The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy.

OBJECTIVE: Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells. METHODS: A miR122 target (122T) sequence was incorporated into the 3' untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined. RESULTS: The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells. CONCLUSION: HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.

Prevalence of neutralizing antibodies against liver-tropic adeno-associated virus serotype vectors in 100 healthy Chinese and its potential relation to body constitutions.

Recombinant adeno-associated virus (rAAV) serotype 2, 3 and 8 vectors are the most promising liver-tropic AAV serotype vectors. Liver diseases are significant problems in China. However, to date, few studies on AAV neutralizing antibodies (Nabs) were working with the Chinese population or with the rAAV3 vectors. The present study aimed to determine the prevalence of Nabs in Chinese population against wild-type AAV2, AAV3 and AAV8 capsids as well as additional two AAV3 variants. In addition, we performed a preliminary analysis to investigate the potential influence of traditional Chinese medicine body constitutions on AAV Nabs. Our work demonstrated that the majority of healthy Chinese subjects were positive for AAV Nabs, with the order of AAV2>AAV3=AAVLK03>AAV8. There was no difference between: 1) AAV3 and its variants; 2) male and female subjects; and 3) different age cohorts (≤35, 36-50, and ≥51 years old). People in the Qi-deficiency constitution had significantly increased AAV8 Nabs than people in the Gentleness constitution. Our studies may have impact on the future clinical design of AAV-based gene therapy in the Chinese population.

The investigation of the correlation between metabolic syndrome and Chinese medicine constitution types in senior retired military personnel of the People's Liberation Army.

OBJECTIVE: To investigate the incidence of metabolic syndrome (MS) and the distribution of constitutional pattern in elderly retired personnel of the People's Liberation Army. METHODS: Adopting the method of cross-sectional field investigation, from June to December in 2008, the investigation questionnaires were completed by the aged over 60 and collected from 69 military retired residences in the 4 cities of Shanghai, Nanjing, Hangzhou and Qingdao. Other data, including demographic characteristics, physiological characteristics, life style and former medical history, were collected and analyzed. The statistical analysis for the database was drawn up by the software Epidata 3.0. RESULTS: A total of 4,502 people were included in this study, and 35.3% of them were diagnosed with MS. There was no obvious difference in mobility among ages (60 to 69, 70 to 79 and over 80, P>0.05). Referring to the MS patients in the 70s age group, both the phlegm-dampness and dampness-heat constitutional types were evidently higher than those in the 60s age group (P=0.019, P=0.008); while MS patients in 80s and older showed a significantly lower incidence of dampness-heat constitution than those in the 60s (P=0.00); and ql-deficiency constitution was obviously higher in the 80s age group than those in the other two groups (P=0.00). The top 3 constitutions in MS people were, respectively, phlegm-dampness, dampness-heat and qi-deficiency constitution; while in non-MS people, the top 3 constitutions were gentleness, qi-deficiency and phlegm-dampness. When the patient's body mass index (BMI) was more than 25 kg/m(2), the rate of phlegm-dampness and dampness-heat constitution significantly increased, while the rate of qi-deficiency constitution declined; the discrepancy was significant (P=0.00). CONCLUSIONS: The prevalence rate of MS in military senior people was 35.3%, which did not vary among the three age groups. Phlegm-dampness, dampness-heat and qi-deficiency constitution were the three dominant constitutional types seen in the MS patients. The distribution of constitution formation was different in MS people and non-MS people. For different dimensions of BMI, the proportion of each kind of constitutions was varied.

[Efficacy of ginsenosides combined with prednisone in patients with systemic lupus erythematosus: a prospective, randomized, double-blind, placebo-controlled trial].

BACKGROUND: The side effects of glucocorticoid in treatment of systemic lupus erythematosus (SLE) have been the focus of debate, and our preliminary study indicates that ginsenosides can enhance the efficacy of dexamethasone. OBJECTIVE: To observe the effects of ginsenosides combined with prednisone in SLE patients. DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: A total of 60 SLE patients from Department of Rheumatology and Immunology, Changhai Hospital, Second Military Medical University, were randomly divided into treatment group and control group, with 30 patients in each group. Patients in the treatment group were given routine treatment with prednisone plus ginsenosides, while those in the control group were given routine treatment with prednisone plus placebo. They were all treated for 3 months. MAIN OUTCOME MEASURES: After three-month treatment, syndrome score in traditional Chinese medicine (TCM), total response rate and symptom improvement rate were measured and evaluated. RESULTS: Twenty-eight cases in treatment group and twenty-seven cases in control group were included in analysis. The total response rates in the treatment group and control group were 89.28% and 66.67% respectively, and there was a significant difference between the two groups (P<0.05). After treatment, the TCM syndrome scores in the two groups were lower than those before treatment (P<0.01), and prednisone plus ginsenosides was better in decreasing the TCM syndrome score than prednisone plus placebo (P<0.05). The symptoms were improved in the treatment group as compared with the control group (P<0.05). CONCLUSION: Prednisone combined with ginsenosides can increase the clinical effective rate and improve the clinical symptoms of SLE patients.

[Efficacy of combined therapy with ginsenosides and prednisone in treating systemic lupus erythematosus--a randomized, controlled and double-blinded trial].

OBJECTIVE: To observe the therapeutic efficacy of combined therapy with ginsenosides (GS) and prednisone on systemic lupus erythematosus (SLE). METHODS: Sixty patients with SLE were assigned to 2 groups randomly by a randomizing digital table, the treated group and the control group, 30 cases in each group. All patients were treated with routine administration of prednisone, but to the treated group GS Capsule (50 mg) was given additionally twice every day, while to the control group placebo capsule of equal dosage was given instead. The total clinical efficacy, and changes of systemic lupus erythematosus disease activity index (SLEDAI), erythrocyte sedimentation rate (ESR), complement 3 (C3) and anti-ds-DNA were observed after 3-month-treatment. RESULTS: The total clinical efficacy rate was 89.28% in the treated group and 66.67% in the control group, showing a significant difference between them (P<0.05). The improvements of SLEDAI, ESR and C3 in the treated group were more significant than those in the control group (P<0.01, P<0.05). CONCLUSION: The combined therapy of GS and prednisone could enhance the clinical efficacy, reduce the SLEDAI and promote the recovery of laboratory indices in SLE patients.

[Regulatory effect of ginsenoside on glucocorticoid receptor in mice with ischemic liver damage].

OBJECTIVE: To study whether ginsenoside (GS) can regulate the glucocorticoid receptor (GR) in mice with ischemic liver damage, and to preliminarily observe its dose-effect relationship for providing an experimental bases in seeking a new way to relieve the damage from view of GR. METHODS: Adult male SD mouse was used to establish liver ischemia model, and different doses (100, 50, and 25 mg/kg) of GS was given via gastric infusion before modeling. The maximal GR binding capacity (Bmax) of liver and the level of GR mRNA expression in liver were dynamically determined at various time points (2 h, 6 h, 12 h and 24 h) after modeling. RESULTS: Compared with the normal control group, GR Bmax and GR mRNA expression in model rats were lower obviously (P < 0.01). As compared with the control group, GR Bmax and GR mRNA expression in model rats treated with 50 mg/kg GS significantly raised at 2 h, 6 h, 12 h (P < 0.01), while the changes in modeling rats treated with other two doses of GS were of no statistical significance. CONCLUSION: GS in dose of 50 mg/kg can elevate the GR Bmax of liver and the level of GR mRNA expression in liver of rats with ischemic damage.

[Ginsenosides and dexamethasone in managing the liver injury and renal function after transcatheter arterial chemoembolization for hepatic carcinoma patient].

OBJECTIVE: To observe the protective effect of ginsenosides (GS) or low dose of glucocorticoid dexamethasone (Dex) alone or combined in managing the liver injury and renal function after transcatheter arterial chemoembolization (TACE). METHODS: 120 patients with primary liver carcinoma were randomly divided into four groups (A, B, C, D) with 30 patients in each. Group A was treated with placebo; group B with Dex; group C with GS and group D with Dex plus GS. The changes in liver and renal function after TACE were observe according to the WHO criteria for side effects of anti-cancer drug. RESULTS: Compared with group A, Dex combined with GS was able to reduce the level of TB, ALT/AST, BUN and Child-grade, which significantly protected the liver and kidney (P < 0. 05). However, Dex or GS alone could also improve some parameters of liver and renal function after TACE (P < 0.05). CONCLUSION: Dex combined with GS is effective in managing the liver injury and renal function after transcatheter arterial

[Clinical study on integrative medicine for preventing and treating post-transcatheter arterial chemoembolization].

OBJECTIVE: To observe the effects of ginsenosides (GS) and low dose glucocorticoid for preventing and treating the post-transcatheter arterial chemoembolization (TACE) syndrome. METHODS: Adopting randomized, double blinded, controlled method, 80 patients with primary liver cancer were divided into 4 groups, placebo group, dexamethasone (Dex) group, Ginsenosides (GS) group, and Dex combined with GS group to observe the clinical effect of the patients after TACE. RESULTS: Dex combined with GS could markedly lower the occurrence of nausea, vomiting, fever and pain, and the median time of symptoms persistence, also alleviate the bone marrow inhibition of chemotherapy. CONCLUSION: Dex combined with GS could effectively prevent and treat TACE syndrome.

[Ginsenosides combined with dexamethasone in preventing and treating postembolization syndrome following transcatheter arterial chemoembolization: a randomized, controlled and double-blinded prospective trial].

OBJECTIVE: To observe the effect of ginsenosides (GS) and low dose glucocorticoid in preventing and treating the postembolization syndrome following transcatheter arterial chemoembolization (TACE). METHODS: Eighty patients with primary liver carcinoma were randomly divided into 4 double-blinded groups, with 20 patients in each group. Patients in groups A, B, C, D were treated with placebo, dexamethasone (Dex), GS, Dex and GS, respectively. The changes of clinical symptoms and laboratory tests after TACE were observed. RESULTS: Dex combined with GS markedly decreased the occurrence ratio and lasting time of the symptoms such as nausea, vomiting, fever and pain, and protected the function of liver as compared with the placebo (P<0.05). Single use of Dex or GS improved some symptoms as compared with the placebo, but it was not as good as the combination of Dex and GS. CONCLUSION: Dex combined with GS can effectively prevent and treat the postembolization syndrome following TACE.