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.

[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.

[Discussion on some basic academic problems in research and development of treatment based on syndrome differentiation].

To implement treatment depending on syndrome differentiation, namely, the syndrome dependent therapy (SDT), is the characteristics of Chinese medicine, also an important field for TCM researches and development in the contemporary era. This paper summarized the common academic problems in this field, involving the relationship between syndrome and disease, body constitution, four diagnostic methods and SDT, the mechanisms of diseases, syndromes and SDT, as well as the relationship between SDT and Western medicine. The thinking path and recommendation for solution were proposed as well.

[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.

[Effects of Erxian Decoction and its separate prescriptions on the levels of luteinizing hormone and follicle-stimulating hormone in anterior pituitary cells from female rats].

OBJECTIVE: To investigate the effects of Erxian Decoction (EXD), a traditional Chinese compound herbal medicine and its separate prescriptions such as Wenshen Yijing Recipe (WSYJR, a recipe for warming kidney and replenishing essence), Ziyin Xiehuo Recipe (ZYXHR, a recipe for nourishing yin and dispersing fire) and Tiaoli Chongren Recipe (TLCRR, a recipe for regulating thoroughfare and conception vessels) and some extracts from EXD on the levels of LH and FSH in the primary cultured anterior pituitary cells from female rats. METHODS: EXD, its separate prescriptions and traditional Chinese herbal extracts from EXD were added directly to the incubators. The levels of LH and FSH were tested by radioimmunoassay. RESULTS: The levels of LH and FSH in the supernatant of anterior pituitary cells treated by EXD or its separate prescriptions including ZYXHR and TLCRR were increased significantly as compared with those in the blank control. There was a tendency for stimulating the secretions of LH and FSH in the WSYJR-treated group too. Dimethyl sulfoxide as the solvent of icariin and curculigoside could interfere with the results. CONCLUSION: EXD and its separate prescriptions such as ZYXHR and TLCRR can increase the levels of LH and FSH significantly.

[Effect of er xian decoction and its subdivisions on granulosa cells secretory function in rats].

OBJECTIVE: To investigate the effects of Er Xian Decoction (EXD) and its three subdivisions "warming Shen" ,"nourshing Yin" and "adjusting Chong and Ren" in regulating the level of estradiol (E2) on the primary cultured granulosa cells. METHODS: Effect of EXD and its three subdivisions, also part of the effective components of this formula, icariin and curculigoside, on level of E2 were carried out using primary cultural granulose cell as the experimental model. RESULT: EXD and its three subdivisions could stimulate the secretion of E2, especially the "warming Shen" group (P <0.05). All the composing of Chinese herbs of this formula could promote the level of E2 in different degree, and the Rhizoma Curculiginis, Radix Moromade Officinalis, and Herba Epimedii have the best effects (P <0. 01). CONCLUSION: The regulation of EXD on the hypothalamus-pituitary-gonad (HPG) axis may be related to promoting the secreting of E2 at the site of granulosa cell. The "warming Shen" subdivision has the better effect in promoting the secretion of E2.