Isolation, physicochemical properties and immunomodulating effects of polysaccharides from Sophorae Flavescentis Radix / 国际药学研究杂志

Objective To investigate the physicochemical properties and immunomodulatory activities of crude polysaccharides and their fractions from Sophorae Flavescentis Radix. Methods The crude polysaccharide (SFP-100) was obtained successively by boiling Sophorae Flavescentis Radix in water, ethanol precipitating, dialyzing and freeze drying. SFP-100 was separated withDEAE-cellulose column to obtain three fractions, and these fractions were fruther separated with Sephadex G-100 column to obtain their sub-fractions. The sugar content was determined by phenol-sulfuric acid method, the molecular distribution was determined with gel filtration chromatography, and the monosaccharide composition was analyzed with capillary electrophoresis after PMP derivatization. The immunobiological activities were estimated by measuring the proliferation of mouse spleen lymphocytes as well as the IFN-secretion in mouse splenocytes and the TNF-α secretion in mouse peritoneal macrophages. Results The yield of SFP-100 from Sophorae Flavescentis Radix was 4.83％ and sugar content was 71.62％. SFP-100 was separated into three fractions SFP-100-A, SFP-100-B and SFP-100-C, whose yields were 3.5％, 25.6％ and 16.7％, and the sugar content was 85.99％, 72.09％ and 24.30％, respectively.The monosaccharide composition and their molar ratio for SFP-100-A, SFP-100-B and SFP-100-C were Ara∶Glc∶Gal=7.16∶91.02∶1.82, Xyl∶Ara∶Glc∶Rha∶Gal∶GalA=0.05∶1.00∶0.85∶0.04∶0.35∶0.43, and Xyl∶Ara∶Glc∶Rha∶Gal∶GlcA∶GalA=0.20∶1.00∶0.33∶0.36∶0.45∶0.55∶14.37, respectively. SFP-100-B was further separated into two sub-fractions SFP-100-B-a and SFP-100-B-b with Sephadex G-100, and the other two sub-fractions SFP-100-C-a and SFP-100-C-b were also obtained with the same Sephadex G-100 from SFP-100-C. SFP-100-B-a showed a main wide peak, which had the relative molecular weight 1.02×105 and monosaccharide composition Ara∶Glc∶Rha∶Gal = 1.00∶0.06∶0.02∶0.29. Meanwhile, the relative molecular weight and monosaccharide composition were 5.43×104 and Xyl∶Glc =1.00∶3.81 for the main peak of SFP-100-C-a, and 2.75×104 and Ara∶Glc∶Rha∶Gal∶GlcA∶GalA=1.00∶2.10∶0.57∶0.74∶1.09∶33.75 for the main peak of SFP-100-C-b, respectively. The crude polysaccharides SFP-100 and its fractions, SFP-100-B and SFP-100-C, as well as the sub-fractions, SFP-100-B-a, SFP-100-B-b and SFP-100-C-a, increased the proliferation of spleen cells, all in a dose-dependent manner. Further, SFP-100 could improve the secretion of IFN-γ and TNF-α in spleen cells, while SFP-100-B, SFP-100-C, SFP-100-B-a and SFP-100-B-b could stimulate the secretion of IFN-γ. Conclusion The crude polysaccharides of Sophorae Flavescentis Radix and their fractions showed a good immunomodulatory activity, which may be related to the clinical use of Sophorae Flavescentis Radix for the anti-HBV and anti-inflammatory therapy.

Research on HBV DNA inhibition of plasmid acute infection mouse with betulinic acid / 中国中药杂志

Betulinic acid is a naturally occurring pentacyclic triterpenoid, which has antiretroviral, antimalarial, and anti-inflammatory properties. The purpose of this study is to investigate the HBV DNA replication inhibition in the mouse model with betulinic acid. Hydrodynamic injection method via the tail vein with the Paywl. 3 plasmid was used to establish the animal mode (n = 15), and the mice were randomly divided into the PBS control group (n = 5), Betulinic acid treatment group (n = 5) and lamivudine control group (n = 5). The day after successful modeling , the mice would have taken Betulinic acid (100 mg x kg(-1)), lamivudine (50 mg x kg(-1)), PBS drugs orally, once daily for 7 days, blood samples were acquired from the orbital venous blood at 3, 5, 7 days after the administering, HBsAg and HBeAg in serum concentration were measured by ELISA and the mice were sacrificed after 7 days, HBV DNA southern detections were used with part of mice livers. The results showed that betulinic acid significantly inhibited the expression of HbsAg in the mice model at the fifth day compared with the control group, and there was no significant differences between the effects of lamivudine and the PBS control group; both the betulinic acid and lamivudine groups had no significant inhibition for the HBeAg expression; the HBV DNA expressions of the liver tissue from the betulinic acid and lamivudine groups were inhibited compared with the control group. Taken together, these results reveal betulinic acid can inhibit the HBsAg expression and replication of the liver HBV DNA in the mouse model.

Characteristic of liver pathology in HBeAg-positive and HBeAg-negative chronic hepatitis B patients with mildly elevated ALT / 中华肝脏病杂志

<p><b>UNLABELLED</b>To analyse the live pathology characteristics in mild ALT-elevated (1 x ULN less than ALT less than 2 x ULN ) HBeAg-positive and HBeAg-negative chronic hepatitis B (CHB) patients, and to explore the influence of the age and HBV DNA level to liver pathology in different HBeAg status patients.</p><p><b>METHODS</b>All the patients who met the inclusion criteria form "eleventh five-year plan" National Science and Technology Major Project, the treatment program of integrative traditional and western medicine for CHB were enrolled in this study between October 2009 and March 2011 .B type ultrasound-guided liver biopsy was carried out in all patients and hepatitis B surface antigen (HBsAg) , HBeAg titer as well as HBV DNA level were detected at the same time. Hepatic tissue inflammation and fibrosis degree of patients according to HBeAg-positive and negative, age ( more than or equal to 40 years and less than 40 years), HBV DNA level (more than or equal to 10

Expression characteristics of some immune effector molecules in CD8+ T lymphocytes from patients with chronic hepatitis B / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expressions of perforin (PF), granzyme B (GrB), granulysin (GNLY), TNF-alpha and IFN-gamma in peripheral CD8+ T lymphocytes and their correlation to infection status in patients with chronic hepatitis B (CHB).</p><p><b>METHODS</b>ALT, AST, TB and HBV DNA copy were detected to evaluate the infection status in CHB patients, with healthy volunteers serving as the control group. According to the infection status, the CHB patients were divided into 4 groups, namely normal hepatic function and high HBV DNA level group, normal hepatic function and low HBV DNA level group, abnormal hepatic function and high HBV DNA level group and abnormal hepatic function and low HBV DNA level group. The expressions of some immune effector molecules in CD8+T cells were detected by flow cytometry, and the correlations between these immune effector molecules and the infection status were analyzed.</p><p><b>RESULTS</b>The expressions of GrB, TNF-alpha and IFN-gamma in normal hepatic function and low HBV DNA level group were significantly higher than those in abnormal hepatic function and high HBV DNA level group (P<0.05). The expression of IFN-gamma in normal hepatic function and high HBV DNA level group was significantly higher than that in abnormal hepatic function and high HBV DNA level group (P<0.05). The expressions of PF and GNLY were similar among all the 4 groups. Positive correlations were noted between GrB, PF, GNLY, TNF-alpha and IFN-gamma.</p><p><b>CONCLUSION</b>GrB, TNF-alpha and IFN-gamma in peripheral CD8+ T cells are inversely correlated to hepatic dysfunction and HBV DNA level in CHB patients.</p>

Effect of bushen recipe on the immune effector molecules of natural killer cells in patients with chronic hepatitis B / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the immunoregulation mechanism of Bushen Recipe (BSR) in patients with chronic hepatitis B (CHB) of Gan-Shen yin-deficiency and lingering damp-heat syndrome (GSS).</p><p><b>METHODS</b>Thirty-five patients with positive HBV DNA and abnormal alanine transaminase (ALT) level were assigned to the treatment group (22 patients) and the control group (13 patients), they were treated with BSR and alpha-2b interferon for 6 months respectively. Blood levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and HBV DNA were measured before and after treatment. And the expressions of immune effector molecules of nature killer (NK) cell, including perforin (PF), granzyme B (GrB), granulysin (GNLY), tumor necrosis factor-alpha (TNF-alpha) and gamma-interferon (gamma-IFN), were detected using flow cytometry.</p><p><b>RESULTS</b>Levels of ALT and AST declined significantly in both groups after treatment (P < 0.05 or P < 0.01), showing insignificant difference between them. And the expressions (%) of PF and GNLY in the treatment group reduced significantly after treatment, from 69.62 +/- 27.58 to 34.86 +/- 31.60 for PF and from 64.54 +/- 25.96 to 25.72 +/- 24.98 for GNLY (both P < 0.05). In the treatment group and the control group, as compared with before treatment, the total scores of Chinese medicine symptoms were significantly declined after treatment (P < 0.01), and the total scores of Chinese medicine symtoms in the treatment group was significantly lower than that of the control group (P < 0.05). As compared with the total effective rate of Chinese medicine syndromes in the control group after treatment, that in the treatment group was significantly increased (P < 0.05).</p><p><b>CONCLUSION</b>The mechanism of BSR in immuneregulating on patients with CHB of GSS is by way of declining the expressions of relative immune effector molecules to promote the recovery of the damaged liver.</p>

Curative effect of Yidu Recipe in treating chronic hepatitis B patients of gan-shen yin-deficiency and damp-heat syndrome type and its influence of T-cell subsets / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of Yidu Recipe (YDR) in treating patients of chronic hepatitis B (CHB) with positive hepatitis B e-antigen (HBeAg) and its influence on the quantity and function of T-cell subsets.</p><p><b>METHODS</b>Fifty-seven CHB patients measured up the inclusive criteria were randomly assigned to the control group and the treated group, treated respectively by entecavir alone and entecavir + YDR for 6 months. Changes of alanine a minotransferase (ALT), aspartate a minotransferase (AST), HBV-DNA, HBV-M, interleukin-4 (IL-4) and Chinese medicine syndrome score, as well as amounts of natural killer (NK) T cell, gamma-interferon (gamma-IFN), Th1, Th2, Tc1 and Tc2 cells in peripheral blood (detected by flow cytometry) before and after treatment were observed. And the liver function normalization rate, negative inversion rates of HBV-DNA and HBeAg were estimated at terminal of the trial.</p><p><b>RESULTS</b>Seven cases were dropped out in the observation period. Compared with the control group, levels of ALT, AST, HBV-DNA and Chinese medicine syndrome score were lower after treatment (P < 0.05), and liver function normalization rate was higher in the treated group, while the difference between groups in negative inversion rates HBV-DNA and HBeAg were insignificant (P > 0.05). Amount of IFN-gamma increased, IL-4 reduced, and Tc1 cell raised after treatment, which led to the rise of Tcl/Tc2 ratio in both groups; while in the treated group, in addition to the above-mentioned changes, the Th1 cell was increased also, and thus to make elevation of Th1/Th2 ratio (P < 0.05).</p><p><b>CONCLUSION</b>The efficacy of entecavir + YDR in treating HBeAg positive CHB patients is better than that of entecavir alone. YDR can effectively improve patients' liver function, inhibit HBV-DNA replication and improve clinical symptoms, its action may be realized by way of increasing the amount of NKT cells, inducing increase of IFN-gamma and decrease of IL-4 secretions, and regulating the balance between Th1/Th2 and Tc1/Tc2.</p>

Expression profile of immune effector molecules in natural killer cells in patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the expression profile of immune effector molecules in peripheral natural killer cells (NK) in patients with chronic hepatitis virus B.</p><p><b>METHODS</b>According to the infection status, patients were divided into four experiment groups: normal hepatic function and high HBV DNA level group, normal hepatic function and low HBV DNA level group, abnormal hepatic function and high HBV DNA level group and abnormal hepatic function and low HBV DNA level group. The expression of perforin (PF), granzyme B (Gr B), granulysin (GNLY), tumor necrosis factor alpha (TNFa) and interferon gamma (IFNr) in NK cells were detected by flow cytometer.</p><p><b>RESULTS</b>Compared with control group (31.50%+/-27.64%), the expression of GNLY was significantly increased in normal hepatic function and high HBV DNA level group (59.74%+/-30.82%) and normal hepatic function and low HBV DNA level group (61.89%+/-33.30%); the expression of IFNr in normal hepatic function and high HBV DNA level group (39.89%+/-21.30%) and abnormal hepatic function and high HBV DNA level group (37.54%+/-18.79%) was lower than that in normal control group (57.38%+/-23.69%); the expression of PF, GrB, GNLY in abnormal hepatic function and high HBV DNA level group (35.47%+/-29.64%, 66.55%+/-22.92%, 42.03%+/-33.17%) was lower than that in normal hepatic function and high HBV DNA level group (56.98%+/-38.34%, 81.53%+/-19.58%, 59.74%+/-30.82%) and normal hepatic function and low HBV DNA level groups (62.95%+/-31.98%, 84.51%+/-14.57%, 61.89%+/-33.3%); there were positive correlations between ef PF, Gr B, GNLY, TNFa, and IFNr.</p><p><b>CONCLUSION</b>The expression of IFNr in NK cells from patients with high HBV DNA replication level is lower than that in normal control group; the expression of PF, Gr B and GNLY in NK cells from patients with normal hepatic function is higher than that in NK cells from patients with abnormal hepatic function.</p>

Effect of spleen-invigorating prescription on dendritic cell function in patients with chronic hepatitis B of TCM Pi-deficiency syndrome type / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the effect of Spleen-invigorating Prescription (SIP) on dendritic cell function in patients with chronic hepatitis B.</p><p><b>METHODS</b>A total of 60 patients with chronic HBV of Pi-deficiency syndrome type were enrolled and randomized to 2 groups, 30 in each group. Patients in the control group were given intramuscular injection with human interferon alpha 1b, 3 times a week, while those in the treated group were given orally with SIP twice a day, the therapy lasted for 6 months. Dendritic cells (DCs) were isolated from peripheral blood and cultured, then the expression of surface markers, HLA-DR, CD86, CD80, CD40, CD14 and CD11c were detected before and after treatment by flow cytometry, and the function of DCs was also evaluated by mixed lymphocyte reaction (MLR) determination once before treatment and once after treatment.</p><p><b>RESULTS</b>The expressions of DCs' surface CD86, CD80, CD40 and CD11c in the treated group were higher (P < 0.05, P < 0.01) and the changes of stimulating index, IFN-gamma and IL-12 were superior in the treated group to those in the control group (P < 0.05).</p><p><b>CONCLUSIONS</b>SIP can significantly improve DC's function, so, one of mechanisms of SIP in improving clinical efficacy may be the regulation of immune function.</p>

Epidemic survey on TCM syndrome typing and preliminary discussion on TCM etiology and pathogenesis of fatty liver / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the common TCM syndrome types of fatty liver by way of epidemic questionnaire, their occurrence ratio, and the correlation between various syndrome types and objective indexes.</p><p><b>METHODS</b>A total of national wide 503 subjects with fatty liver were enrolled, the TCM syndromes, body mass index (BMI), abdominal perimeter/hip circumference, liver function, blood lipids, B ultrasonic examination and CT in them were observed and recorded.</p><p><b>RESULTS</b>In the 46 symptoms investigated in total, the first ten symptoms in order of appearing rate were lassitude, obese, oral dryness, vertigo, hypochondriac distending pain, soreness and pain in loin, spiritlessness, oral bitterness, aching and weakness in knee and abdominal distention. The mostly appeared tongue figures were pale and corpulent or pale dim tongue proper, white greasy or yellow greasy tongue coating, and the mostly appeared pulse figures were taut, taut-thin and taut slippery. Statistical cluster analysis showed that syndromes of fatty liver could be typed into 4 TCM types, the asthenia Pi-Shen with Gan-stagnation type, the asthenia Pi-Shen type, the asthenia Pi with phlegm-heat type and the unclassified type. Among them the asthenia Pi-Shen with Gan-stagnation type was the commonest one, which accounted to 62.32%.</p><p><b>CONCLUSION</b>The mostly appeared syndrome type of fatty liver was asthenia Pi-Shen with Gan-stagnation type. The TCM pathogenesis of fatty liver was deficiency of origin, mainly deficiency of Shen, involving Pi, with excess superficiality, the turbid-phlegm and blood stasis.</p>

Effect of Bushen recipe on T-cell subsets and their function in patients with chronic hepatitis B / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the regulatory effect of Bushen recipe (BSR) on immune function in patients with chronic hepatitis B.</p><p><b>METHODS</b>Thirty patients with HBeAg+ and abnormal alanine transaminase (ALT) level were treated with BSR for 6 months. The following parameters before treatment, 3 months and 6 months after treatment were tested: (1) HBV DNA by fluorescence quanitiative PCR; (2) Levels of interleukin-10 (IL-10) and interferon-gamma in supernatant of cultured peripheral blood mononuclear cell (PBMC) after had been cultured with HBeAg, HBcAg and phytohemagglutinin (PHA) for 48 hrs; (3) Blood levels of T-cells, including CD4+ , CD8+ , CD8+ CD28+ , CD8+ CD28- and CD28+ , and their ratio, as well as the percentage of CD8+ CD28+ in the cell culture after being co-cultured with PHA for 48 hrs.</p><p><b>RESULTS</b>In patients effectively treated by BSR, after 3 months treatment, level of IFN-gamma in PBMC culture significantly increased, CD8+ CD28+ , CD28+ T-cells significantly enhanced and IL-10 and CD8 + CD28+ T-cells significantly decreased (P < 0.05, P < 0.01).</p><p><b>CONCLUSION</b>BSR could significantly strengthen the expression of Th1 type cytokine, decrease the expression of Th2 type cytokine and promote the expression of CD8+ cytotoxic T-lymphocyte, which may be one of mechanisms in suppressing HBV DNA replication.</p>