory effect of NCTD on pre-RC components in HepG2 cells. We showed that NCTD induced degradation of Cdc6 and Mcm2 in a dose-dependent manner. Under 100 μM NCTD concentration, about 70% of Cdc6 and 50% of Mcm2 were degraded. In addition, the nuclear translocation of Mcm6 was inhibited by NCTD. Further studies aiming at G1 synchronous cells showed that, NCTD reduced the chromatin-bound Cdc6, Mcm2 and Mcm6. Moreover, the cells were blocked from entering the S phase and accumulated at the G1 phase when released synchronously into the cell cycle. Consistently, the DNA replication was inhibited by NCTD. Finally, the combination NCTD with Cdc6 depletion lead to more severe cytotoxicity (88%) than NCTD (52%) and Cdc6 depletion (39%) alone. A synergic cytotoxicity was observed between Cdc6 depletion and NCTD. In conclusion, our results demonstrate that NCTD inhibits pre-RC assembly; subsequently blocks the G1 to S transition; and inhibits DNA replication in HepG2 cells. Pre-RCs are an intriguing target for cancer therapy, which merits further investigations for anticancer development.

Oral cancer is a common malignancy associated with high morbidity and mortality. While p38 MAPK is reported to be involved in different cellular activities such as proliferation and differentiation, reports rarely define the roles of the individual members of the p38 MAPK family in cancer. We used two unique cell lines developed by our lab representing chemically induced oral cancer cells (T28) and non-tumor cells (N28) obtained from tissues surrounding the induced cancer as a model to screen out whether p38 MAPK is involved in the malignant transformation processes. The results suggest an association between p38β not p38α and oral cancer development. Additionally, the anti-cancer activity of thymoquinone (TQ) was screened out and we found evidences suggesting that the anti-tumor activity of TQ may be attributed to the downregulation of p38β MAPK.

We developed a sensitive and rapid method for determination of ferulic acid, caffeic acid, vanillic acid, and paeoniflorin in rat plasma based on ultra high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS). The separation of the four compounds was carried out on an AcQuity UHPLC™ BEH C₁₈ column using a mobile phase consisting of acetonitrile and water (containing 0.1% formic acid). Electrospray ionization in positive and negative ion mode and multiple reaction monitoring was used to identify and quantify active components. All calibration curves gave good linearity (r > 0.991) over the concentration range from 4.24–2875 ngmL^-1 for all components. The precision of the in vivo study was evaluated by intraday and interday assays and the percentages of RSD were all within 10.6%. The recovery ranged from 60.2 to 77.9%. The method was successfully applied to pharmacokinetic study of all three aromatic acids and one monoterpene in rat plasma. Furthermore, we compared the pharmacokinetics profile of the four compounds in normal and primary dysmenorrhea rats' plasma following oral administration of Shaofu Zhuyu decoction (SFZYD) and its ethanol supernatant extract (SFE).