Effect of Shaoyao Tang on ulcerative colitis in rats via regulation of TLR4/NF-κB signal pathway / 中国中药杂志

The aim of this paper was to investigate the effect of Shaoyao Tang on ulcerative colitis(UC) in rats via regulation of TLR4/NF-κB signal pathway. A total of 56 Wistar rats were randomly divided into 6 groups: normal control group(double distilled water), model group(double distilled water), mesalazine group(10 mL·kg~(-1)), high dose, middle dose and low dose Shaoyao Tang groups(2.4, 1.2, and 0.6 g·mL~(-1)). After UC rat models were established by 2, 4-dinitrochlorobenzene(DNCB)/ethanol enema, the rats received double distilled water or corresponding drugs twice a day for 7 days. After the treatment cycle, the general performance and disease activity index(DAI) of rats were observed on the next day. Then the rats were sacrificed. The length of colon was measured. Macroscopic and histological score of colon were evaluated. Histopathological changes of colon were observed by HE staining. Ultraviolet spectrophotometry detection was used to detect the content of myeloperoxidase(MPO) in blood and colon tissues. The levels of P-selectin, macrophage migration inhibitory factor(MIF) and thromboxane B_2(TXB_2) in blood and colon tissues were determined by ELISA. Immunohistochemistry and Western blot analysis were performed to detect the protein expressions of TLR4 and NF-κB in colon tissues. The results showed that as compared with the model group, Shaoyao Tang of different doses improved the general performance of UC rats. Moreover, high-dose Shaoyao Tang group showed the most obvious effect in scoring of disease activity index(P<0.001); both medium and high doses of Shaoyao Tang significantly inhibited the colon shortening and pathological injury, with significantly decreased expression levels of MPO, P-selectin, MIF and TXB_(2 )in serum and colon tissues of UC rats(P<0.001). Immunohistochemistry and Western blot assay showed that the levels of TLR4 and NF-κB protein expression in the colon tissues of Shaoyao Tang high-dose group were remarkably lower than that in the model group(P<0.001). This study shows that Shaoyao Tang has protective and repairing effects on UC, and its possible mechanism is achieved probably by regulating the TLR4/NF-κB pathway and inhibiting the expressions of MPO, P-selectin, MIF and TXB_2.

Efficacy and Immune Mechanism of Baitouweng Tang on Ulcerative Colitis in Rats / 中国实验方剂学杂志

Objective: To investigate the therapeutic effect and immune mechanism of Baitouweng Tang on ulcerative colitis(UC) rats. Method: The mode of UC rats was made of 2, 4-dinitrochlorobenzene (DNCB)/ethanol enema. Rats were randomly divided into control group, model group, mesalazine group, and high-dose, middle-dose and low-dose Baitouweng Tang groups. The mesalazine group were administered with mesalazine (0.5 g·kg-1). Baitouweng Tang groups were given Baitouweng Tang (10, 5, 2.5 g·kg-1), while the other groups were given double steaming water. After 7 days of continuous administration, the general condition and disease activity index of rats in each group were observed. After anesthesia in rats, blood was taken from the abdominal aorta. Then the rats were put to death, and the length and morphological observation of the colon were measured. Ultraviolet spectrophotometry detection was used to detect the activities of myeloperoxidase (MPO) in blood and colon tissue. The levels of P-selectin, macrophage migration inhibitory factor (MIF) and thromboxane B2 (TXB2) in blood and colon tissues were detected by enzyme-linked immunosorbent assay(ELISA). Immunohistochemistry and Western blot methods were undertaken to determine the expressions of Toll-like receptor 4 (TLR4) and nuclear transcription factor-κB (NF-κB) proteins in colon tissue. Result: Compared with the model group, the rats in model group showed severe symptoms, such as loose stools, diarrhea and bloody stools, while Baitouweng Tang obviously ameliorated them. Moreover, Baitouweng Tang significantly reduced DAI, colon general and pathological scores, which were high in model group(P2 in the serum and colon tissues of the model group were obviously increased(PκB in colons of model group were markedly higher than those in control group(PPConclusion: Baitouweng Tang could inhibit TLR4/NF-κB signaling pathway in treatment of ulcerative colitis, and reduce the expressions of P-selectin, MPO, MIF and TXB2, and thus promoting intestinal mucosal repair and improving intestinal function.

Study of isobutyrylshikonin inhibiting proliferation of colon carcinoma cells through PI3K/Akt/m-TOR pathway / 中国中药杂志

To investigate the inhibitory effect of isobutyrylshikonin on the growth of human colon carcinoma cells and its effect on the PI3K/Akt/m-TOR pathway. MTT assay was used to detect the inhibitory effect of different concentrations (0, 6.25, 12.5, 25, 50, 100 mg·L⁻¹) of isobutyrylshikonin on the proliferation of human colon carcinoma cell HT29 at 24, 48 h. CCK-8 method was used to detect the inhibitory effect of isobutyrylshikonin on HT29, HCT116, DLD-1 and Caco-2 at 48 h. AnnexinV/propidium iodide staining was applied in detecting the apoptoticrate of HT29 cells treated with different concentrations of isobutyrylshikonin at 24 h and 48 h. Cycletest plus DNA was employed to analyze HT29 apoptosis and cell cycle after 48 h treatment with isobutyrylshikonin at different concentrations. Western blot and RT-PCR assay were used to examine the protein and mRNA expressions of PI3K, p-PI3K, Akt, p-Akt and m-TOR. The results showed that isobutyrylshikonin inhibited the proliferation of different human colon carcinoma cells, and the inhibition rate was in a dose-dependent manner. Isobutyrylshikonin induced apoptosis mainly in the early stage and blocked cells in the G₀/G₁ or G₂/M phase. Isobutyrylshikonin reduced the protein expressions of PI3K, p-PI3K, Akt, p-Akt, m-TOR and the mRNA expressions of PI3K, Akt, m-TOR in a dose-dependent manner. Isobutyrylshikonin can significantly inhibit the proliferation, induce the early apoptosis and change the cycle distribution in colon carcinoma cells.This biological effect may be correlated with the inhibition of PI3K/AKT/m-TOR pathway.

Effect of baicalin on signal transduction and activating transcription factor expression in ulcerative colitis patients / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the intervention of baicalin on signal transduction and activating transcription factor expression of ulcerative colitis (UC) patients.</p><p><b>METHODS</b>Recruited were UC patients at Outpatient Department of Digestive Disease, Inpatient Department of Digestive Disease, Center for Digestive Endoscopy of College City Branch, Guangdong Provincial Hospital of Traditional Chinese Medicine, and Southern Hospital affiliated to Southern Medical University from June 2010 to January 2011. They were assigned to the UC group (33 cases) and the diarrhea-predominant irritable bowel syndrome (IBS-D) group (30 cases). Another 30 healthy subjects were recruited as a healthy control group. Peripheral blood mononuclear cells (PBMCs) in vitro intervened by different concentrations baicalin were taken from UC patients. IL23R gene expressions in vitro intervened by different concentrations baicalin were detected using Q-PCR. Expressions of signal transducer and activator of transcription 4 (STAT4) , STAT6, phosphorylated-STAT4 (p-STAT4), and p-STAT6 were detected using Western blot. Serum levels of IFN-γ, IL-4, IL-6, and IL-10 were measured by ELISA. Effects of different concentrations baicalin on expressions of PBMCs, and levels of IFN-γ, IL-4, IL-10 of UC patients were also detected.</p><p><b>RESULTS</b>Compared with the negative control group, 40 µmol baicalin obviously decreased IL23R gene expression of UC patients (P <0. 01). Compared with the healthy control group and the IBS-D group, p-STAT4/STAT4 ratios increased, p-STAT6/STAT6 ratios decreased, levels of IFN-γ, IL-4, IL-10 all increased in the US group (all P <0. 05). Compared with the negative control, 5 and 10 µmol baicalin groups, 20 and 40 moL baicalin obviously decreased p-STAT4/STAT4 ratios (all P <0. 05); 20 and 40 µmoL baicalin obviously increased p-STAT6/STAT6 ratios (all P <0. 05); 20 and 40 µmoL baicalin obviously lowered levels of IFN-γ and IL-4, and elevated IL-10 levels (all P <0. 05).</p><p><b>CONCLUSION</b>40 µmoL baicalin could in vitro inhibit p-STAT4/STAT4 ratios, adjust p-STAT6/STAT6 ratios and related cytokines, thereby balancing the immunity and relieving inflammatory reactions of UC.</p>

Soluble expression of A/H1N1 influenza virus HA with Drosophila S2 cell line and its bio-activity identification / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To express soluble HA of A/H1N1 influenza virus in drosophila S2 cell line and identify its bio-activity.</p><p><b>METHODS</b>HA gene was amplified from A/Shenzhen/71/09 virus strain using RT-PCR, then we constructed pAC5.1-HA expression vector, which was co-transfected into S2 cell with pCoblast vector. After transfection, stable S2 cell was selected through Blasticindin. HA in the supernatant was identified with Western Blot assay and purified with Ni-column. Recombinant HA was immunized into BALB/c mice 3 times, and the Abs titers were evaluated with ELISA.</p><p><b>RESULTS</b>We successfully cloned HA gene with 1.7 x 10(3) bp of A/Shenzhen/71/09 virus strain and got recombinant pAC5. 1-HA expression vector. Stable S2 cell line was established after transfection and selection, which continuously expressed HA with molecular weight 75 x 10(3) D. After immunization with HA, the Abs titers were 1:1280 and 1: 5120 respectively on 10 d, 30 d.</p><p><b>CONCLUSION</b>We expressed soluble HA with good bio-activity, which contributed to research on immune diagnosis, subunit vaccine, and monoclonal Abs for influenza.</p>

Protective effect of baicalin against LPS-induced intestinal injury / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the protective effect of baicalin on the intestinal mucosal injury caused by endotoxin-lipopolysaccharide (LPS) and the anti-oxidative injury in colonic and ileal mucosa of rats with septicopyemia.</p><p><b>METHOD</b>Fifty healthy male BALB/c mice were randomly divided into 5 groups: the normal control group, the model group, and baicalin high-dose, medium-dose and low-dose groups. They were orally administered with double distilled water, 100 mg x kg(-1) of baicalin, 50 mg x kg(-1) of baicalin, and 25 mg x kg(-1) of baicalin respectively for three days, once a day. 1 h after the oral administration on 3 d, they were intraperitoneally injected with normal saline or LPS (17 mg x kg(-1)). At 20 h after the injection of LPS, all of the mice were sacrificed, and their colonic and ileal tissues were collected. The mental status, life state and death rate of mice in each group were observed, and the lengths of colonic were measured. Chiu's scoring method was used to assess the intestinal mucosal injury. Histopathological changes of intestinal tissues were tested by HE staining. The ultraviolet spectrophotometry was used to detect total antioxidant capacity (T-AOC), superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-PX) of intestinal homogenate. The immunohistochemical method was used to analyze the expression of PCNA in intestinal tissues of each group.</p><p><b>RESULT</b>The death of mice was observed after the intraperitoneal injection of LPS. The death rates of baicalin groups were remarkably lower than the death rate of the model group. The colons in the medium-dose baicalin group were much longer than that in the model group (P < 0.05), with a much lower intestinal mucosa injury degree than the model group. Colonic and ileal injuries in the high-dose baicalin group significantly (P < 0.05). Colonic and ileal injuries in the medium-dose baicalin group and the low-dose baicalin group significantly reduced compare with the model group (P < 0.000 1). The medium-dose baicalin group showed no significant increase in homogenate's T-AOC, T-SOD and GSH-PX compare with the model group (P < 0.05). There was no significant difference between baicalin groups and the model group in PCNA.</p><p><b>CONCLUSION</b>Baicalin can protect intestinal epithelial cells suffering from injury from oxygen radicals, and relieve the intestinal injury caused by LPS by improving the intestinal mucosa structure and functions.</p>

Huangqin decoction lowers the number of mast cells in ulcerative colitis in rats with dampness and heat syndrome / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of mast cells in the pathogenesis of ulcerative colitis in rats with dampness and heat syndrome, and observe the regulatory effect of Huangqin decoction on the mast cells.</p><p><b>METHODS</b>Rat models of dampness and heat syndrome were established by feeding with high-fat and-sugar chow, maintenance of a hot and humid environment, and intrarectal administration of 2,4,6-trinitro-benzene-sulfonic acid. The model rats were then randomized into the model group (n=12), Huangqin decoction group (n=13) and mesalazine group (n=12). After a one-week treatment, the inflammatory cell infiltration was observed using HE staining, and the number of mast cells was determined using toluidine blue staining. Immunohistochemistry was used to detect the expression of tryptase, and serum IL-4 and IL-6 levels were measured using ELISA.</p><p><b>RESULTS</b>Compared with the normal control rats (n=15), the rats in the model group showed obvious inflammatory cell infiltration at the lesion site with significantly increased mast cells and serum IL-6 level (P<0.05). Huangqin and mesalazine significantly lessened inflammatory cell infiltration and decreased the mast cell number and serum IL-6 level after a one-week treatment.</p><p><b>CONCLUSION</b>The intestinal mucosal immune cells such as the mast cells play an important role in the pathogenesis of ulcerative colitis associated with dampness and heat syndrome. Huangqin decoction can ameliorate the inflammation, decrease mast cell number and tryptase release, and inhibit IL-6 secretion for treatment of ulcerative colitis in rats with dampness and heat syndrome.</p>