[Clinical study of type â ¢ prostatitis treated with "warming and promoting technique" of acupuncture combined with extracorporeal shock wave].

OBJECTIVE: To observe the clinical curative effect of wentongzhenfa (warming and promoting technique of acupuncture) combined with extracorporeal shock wave in treatment of type Ⅲ prostatitis. METHODS: A total of 96 patients with type Ⅲ prostatitis were randomly divided into an observation group (48 cases) and a control group (48 cases). In the control group, the extracorporeal shock wave was combined with even-needling technique of acupuncture at Guanyuan (CV4), Mingmen (GV4), Zhongji (CV3), Zusanli (ST36), etc. In the observation group, the extracorporeal shock wave was combined with "warming and promoting technique" of acupuncture at the same acupoints as the control group. The treatment lasted 30 min each time, once daily in either group. There were 2 days of interval after consecutive treatment for 5 days. Totally, the duration of treatment was 1 month in two groups. The clinical curative effect was assessed after treatment. Before and after treatment, the changes in the concentrations of tumor nerosis factor-α (TNF-α), interleukin-1ß(IL-1ß) and IL-6 in prostatic fluid were determined; and the symptoms were scored, i.e. frequent, urgent and burning painful urine, difficulty in urination, dribbling urine, distending pain in perineum, bitter taste and dry mouth, and scrotal dampness. The changes in the scores of National Institute of Health chronic prostatitis symptom index (NIH-CPSI), international index of erectile function (IIEF), visual analogue scale (VAS) were evaluated befroe and after treatment. Successively, before treatment, after treatment, as well as 1 and 3 months after treatment, the quality of life was evaluated by Karnofsky in the patients of two groups. RESULTS: After treatment, the total effective rate was 89.6% (43/48) in the observation group, higher than 70.8% (34/48) in the control group (P<0.05). Compared with those before treatment, the concentrations of TNF-α and IL-1ß in the prostatic fluid were all decreased (P<0.05) and the concentration of IL-6 was increased (P<0.05), the scores of symptoms, NIH-CPSI, IIEF and VAS were all reduced (P<0.05) in two groups. The changes of the above indexes were more obvious in the observation group than those in the control group (P<0.05). After treatment and 1 and 3 months after treatment, Karnofsky scores all increased (P<0.05) in two groups，and the increases were more significant in the observation group (P<0.05). CONCLUSION: "Warming and promoting technique" of acupuncture combined with extracorporeal shock wave promotes the elimination of local inflammatory factors, relieves clinical symptoms, improves the quality of life, as well as has a satisfactory short-term and medium-term curative effect on type Ⅲ prostatitis.

Diterpenoids with herbicidal and antifungal activities from hulls of rice (Oryza sativa).

Diterpenoids are the main secondary metabolites of plants and with a range of biological activities. In the present study, 7 compounds were isolated from the hulls of rice (Oryza sativa L.). Among them, 3 diterpenoids are new namely, 3,20-epoxy-3α-hydroxy- 8,11,13-abietatrie-7-one (1), 4,6-epoxy-3ß-hydroxy-9ß-pimara-7,15-diene (2) and 2-((E)-3- (4-hydroxy-3-methoxyphenyl) allylidene) momilactone A (3). While, 4 terpenoids are known, namely momilactone A (4), momilactone B (5), ent-7-oxo-kaur-15-en-18-oic acid (6) and orizaterpenoid (7). The structures of these diterpenoids were elucidated using 1D and 2D NMR in combination with ESI-MS and HR-EI-MS. Furthermore, all isolated compounds displayed antifungal activities against four crop pathogenic fungi Magnaporthe grisea, Rhizoctonia solani, Blumeria graminearum and Fusarium oxysporum, and phytotoxicity against paddy weed Echinochloa crusgalli. The results suggested that rice could produce plenty of secondary metabolites to defense against weeds and pathogens.

Akebia trifoliata (Thunb.) Koidz Seed Extract inhibits human hepatocellular carcinoma cell migration and invasion in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: The high recurrence rate postoperative and extensive metastases have become the obstacle of Hepatocellular Carcinoma (HCC) efficacy improvements, which contribute to most of the patient mortality. Akebia trifoliata (Thunb.) Koidz has been shown pharmacological activities in clinical and anti-HCC biological activity in previous research, but its potential function of anti-metastasis remains unknown. AIM OF THIS STUDY: To make sure whether ATKSE inhibits migration and invasion in HCC cell lines in vitro and the potential mechanism. MATERIALS AND METHODS: A UHPLC-HRMS analysis was adopted to identify and control the quality of the ethanol extract of Akebia trifoliata (Thunb.) Koidz Seed (abbreviated ATKSE). Cell viability of three kinds of HCC cell lines (HEPG2, HUH7, and SMMC7721) was detected using MTT assay and Flow cytometry. Adhesion capacity was measured by cell-matrigel adhesion assay. Wounded healing and Matrigel-transwell invasion assays were performed to assess cell migration and invasion, respectively. Western blot assay was used to detect several metastasis-related protein molecules, including FAK adhesion signaling, cadherin molecules, and MMPs. ELISA assay was used to evaluate the secreted MMP9 level. RESULTS: ATKSE significantly suppressed HCC cells viability and proliferation (from 0.9 up to 3.0 mg/ml); then under sub-lethal concentration (from 0.25 up to 1.0 mg/ml), ATKSE inhibited cell adhesion, migration, and invasion in a way of dose-dependent. Several metastatic-related molecules or pathway, including FAK adhesion signaling, cadherin molecules, and MMPs, took part in this process. There are both differences and commonalities in various cell lines: typically such as p-FAK was down-regulated by ATKSE in both HEPG2 and SMMC7721, while was raised in HUH7; Further attempts on the combination of ATKSE and FAK inhibitors, provide us with the enhanced inhibitory effects of invasion and migration in HEPG2 and HUH7 cells, as well as antagonistic effects in SMMC7721. As a target or potential mechanism, it may be more valuable to concern FAK inhibition by ATKSE in HEPG2 cells than in the other two cells. CONCLUSIONS: These results suggest that ATKSE has anti-metastasis potency in HCC cells.

Downregulation of miR-219 enhances brain-derived neurotrophic factor production in mouse dorsal root ganglia to mediate morphine analgesic tolerance by upregulating CaMKIIγ.

BACKGROUND: Increasing evidence suggests that microRNAs are functionally involved in the initiation and maintenance of pain hypersensitivity, including chronic morphine analgesic tolerance, through the posttranscriptional regulation of pain-related genes. We have previously demonstrated that miR-219 regulates inflammatory pain in the spinal cord by targeting calcium/calmodulin-dependent protein kinase II gamma (CaMKIIγ). However, whether miR-219 regulates CaMKIIγ expression in the dorsal root ganglia to mediate morphine tolerance remains unclear. RESULTS: MiR-219 expression was downregulated and CaMKIIγ expression was upregulated in mouse dorsal root ganglia following chronic morphine treatment. The changes in miR-219 and CaMKIIγ expression closely correlated with the development of morphine tolerance, which was measured using the reduction of percentage of maximum potential efficiency to thermal stimuli. Morphine tolerance was markedly delayed by upregulating miR-219 expression using miR-219 mimics or downregulating CaMKIIγ expression using CaMKIIγ small interfering RNA. The protein and mRNA expression of brain-derived neurotrophic factor were also induced in dorsal root ganglia by prolonged morphine exposure in a time-dependent manner, which were transcriptionally regulated by miR-219 and CaMKIIγ. Scavenging brain-derived neurotrophic factor via tyrosine receptor kinase B-Fc partially attenuated morphine tolerance. Moreover, functional inhibition of miR-219 via miR-219-sponge in naive mice elicited thermal hyperalgesia and spinal neuronal sensitization, which were both suppressed by CaMKIIγ small interfering RNA or tyrosine receptor kinase B-Fc. CONCLUSIONS: These results demonstrate that miR-219 contributes to the development of chronic tolerance to morphine analgesia in mouse dorsal root ganglia by targeting CaMKIIγ and enhancing CaMKIIγ-dependent brain-derived neurotrophic factor expression.

[Effect of Osthole on Adrenocortical Function in Y1 Mouse Adrenocortical Tumor Cells].

OBJECTIVE: To study the effect of osthole (Ost) on adrenocortical function in Y1 mouse adrenocortical tumor cells. METHODS: Y1 mouse adrenocortical tumor cells were taken as subjects in this experiment. In 10.0%, 1.0%, and 0.1% serum DMEM-F12 medium, Y1 cells were treated with 1, 10, 25, 50, 100, and 200 micromol/L Ost for 24 and 48 h. 0.1% Dimethyl Sulfoxide (DMSO) was taken as negative control group and 1 mmol/L (Bu) 2cAMP as positive control group. Cell growth morphology was observed under inverted microscope. Contents of corticosterone were tested by ELISA. Expression levels of steroids synthase such as Star, Cyp11a1, Cyp21a1, Hsd3b2, Cyp11b1, Cyp11b2, Cyp17a1, and Hsd17b3 mRNA were detected by Real time quantitative PCR (RT-qPCR). RESULTS: Y1 cell proliferation was obviously inhibited by 100 and 200 micromol/L Ost, and its inhibitory effect was more significant in 0.1% serum medium. Compared with the negative control group, gene expressions of Star, Cyp11a1 , Cyp21a1, Hsd3b2, Cyp11b1, Cyp17a1, and Hsd17b3 were significantly enhanced in the posi- tive control group (P < 0.05). Y1 cell corticosterone levels significantly increased in 50 micromol/L Ost treatment group after 24-and 48-h intervention (P < 0.05). Contents of corticosterone increased more obviously in 25 and 50 +/- mol/L Ost treatment groups after 48-h intervention, as compared with 24-h intervention (P < 0.01). After 24-h intervention, expression levels of Star, Cyp21a1, and Hsd3b2 genes were significantly up-regulated in 25 and 50 lLmol/L Ost groups (P < 0.05). Star gene expression was further enhanced after 48-h intervention (P < 0.05). However, Ost showed no effect on Cyp11a1 (P > 0.05). Additionally, gene expressions of Cyp11b1 and Cyp17a1 were significantly enhanced by 10, 25, and 50 pLmolIL Ost after treatment for 24 and 48 h (P < 0.05). Ost showed no obvious effect on Cyp11b2 and Hsd17b3 expressions. CONCLUSION: Ost could regulate adrenal cortex function and promote corticosterone synthesis and secretion through strengthening gene expressions of steroidogenic enzymes.

Akebia trifoliate (Thunb.) Koidz Seed Extract Inhibits the Proliferation of Human Hepatocellular Carcinoma Cell Lines via Inducing Endoplasmic Reticulum Stress.

Akebia Fructus has long been used for hepatocellular carcinoma (HCC) in China, while the molecular mechanism remains obscure. Our recent work found that Akebia trifoliate (Thunb.) Koidz seed extract (ATSE) suppressed proliferation and induced endoplasmic reticulum (ER) stress in SMMC-7721. The present study aimed to throw more light on the mechanism. ER stress occurred after ATSE treatment in HepG2, HuH7, and SMMC-7721 cells, manifested as ER expansion, and SMMC-7721 was the most sensitive kind in terms of morphology. Cell viability assay showed that ATSE significantly inhibited cells proliferation. Flow cytometry analysis indicated that ATSE leads to an upward tendency of G0/G1 phase and a reduced trend of the continuous peak after G2/M phase in HepG2; ATSE promoted apoptosis in HuH7 and a notable reduction in G0/G1 phase; ATSE does not quite influence cell cycles of SMMC-7721. Western blot analysis showed an increased trend of the chosen ER stress-related proteins after different treatments but nonsignificantly; only HYOU1 and GRP78 were decreased notably by ATSE in HuH7. Affymetrix array indicated that lots of ER stress-related genes' expressions were significantly altered, and downward is the main trend. These results suggest that ATSE have anticancer potency in HCC cells via partly inducing ER stress.

[Analysis of the dynamic changes of blood hormone levels in H22 liver cancer mice of poisonous pathogenic factors syndromes to different degrees].

OBJECTIVE: To study the dynamic changes of blood hormone levels in H22 liver cancer mice of poisonous pathogenic factors syndromes (PPFS) to different degrees. METHODS: Two hundred and twenty mice were injected with H22 tumor cells from their armpits. On the ninth day after inoculation the mice of severe poisonous pathogenic factors syndrome (SPPFS) and of mild poisonous pathogenic factors syndrome (MPPFS) were screened. Besides, another normal control group consisting of 30 mice was set up. The mice were killed on the tenth and eleventh day after inoculation (as the 1st and 2nd time window). The weight of the tumor, the wet weight of the thymus and the spleen were weighed. The plasma adrenocorticotropic hormone (ACTH), corticosterone, aldosterone, thyroid hormone T3 and T4, testosterone, TNF-alpha, and IFN-gamma were detected by ELISA. All the aforesaid laboratory parameters were analyzed. RESULTS: The tumor weight was obviously larger in mice of the SPPFS group than in those of the MPPFS group at the same time window (P < or = 0.05). Compared with the normal control group, the thymus was obviously atrophied (P < or = 0.05), the spleen was significantly enlarged (P < or = 0.05), the plasma ACTH significantly increased (P < or = 0.05) in the SPPFS group at the two time windows. But the increment of ACTH was less in the MPPFS group. The plasma corticosterone showed similar tendency as that of ACTH. At the 1st time window the plasma testosterone significantly increased in the two groups (P < or = 0.05). The plasma testosterone and T4 showed a decreasing tendency in the SPPFS group. The plasma TNF-alpha and IFN-gamma levels showed an increasing trend in the two groups. Correlation study showed that the degree of PPFS was negatively correlated with qi deficiency (r = -0.766, P < or = 0.05) and T4 (r = -0.738, P < or = 0.05). The degrees of PPFS was positively correlated with the plasma ACTH level (r = 0.635, P < or = 0.05). The degree of qi deficiency was positively correlated with yang heat syndrome (r = 0.632, P < or = 0.05). The plasma ACTH was negatively correlated with T4 (r = -0.504, P < or = 0.05). The plasma testosterone was positively correlated with TNF-alpha (r = 0.619, P < or = 0.05). CONCLUSIONS: PPFS occurs naturally and shows difference to different degrees in the development of H22 liver cancer. The disorders of neuroendocrine hormones and the suppression of the immune function show dynamic changing trends.

[The correlation between adrenal corticosteroids and cytokines expressions in mice of different syndromes].

OBJECTIVE: To study the cytokines expressions in the adrenal gland and its correlation with serum adrenal corticosteroids in mice of different syndromes. METHODS: Using the quantitative four diagnosis and syndrome differentiation methods, 60 normal mice and 190 H22 liver cancer bearing mice were syndrome typed. Serum corticosterone and aldosterone were tested by ELISA, and mRNA expressions of cytokines in the adrenal gland were detected using Real-time PCR. RESULTS: Mice of different syndromes were obtained, such as normal mice of no syndrome, normal mice of vigorous qi syndrome, normal mice of qi deficiency syndrome, liver cancer bearing mice of excessive evil toxic syndrome, liver cancer bearing mice of evil lying in the middle syndrome, liver cancer bearing mice of weak evil toxic syndrome, and liver cancer bearing mice of poisonous pathogenic factors and qi deficiency syndrome. The serum corticosteroids were significantly higher in the liver cancer bearing mice than in the normal mice (P < 0.05). The cortex hormones increased most significantly in the liver cancer bearing mice of excessive evil toxic syndrome (P < 0.05). Compared with the normal mice, IL-1beta, IL-2, IL-6, IL-10, IL-12alpha, IL-12beta, and TNF-alpha gene expressions increased in the liver cancer bearing mice, while only expressions of IL-1alpha and IL-5 decreased. But the expressions of IL-13 and transforming growth factor beta1 (TGF-beta1) showed no regularity. The expressions of IL-4 and INF-alpha were not detected in all mice. It is notable that the more severe degree of poisonous pathogenic factors, the higher the expressions of serum corticosterone and aldosterone levels as well as IL-6, the lower expressions of IL-1beta, IL-2, IL-5, IL-12alpha, IL-12beta, and TNF-alpha. CONCLUSIONS: The increased serum corticosteroid level in liver cancer bearing mice could possibly be induced by chronic tumor stress, partial cytokines were involved in the synthesis and secretion of the adrenal hormone. Of them, IL-6 might positively regulate the secretion of corticosteroids, while IL-1beta, IL-2, IL-5, IL-12alpha, IL-12beta, and TNF-alpha might negatively regulate their secretions.

[Characteristics of gene expression of adrenal cortical steroid synthetase and its regulatory factor in mice with H22 liver cancer of different patterns].

OBJECTIVE: To study the characteristics of gene expression of adrenal cortical steroid synthetase and its regulatory factor in mice with H22 liver cancer of different patterns. METHODS: Syndromes revealed in mice with H22 tumor were differentiated by quantified four diagnostic methods and syndrome differentiation, and mice with commonly encountered patterns (A: evil-toxin accumulation pattern, B: qi-deficiency pattern, C: yang-qi deficiency pattern and D: qi-yin-yang deficiency pattern) were screened out for subjecting to the study. Two batches of GeneChip Mouse Exon 1.0 ST Array detection were performed in the selected mice for detecting the gene expressions of adrenal cortical steroid synthetase and its regulatory factor, with the analysis performed put stress on the differential expressions in mice of various syndrome patterns. RESULTS: Data obtained from the two batches detection showed well repeatability, in which similar genes of high or low expression emerged. The adrenal cortical steroid synthetase genes, such as Cyp11a1, Star, Cyp11b2, Cyp21a1, Hsd3b and Hsd17b were highly expressed, with few difference among the four patterns. However, Cyp11a1 was down-regulated and Cyp1b2 up-regulated in all patterns; Hsd3b1 and Cyp21a1 down-regulated in pattern A and B, but up-regulated in pattern C and D. As for the expressions of the relative regulatory factors, Cyb5b and Wnt4 were down-regulated but Fdx1, Fdxr, Hsd11b1, Por, Agt and Nr 0b1 were up-regulated in all patterns; Nr5al down-regulated in pattern A but up-regulated in other three patterns; Nr4al and Nr4a2 up-regulated in pattern A and down-regulated in the others. CONCLUSIONS: The adrenal cortical steroid synthetase genes are rather conservative and stable in mice bearing H22 liver cancer, part of the expression might be correlated to the condition of disease and essence of syndromes, embodying the differences among different patterns in the same disease.

[Differential gene expressions of steroid- and catecholamine-synthesizing enzymes in adrenal gland of rats with different diseases but the same syndromes].

OBJECTIVE: To study the gene expression characteristics of steroid- and catecholamine-synthesizing enzymes in adrenal gland of spontaneously hypertensive rats (SHRs), Goto-Kakizaki (GK) rats and normal Wistar rats with the same traditional Chinese medicine syndromes. METHODS: Sixteen-week-old Wistar rats, SHRs and GK rats were used. By the quantitative four diagnosis and syndrome differentiation methods and GeneChip Mouse Exon 1.0 ST Array, we observed adrenal gland gene expressions in normal Wistar rats, qi deficiency Wistar rats, SHRs with qi deficiency and qi excess, GK rats with qi deficiency and qi excess. Differentially expressed genes of steroid- and catecholamine-synthesizing enzymes and their regulatory factors were analyzed. RESULTS: Thirty-one genes were differentially expressed among all syndromes. Hsd3b6 was down-regulated significantly 6.0-fold in GK rats with qi deficiency syndrome and qi excess syndrome, and Cyp11b2 was up-regulated 1.5 times in GK rats with qi deficiency syndrome. Por, Hsd11b2, and Nr2f6 were up-regulated in all syndromes, and Cyp2c23, Cyp4a3, Cyp4a8 and Cyp2e1 were down-regulated. However, Srd5a1 and Nr4a1 were up-regulated only in GK rats, and Lss was down-regulated only in SHRs. Th was up-regulated 1.5 times in SHRs with qi deficiency syndrome, GK rats with qi deficiency syndrome and GK rats with qi excess syndrome. Ddc was up-regulated 1.5 times in GK rats with qi excess syndrome. Dbh was up-regulated 3.0 times in GK rats with qi deficiency syndrome and qi excess syndrome. However, Comt was down-regulated 1.5 times in GK rats with qi deficiency syndrome and qi excess syndrome, and Mao was up-regulated 1.5 times in SHRs with qi deficiency syndrome and qi excess syndrome. CONCLUSION: Some genes associated with steroid- and catecholamine-synthesizing pathways were differentially expressed in SHRs and GK rats, and the differentially expressed genes may be related to the development of traditional Chinese medicine syndromes.

[Methodology and purposes of establishing mouse and rat models for syndrome differentiation and treatment].

Methodology of syndrome differentiation and syndrome-based treatment in rats and mice has professional characteristics and caters to the research and development of traditional Chinese medicine (TCM). In this paper, the authors introduced their systematic research in five aspects. 1) Rats and mice can be used to simulate TCM clinical practice. Diagnosis and syndrome differentiation can be done to the rats and mice, and information collected by the four diagnostic methods from the experimental animals meets the requirements of treatment based on syndrome differentiation. 2) Standardized and quantified four diagnostic methods and syndrome differentiation for rats and mice can be established, and are operational and applicable for general use. 3) There exists constitution and syndrome diversity in normal rats and mice. A spontaneous syndrome can develop in diseased rats and mice, and it can be accompanied by or even change to another syndrome, similar to that in human beings. 4) There is a complicated material base for syndromes inferred from the different gene expressions and splices in neuroendocrine-immune network. 5) Individualized treatment based on syndrome differentiation, as well as quantified evaluation and comparison of the treatment efficacy can be done in the rat and mouse models of syndromes. The established methodology and criteria for syndrome differentiation and syndrome-based treatment in rats and mice can be used in the following four research fields: 1) syndrome identification on rat or mouse models; 2) research on the basic theories of TCM, such as the research on the viscera manifestation theory, the material base of syndromes, function mechanisms of the treatment based on syndrome differentiation, and the diagnostics of TCM; 3) study in clinical subject of TCM, such as evaluation and comparison of the efficacy of treatment based on syndrome differentiation, protocol optimization of syndrome differentiation and treatment, and preventive treatment of diseases; 4) study in traditional Chinese drugs, such as the research on properties of Chinese herbal drugs, and pharmacological research on Chinese herbal medicines and formulas.

[Differentially expressed genes in adrenal gland of H22 liver cancer mice with different syndromes and in different stages].

OBJECTIVE: To reveal the characteristics of gene expression in adrenal gland of H22 tumor mice with typical syndromes and in different liver cancer stages. METHODS: By the quantitative four diagnosis and syndrome differentiation methods and GeneChip Mouse Exon 1.0 ST Array, we observed adrenal gland gene expression in H22 tumor mice with pathogenic factor-toxin predominance syndrome and qi deficiency syndrome in the earlier stage, yang-qi deficiency syndrome in the intermediate stage, and qi-yin-yang deficiency syndrome in the advanced stage. Genes highly expressed and remarkably different were analyzed in this study. RESULTS: A total of seventy-three up-regulated coincident genes and twenty-six down-regulated coincident genes in different stages were investigated in the study. Up-regulated coincident genes included Hp, C3, Anxa1, Procr, C2, Il4ra, Cd14, Ptprc, Cd52, C4b, Eno3, Xdh, Gpx3, and so on. Down-regulated coincident genes included nervous system function-related genes such as Plp1, Mbp, Aldh1a1, Cck, Atn1, genes associated with electrolyte metabolism such as Aldh1a1 and Slc22a17, genes related to signal transduction such as Cxcr4, Spag5 and Stmn3, etc, and genes related to transcriptional control and protein biosynthesis such as Hspa1a, Dnajb1, Thra, Hhex and so on. CONCLUSION: With the development of the tumorigenesis, the symptoms and signs and differentially expressed genes in adrenal gland of H22 tumor mice can be measured. Up-regulated and down-regulated coincident genes may be the features of H22 tumor mice different from those of normal mice.