A Mechanism Study of Electroacupuncture for Dry Eye Syndrome by Targeting Conjunctival Cytokine Expressions.

Aim: To discuss the immunological mechanism in electroacupuncture (EA) treatment of dry eye syndrome (DES) by targeting the changes in conjunctival cytokine expression profile.Method: Eligible DES patients were randomized into an EA group (EAG) or an acupuncture group (AG). The ocular surface disease index (OSDI), amount of tear production, and tear film break-up time (BUT) were observed to evaluate the efficacy. Conjunctival cells were collected from both effective and invalid cases to observe the expressions of cytokines by protein microarray. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for functional cluster and signaling pathway analysis of the differentially expressed proteins. Enzyme-linked immunosorbent assay (ELISA) was used to verify the specific differential proteins.Result: After treatment, OSDI dropped and BUT extended in both groups, and the tear production increased only in the EAG (all P < .01). Compared with the AG, the improvement in tear production was more significant in the EAG (P < .01). There were 17 differentially expressed conjunctival cytokines between the effective and invalid cases in the EAG, and those expressed higher than the limit of detection (LOD) included monocyte chemoattractant protein 1 (MCP-1), macrophage colony-stimulating factor (M-CSF), regulated on activation in normal T-cell expressed and secreted (RANTES) and tissue inhibitor of metalloproteinases 1 (TIMP-1). GO analysis showed that the differential cytokines were mainly involved in cellular interaction, signaling pathways and reactions to stimuli. KEGG analysis revealed that the signaling pathways of these cytokines were mainly responsible for interactions between cytokines or between cytokines and their receptors, such as Jak-STAT signaling pathway, chemokine signaling pathway, and tumor necrosis factor signaling pathway.Conclusion: EA can effectively treat DES by improving the symptoms, increasing tear secretion and extending BUT, which is possibly related to its regulation on the conjunctival cytokine expressions.

The effect of RHIZOMA COPTIDIS and COPTIS CHINENSIS aqueous extract on radiation-induced skin injury in a rat model.

BACKGROUND: Radiation-induced skin injury is a common complication of radiotherapy. The RHIZOMA COPTIDIS and COPTIS CHINENSIS aqueous extract (RCE) can ameliorate radiation-induced skin injury in our clinical observation. But, the protective mechanism of RHIZOMA COPTIDIS and COPTIS CHINENSIS in radiation-induced skin injury remains unclear. METHODS: In this experiment, we developed a radiation-induced skin injury rat model to study the mechanism. The animals were randomly divided into control group, treatment group, radiation group, and treatment and radiation group. 5 rats in each group were separately executed on 2 d and 49 d post-radiation. The semi-quantitative skin injury score was used to measure skin reactions by unblinded observers, and hematoxylin and eosin staining was used to evaluate the damage areas by irradiation. The MDA content, SOD activity of skin and serum were measured to detect the oxidative stress. RESULTS: Acute skin reactions were caused by a single dose of 45 Gy of ß-ray irradiation, and the skin injury could be found in all rats receiving irradiation based on the observation of HE staining of skin at different time-points, while RCE could significantly ameliorate those changes. The MDA content in serum and skin of control rats was 4.13±0.12 mmol/ml and 4.95±0.35 mmol/mgprot on 2 d post-radiation. The rats receiving radiation showed an increased content of MDA (5.54±0.21 mmol/ml and 7.10±0.32 mmol/mgprot), while it was 4.57±0.21 mmol/ml and 5.95±0.24 mmol/mgprot after treated with RCE (p<0.05). Similar changes of the MDA content could be seen on 49 d post-radiation. However, the SOD activity of rats receiving radiation decreased compared with control group on both time-points, which was inhibited by RCE (p<0.05). Meanwhile, no valuable changes could be found between control group and treatment group on 2 d and 49 d. CONCLUSIONS: Our study provides evidences for the radioprotective role of RCE against radiation-induced skin damage in rats by modulating oxidative stress in skin, which may be a useful therapy for radiation-induced skin injury.

[Inhibitory effects of Scutellaria barbate extracts on diethylnitrosamine-induced hepatocarcinoma in rats].

OBJECTIVE: To investigate the inhibitory effects of Scutellaria barbate extracts on diethylnitrosamine-induced hepatocarcinoma in rats. METHODS: Hepatocarcinoma model rats were induced by diethylnitrosamine (DEN). Sixty SD male rats were randomly divided into 4 groups: normal control group, hepatocarcinoma model group, ESB of high dose group and ESB of low dose group. All rats were killed in the 18th week, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), alkaline phosphatase (ALP), gamma-glutamyltransferase (gamma-GT) and alpha-L-fucosidase (AFU) in serum were measured by biochemical examinations; Hematoxy and eosin (HE) methods were used to examine the changes of liver pathology. RESULTS: The levels of ALT, AST, TBIL, ALP, gamma-GT, AFU in hepatocarcinoma model group and ESB groups were higher than that of control group (P < 0.05). ESB could relieve hepatic injures. The levels of liver function indexes in ESB groups were lower than that of model group. Histological examination demonstrated that the number of liver cancer nodus in ESB groups were lower than that of model group. Furthermore, ESB could attenuate the grade of cancer cell differentiation. CONCLUSION: ESB could inhibit experimental hepatocarcinoma and relieve hepatic injures in rats.

Matrine induces apoptosis in gastric carcinoma cells via alteration of Fas/FasL and activation of caspase-3.

AIM OF THE STUDY: Matrine, an alkaloid purified from the chinese herb Sophora flavescens Ait, is well known to possess activities including anti-inflammation, anti-fibrotic and anticancer. In this study, the mechanism of matrine inducing the apoptosis of gastric carcinoma cells was investigated. MATERIALS AND METHODS: Proliferation of SGC-7901 cells was examined by MTT assay. Cellular morphology was observed under transmission electron microscope. Flow cytometry (FCM) was used to observe the apoptosis of SGC-7901 cells by staining with annexinV-FITC/PI. The expression levels of Fas/FasL in SGC-7901 cells were monitored by FCM analysis using an indirect immunofluorescence method. Activity of caspase-3 enzyme was measured by spectrofluorometry. RESULTS: MTT assay showed that matrine inhibited SGC-7901 cells proliferation in a dose-dependent and time-dependent manner. Apoptosis induction was demonstrated by morphological changes under electron microscope and FCM analysis. Fluorescence intensity levels of Fas and FasL were found to be equally up-regulated after matrine treatment, which were both correlated with apoptosis rate. The activity of caspase-3 enzyme increased in matrine groups, positively correlated with apoptosis rate. CONCLUSIONS: Matrine could inhibit cell proliferation and induce apoptosis of SGC-7901 cells in vitro. The apoptosis induction appears to proceed by up-regulating Fas/FasL expression and activating caspase-3 enzyme.

Stealth tanshinone IIA-loaded solid lipid nanoparticles: effects of poloxamer 188 coating on in vitro phagocytosis and in vivo pharmacokinetics in rats.

Stealth tanshinone IIA-loaded solid lipid nanoparticles (TA-SSLNs) have been prepared and the influence of poloxamer 188 coating on in vitro phagocytosis and in vivo pharmacokinetics in rats were evaluated. TA-SSLNs have been prepared by a nanoprecipitation/solvent diffusion method. Poloxamer 188 was used as a stealth agent. The physicochemical parameters of TA-SSLNs were characterized in terms of particle size, zeta potential, transmission electron microscopy and stability. In vitro, phagocytosis was investigated by incubating TA-SSLNs and non-stealth tanshinone IIA-loaded solid lipid nanoparticles (TA-NSLNs) with murine macrophages. In vivo, pharmacokinetics of TA-SSLNs and TA-NSLNs after a single dose intravenous injection to rat has been studied. The control was tanshinone IIA solution (TA-SOL). The results showed that TA-SSLNs had an average diameter of (91.3 +/- 3.4) nm, zeta potential of (-19.7 +/- 1.6) mV, drug loading of (4.7 +/- 0.5) % and entrapment efficiency of (92.5 +/- 2.1) %. Phagocytosis studies showed significant differences between TA-SSLNs and TA-NSLNs and demonstrated that the poloxamer 188 coating could decrease the macrophage uptake. In vivo experiments showed that the plasma concentration data of TA-SSLNs, TA-NSLNs and TA-SOL were all fitted to a two-compartment model. Areas under curve (AUCs) of TA-NSLNs and TA-SSLNs were 1.28 and 3.70 times than that of TA-SOL, respectively. TA-SSLNs had generated a long circulating time in blood with a mean residence time (MRT) of 5.286 h, compared to 3.051 h of TA-NSLNs and 0.820 h of TA-SOL. Poloxamer 188 modification on solid lipid nanoparticles (SLNs) reduced opsonization by serum proteins and the macrophage uptake. AUC of tanshinone IIA increased as a function of SLNs. In addition, TA-SSLNs exhibited much longer circulation lifetimes for tanshinone IIA than TA-NSLNs. The pharmacokinetic behavior of the incorporated drug can be modified by changing the surface characteristics of SLNs with the use of poloxamer 188.

Scutellaria barbate extract induces apoptosis of hepatoma H22 cells via the mitochondrial pathway involving caspase-3.

AIM: To study the growth inhibitory and apoptotic effects of Scutellaria barbata D.Don (S. barbata) and to determine the underlying mechanism of its antitumor activity in mouse liver cancer cell line H22. METHODS: Proliferation of H22 cells was examined by MTT assay. Cellular morphology of PC-2 cells was observed under fluorescence microscope and transmission electron microscope (EM). Mitochondrial transmembrane potential was determined under laser scanning confocal microscope (LSCM) with rhodamine 123 staining. Flow cytometry was performed to analyze the cell cycle of H22 cells with propidium iodide staining. Protein level of cytochrome C and caspase-3 was measured by semi-quantitive RT-PCR and Western blot analysis. Activity of caspase-3 enzyme was measured by spectrofluorometry. RESULTS: MTT assay showed that extracts from S. barbata (ESB) could inhibit the proliferation of H22 cells in a time-dependent manner. Among the various phases of cell cycle, the percentage of cells in S phase was significantly decreased, while the percentage of cells in G(1) phase was increased. Flow cytometry assay also showed that ESB had a positive effect on apoptosis. Typical apoptotic morphologies such as condensation and fragmentation of nuclei and blebbing membrane of apoptotic cells could be observed under transmission electron microscope and fluorescence microscope. To further investige the molecular mechanism behind ESB-induced apoptosis, ESB-treated cells rapidly lost their mitochondrial transmembrane potential, released mitochondrial cytochrome C into cytosol, and induced caspase-3 activity in a dose-dependent manner. CONCLUSION: ESB can effectively inhibit the proliferation and induce apoptosis of H22 cells involving loss of mitochondrial transmembrane potential, release of cytochrome C, and activation of caspase-3.

[Antitumor and immune-modulating effects of Scutellaria barbata extract in mice bearing hepatocarcinoma H22 cells-derived tumor].

OBJECTIVE: To investigate the effects of Scutellaria barbata extract (ESB) in suppressing tumor growth and modulating the immune functions in mice bearing tumors derived from hepatocarcinoma H22 cells. METHODS: Fifty mice inoculated subcutaneously with H22 cells were equally divided into the model group, high-, moderate-, and low-dose ESB groups, and 5-Fu group, with corresponding treatments for 10 days. Another 10 mice with only saline injection served as the normal control group. The body weight, tumor mass, thymus index and spleen index of the mice were measured, and the lymphocyte proliferation activity, NK cell activity and interleukin-2 (IL-2) production by the splenocytes were detected. RESULTS: Moderate- and high-dose ESB significantly suppressed the tumor growth with tumor inhibition rate of 28.68% and 36.98%, respectively. ESB treatment at moderate and high doses significantly increased the thymus index and spleen index (P < 0.01), which were decreased significantly in 5-Fu group. The lymphocyte proliferation activity, NK cell activity and IL-2 production by the splenocytes were significantly lower in the model group than in the normal group (P < 0.05). Compared with the model group, ESB at the high dose obviously increased the three indexes above mentioned. The NK cell activity was also significantly improved in moderate-dose ESB group (P < 0.05). CONCLUSION: ESB can suppress the growth of H22 implant tumor and enhance the immune function of the tumor-bearing mice.

[Anti-proliferative and apoptosis-inducing activity of Scutellaria barbate containing serum on mouse's hepatoma H22 cells].

OBJECTIVE: To investigate the effects of Scutellaria barbate extract (ESB) on suppressing proliferation and inducing apoptosis of mouse hepatoma H22 cells. METHODS: H22 cells cultured in vitro were divided into 5 groups: blank control group, ESB in high, medium, low dose groups and 5-Fu group. H22 cells were cultured in media with serum containing different concentrations of ESB and blank serum. The proliferation of H22 cells was determined by microculture tetrazolium (MTT) assay. Fluorescence microscopy was utilized to observe the apoptosis of H22 cells by staining with Hoechst 33258. The cell cycle and apoptosis were analyzed by flow cytometry (FCM). RESULTS: The inhibition of serum containing ESB on the proliferation of H22 cells in vitro was observerd in a dose and time dependent manner. The typical morphological changes of apoptosis were observed after incubation with ESB-containing serum in high dose for 48 hours. Among the various phases of cell cycle, the percentage of cells in S phase decreased significantly, while the percentage of cells in G1 phase increased. Drug-containing serum showed positive effect on cell apoptosis. The apoptosis rate of blank control group, ESB in low, medium, high dose groups and 5-Fu group were 0.51%, 1.07%, 3.15%, 7.83%, 11.26%, respectively. CONCLUSION: ESB containing serum can inhibit proliferation and induce apoptosis of H22 cells in vitro.

[Effects of Scutellaria Barbata drug-containing serum on apoptosis and mitochondrial transmembrane potential of hepatoma H22 cells].

OBJECTIVE: To investigate the effects of serum containing Scutellaria Barbata extract (ESB) on apoptosis rate and mitochondrial transmembrane potential (MTP) of liver cancer cell line H22 from mice in vitro. METHODS: H22 cells were cultured in vitro and divided into 5 groups: blank control group, low-dose ESB group, medium-dose ESB group, high-dose ESB group and fluorouracil (5-Fu) group. Methyl thiazolyl tetrazolium assay was utilized to determine the proliferation rates of H22 cells. Cellular morphology was observed under a transmission electron microscope (EM). The rhodamine 123 was used as a fluorescence probe to label the H22 cells, and the fluorescence intensities were observed with a laser scanning confocal microscope. The fluorescence intensity of H22 cells indicated the MTP of H22 cells. RESULTS: The inhibition of serum containing ESB on the proliferation of H22 cells in vitro was observed in a time-dependent manner. The typical morphological changes of apoptosis were observed after incubation with ESB-containing serum in high dose for 48h. The apoptosis rates of blank control group, 5-Fu group, low-dose ESB group, medium-dose ESB group and high-dose ESB group were (0.51+/-0.32)%, (11.26+/-2.97)%, (1.07+/-0.46)%, (3.15+/-1.12)%, (7.83+/-2.25)% respectively. ESB could reduce the MTP of H22 cells from mice as compared with the untreated group. The MTPs of the blank control group, 5-Fu group, and low-, medium- and high-dose ESB groups were (245.45+/-67.37), (127.42+/-41.35), (213.68+/-65.52), (186.34+/-56.37) and (142.65+/-39.44) respectively, which were negatively correlated with the apoptosis rates. CONCLUSION: ESB-containing serum effectively induces apoptosis, which may be related to the decrease of MTP in H22 cells.

[Effect of matrine injections on invasion and metastasis of gastric carcinoma SGC-7901 cells in vitro].

OBJECTIVE: To investigate the effect of matrine injections on invasion and metastasis of gastric carcinoma SGC-7901 cells in vitro. METHODS: MTT assay was used to examine the effect of matrine injections on proliferation of SGC-7901 cells after 24, 48, 72, 96 hours treatment; Transwell chamber assay was performed to determine the effect of matrine injection on invasion and migratory capacity of the cells; Effect on adhesion potential of SGC-7901 cells was tested by cell matrigel adhesion assay. RESULTS: Matrine injec-tions could inhibit the proliferation of SGC-7901 cells, with obvious dose-dependent and time-dependent effects. Matrine injections sig-nificantly inhibited adhesion, invasion and migration capacity of SGC-7901 cells in vitro. The inhibitory rate of them after treatment with 1.5 mg/ml matrine injections for 24 h were (45.32 +/- 3.10)%, (32.66 +/- 2.82)%, (38.35 +/- 3.41)% respectively. After treat-ment of matrine injections (1.5 mg/ml)for 24 h, the expression level of CD44(V6) in SGC-7901 cells was decreased compared with the untreated group. CONCLUSION: Matrine injections can inhibit the migration, invasion and adhesion capacity of SGC-7901 cells in vitro. The inhibition effect may be related to down-regulating the expression of CD44(V6) protein.