[Clinical efficacy of entecavir combined with Biejiajian pills and its influence on TCM syndrome scores during the treatment of chronic hepatitis B with hepatic fibrosis and blood stasis syndrome].

Objective: To investigate the clinical efficacy of entecavir combined with Biejiajian pills and its influence on TCM syndrome scores during the treatment of chronic hepatitis B with hepatic fibrosis and blood stasis syndrome by prospective, randomized and controlled study. Methods: Patients with chronic hepatitis B with hepatic fibrosis and blood stasis syndrome were selected as the research subjects and randomly divided into a treatment group and a control group. Entecavir plus Biejiajian pills or entecavir plus a simulant of Biejiajian pills were given for 48 weeks. The changes in liver stiffness measurement (LSM) and TCM syndrome scores before and after treatment were compared between the two groups to analyze the correlation. The data between groups were analyzed by t-test/Wilcoxon rank sum test or χ(2) test. Pearson correlation coefficient was used to analyze the correlation between TCM syndrome scores and LSM values. Results: After 48 weeks of treatment, the LSM values of the two groups were significantly lower than those of the baseline (P < 0.001), liver fibrosis was significantly improved, and the LSM values of the treatment group were lower than those of the control group [(8.67 ± 4.60) kPa and (10.13 ± 4.43) kPa, t = -2.011, P = 0.049]. After 48 weeks of treatment, the TCM syndrome scores of the two groups were significantly reduced compared with the baseline (P < 0.001), and the clinical symptoms were significantly relieved, and the total effective rates of the improvement of the TCM syndrome scores in the two groups were 74.19% and 72.97%, respectively, but the differences between the groups were not statistically significant (χ(2) = 0.013, P = 0.910). Correlation analysis showed that there was no obvious trend between TCM syndrome scores and LSM values. There were no serious adverse reactions associated with the drug during the observation period of this study. Conclusion: Based on antiviral treatment with entecavir, regardless of whether it is combined with the Biejiajian pill, it can effectively reduce the LSM value, improve liver fibrosis, reduce TCM syndrome scores, and alleviate symptoms in patients with chronic hepatitis B with liver fibrosis and blood stasis syndrome. Compared with entecavir alone, the combined Biejia pill has greater efficacy in improving liver fibrosis and a favorable safety profile, meriting its implementation and widespread application.

Computational identification and characterization of conserved miRNAs and their target genes in beet (Beta vulgaris).

Highly conserved endogenous non-coding microRNAs (miRNAs) play important roles in plants and animals by silencing genes via destruction or blocking of translation of homologous mRNA. Sugar beet, Beta vulgaris, is one of the most important sugar crops in China, with properties that include wide adaptability and strong tolerance to salinity and impoverished soils. Seedlings of B. vulgaris can grow in soils containing up to 0.6% salt; it is important to understand the molecular mechanisms of salt tolerance to enrich genetic resources for breeding salt-tolerant sugar beets. Here, we report 13 mature miRNAs from 12 families, predicted using an in silico approach from 29,857 expressed sequence tags and 279,223 genome survey sequences. The psRNATarget server predicted 25 target genes for the 13 miRNAs. Most of the target genes appeared to encode transcription factors or were involved in metabolism, signal transduction, stress response, growth, and development. These results improve our understanding of the molecular mechanisms of miRNA in beet and may aid in the development of novel and precise techniques for understanding post-transcriptional gene-silencing mechanisms in response to stress tolerance.

Involvement of AP-1 in p38MAPK signaling pathway in osteoblast apoptosis induced by high glucose.

We investigated the effect of p38MAPK/AP-1 (activator protein-1) signaling on the apoptosis of osteoblasts induced by high glucose. A lentivirus vector of small hairpin RNA (shRNA) targeting p38MAPK was constructed in vitro. Osteoblasts MC3T3-E1 cultured in vitro were treated with vehicle, high glucose, p38MAPK-shRNA transfection, p38MAPK inhibitor, and unrelated shRNA transfection. Apoptosis, protein levels of p38MAPK, and activities of AP-1 in MC3T3-E1 osteoblasts were measured using TUNEL and flow cytometry, Western blot analysis, and an electrophoretic mobility shift assay. Compared with the vehicle group, high glucose induced apoptosis of MC3T3-E1 osteoblasts and activated p38MAPK and AP-1. p38MAPK-shRNA transfection blocked the effect of high glucose stimulation, and the p38MAPK inhibitor showed similar effects as those observed in p38MAPK transfection. Unrelated shRNA had no effect on these changes in MC3T3-E1 osteoblasts induced by high glucose. Therefore, our results suggest that p38MAPK-shRNA reduce apoptosis of MC3T3-E1 osteoblasts induced by high glucose by inhibiting the p38MAPK-AP-1 signaling pathway.

Distance constraints and stereospecific alignment requirements characteristic of p53 DNA-binding consensus sequence homologies.

We present evidence in favor of the position that some mutant p53 proteins retain the ability to trans-activate downstream genes through p53 DNA-binding consensus sequence (CS) homologies. We tested one cell line possessing high levels of mutant p53 and found that this mutant p53 is highly active in trans-activating one CS homology, moderately active in trans-activating a second sequence and inactive in modulating a third sequence. We tested a second cell line, also possessing high levels of mutant p53 and found the same pattern of activation. In addition we find that inter-motif distance [represented by N in RRRCWWGYYY(N)RRRCWWGYYY] is very important in determining the relative binding affinity of a given CS homology for wild-type or mutant p53. Our studies suggest that stereospecific alignment of the DNA-binding motifs within the CS may favor binding of wild-type p53 while misalignment may favor binding of mutant p53. Furthermore, we find that the maximum distances at which p53 DNA-binding CS homologies are functionally active vary for different sequences. Introduction of as few as 200 bp between one CS homology and the downstream TATA box can eliminate a 45-fold p53-mediated transactivation. We present evidence that the composition of the DNA which flanks a p53 DNA-binding consensus sequence may also modulate trans-activation.

In vivo and in vitro gene transfer and expression in rat intestinal epithelial cells by E1-deleted adenoviral vector.

The intestine is proposed to be an attractive target site for somatic gene therapy due to a large mass of proliferating tissue and stem cells in the crypts. Previous studies using a retroviral vector have shown that a reporter gene, bacterial beta-galactosidase (beta-Gal), can be transferred and expressed in the small intestinal epithelial cell. However, transduction efficiency is relatively low in rat and mice intestines. In the present study, we employed an E1-deleted adenoviral vector (which encodes the beta-Gal gene) to investigate the feasibility of gene transfer into rat small intestinal epithelial cell lines and small intestines in male Sprague-Dawley rats. In in vitro studies, expression of AdCMV beta gal was quantitatively measured in IEC-6 and IEC-18 cell cultures using X-Gal histochemistry and chemiluminescent reporter gene assays. The results indicate that AdCMV beta gal can be efficiently transferred into intestinal epithelial cell lines and transgene expression is virus concentration dependent. In in vivo studies, a 5F intestinal feeding tube was used to deliver the vector to the duodenal segment of the rat. Expression of AdCMV beta gal was primarily localized to the epithelium of the intestinal tract. Transduction efficiency of the transgene was seen in the duodenum, jejunum, ileum, and, to a lesser extent, the colon. Moreover, following a single or secondary administration of recombinant adenovirus, efficient expression of AdCMV beta gal in the intestinal tract peaked at 3 days and decreased by 7 and 14 days. No antiadenoviral antibody response was detected in the serum after a single or secondary challenge with this virus. These findings demonstrate that an E1-deleted adenoviral vector, when administered through an oral-duodenal tube, transfers genetic material more successfully in the intestinal epithelium in the small intestine when compared to the large intestine. A single or secondary challenge with adenoviral vector does not cause enhanced host immune responses to this virus. It suggests that successful gene transduction by the repeat administration of the adenoviral vector makes it an alternative candidate for gene therapy applications in intestinal diseases and metabolic deficiencies.

Analysis of cardiovascular responses to PACAP-27, PACAP-38, and vasoactive intestinal polypeptide.

Responses to pituitary adenylate cyclase polypeptide (PACAP)-27, PACAP-38, and vasoactive intestinal peptide (VIP) were compared in the peripheral and pulmonary vascular beds of the cat and in the isolated perfused neonatal pig heart. Intravenous injections of PACAP-27 and PACAP-38 produced biphasic changes in systemic arterial pressure whereas iv injections of VIP caused only decreases in arterial pressure. When blood flow to the hind limb and mesenteric vascular beds was maintained constant, PACAP-27 and PACAP-38 caused dose-related biphasic changes in perfusion pressure, whereas VIP only decreased perfusion pressure. PACAP-27 was approximately threefold more potent than PACAP-38, and the pressor component of the biphasic response was blocked by alpha-adrenergic antagonists and adrenalectomy. PACAP-27, PACAP-38, and VIP produced decreases in pulmonary vascular resistance, and all three peptides had significant vasodilator activity in the isolated perfused neonatal pig heart. Although all three peptides decreased coronary vascular resistance, only PACAP-27 and PACAP-38 increased left ventricular contractility, with PACAP-27 approaching isoproterenol in potency. The results of these experiments show that PACAP-27, PACAP-38, and VIP have significant effects on vasomotor tone that depend on the vascular bed studied and the contribution of adrenal catecholamines.

Chronic estrogen alters contractile responsiveness to angiotensin II and norepinephrine in female rat aorta.

Sex hormones have been postulated to play an important role in the modulation of vascular responsiveness to endogenous vasoactive substances. This study was designed to establish the effects of chronic treatment with estrogen in vivo on subsequent contractile responsiveness of aortic rings to angiotensin II or norepinephrine in vitro. Concentration-response curves for angiotensin II were compared in aortic rings with or without endothelium isolated from ovariectomized rats (275-299 g) pretreated with 17 beta-estradiol (approximately 1 mg/kg per day) or placebo for 14 days and from normal prepubertal female rats (125-149 g) pretreated with 17 beta-estradiol (approximately 5 mg/kg per day) or placebo for 14 days. Whether or not endothelium was present, angiotensin II-induced contraction was significantly depressed in rings from 17 beta-estradiol-treated ovariectomized or prepubertal rats when compared to controls, and the pattern of the effects of 17 beta-estradiol-treatment on the concentration-response curves for angiotensin II was similar in the two models. In contrast to angiotensin II-induced contraction, norepinephrine-induced contraction was significantly enhanced in aortic rings with or without endothelium from ovariectomized rats pretreated with 17 beta-estradiol (approximately 1 mg/kg per day, 14 days) and from normal prepubertal female rats pretreated with 17 beta-estradiol (approximately 5 mg/kg per day, 14 days) when compared to controls. The results demonstrate that chronic treatment with 17 beta-estradiol selectively reduced and enhanced contractile responsiveness of aortic rings to angiotensin II and norepinephrine, respectively. Further, the results indicate that the normal prepubertal female rat is an efficient model to investigate modulation by estrogen of aortic responsiveness to vasoactive agents in vitro.

Analysis of responses to angiotensin IV in the pulmonary vascular bed of the cat.

Responses to angiotensin IV were investigated and compared with responses to angiotensin II in the pulmonary vascular bed of the intact-chest cat under constant flow conditions. Under low resting tone conditions, intralobar injections of angiotensin IV caused dose-related increases in lobar arterial pressure. Angiotensin II also increased lobar arterial pressure and was 100 fold more potent than angiotensin IV. Dose-response curves for both peptides were parallel, and the time-to-peak increase in lobar arterial pressure in response to angiotensin IV and angiotensin II was similar whereas the duration of the response to angiotensin IV was significantly shorter. Following administration of the angiotensin receptor subtype 1 (AT1) antagonist, DuP 532 (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphe nyl-4- yl)-methyl]imidazole), responses to angiotensin IV and angiotensin II were reduced in a similar manner, whereas pulmonary pressor responses to serotonin were not altered. In contrast to the inhibitory effects of the angiotensin AT1 receptor antagonist, administration of PD 123,319 ((S)-1-[[4-(dimethylamino)-3-methyl-phenyl]methyl-5-(diphenylacetyl++ +)- 4,5,6,7-tetrahydro-1H-imidazol[4,5-c]pyridine-6-carboxylic acid), an angiotensin AT2 receptor antagonist, did not change responses to angiotensin II and angiotensin IV. The results of the present study demonstrate that angiotensin IV has significant vasoconstrictor activity in the pulmonary vascular bed, and suggest that pressor responses to angiotensin IV are mediated by the activation of angiotensin AT1 receptors. These data indicate that angiotensin IV is 100-fold less potent than angiotensin II and suggest that the hexapeptide may have a lower apparent affinity for the angiotensin AT1 receptor than does angiotensin II in the pulmonary vascular bed of the cat.

Influence of N omega-nitro-L-arginine methyl ester, LY83583, glybenclamide and L158809 on pulmonary circulation.

The effects of N omega-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase; 6-anilino-5,8-quinolinedione (LY83583), an inhibitor of soluble guanylate cyclase; glybenclamide, a ATP-sensitive K+ channel blocking agent; and 5,7-dimethyl-2-ethyl-3-[[2'-(1H-tetrazol-5-yl)-[1,1']-biphenyl-4- yl]methyl]-3H-imidazo[4,5-b]pyridine (L158809), an angiotensin II type I receptor antagonist, on the response to ventilatory hypoxia were investigated in the isolated blood-perfused rat lung. Under conditions of controlled pulmonary blood flow, and constant left atrial pressure, injections of glybenclamide into the pulmonary arterial perfusion circuit significantly increased baseline pulmonary arterial perfusion pressure, whereas administration of N omega-nitro-L-arginine methyl ester produced smaller increases in baseline tone. Ventilatory hypoxia (3% O2-5% CO2-92% N2) significantly increased pulmonary arterial perfusion pressure and the response was reproducible with respect to time. Following administration of N omega-nitro-L-arginine methyl ester or LY83583, the response to hypoxia was significantly increased, whereas the response to hypoxia was not changed by glybenclamide or atropine. N omega-Nitro-L-arginine methyl ester also significantly enhanced pressor responses to angiotensin II, but had no effect on the pressor response to serotonin. When pulmonary vascular tone was increased with hypoxia, vasodilator responses to acetylcholine were inhibited by N omega-nitro-L-arginine methyl ester and vasodilator responses to levcromakalim were reduced by glybenclamide. In addition, L158809 did not alter the pressor response to hypoxia, whereas responses to angiotensin II were reduced in a selective manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of responses to adrenomedullin and calcitonin gene-related peptide in the pulmonary vascular bed of the cat.

Pulmonary vascular responses to the newly discovered hypotensive peptide, adrenomedullin, were compared with responses to the structurally related peptides, calcitonin gene-related peptide (CGRP) and amylin, in the intact-chest cat. Under conditions of controlled blood flow, when tone in the pulmonary vascular bed had been raised to a high steady level, intralobar injections of adrenomedullin (0.03-1 nmol), CGRP (0.1-3 nmol), and amylin (0.1 and 0.3 nmol) caused dose-related decreases in lobar arterial pressure without changing left atrial pressure. In terms of relative vasodilator activity in the pulmonary vascular bed, the dose of the peptide that decreased lobar arterial pressure 7.5 mm Hg (ED7.5 mm Hg) was significantly lower for adrenomedullin than for CGRP. The duration of the pulmonary vasodilator responses to CGRP was longer than for adrenomedullin, and both peptides decreased systemic arterial pressure when injected into the perfused lobar artery in the higher doses studied. The present data demonstrate that synthetic human adrenomedullin and CGRP have potent but relatively short-lasting vasodilator activity in the pulmonary vascular bed. These data show also that amylin, a structurally related pancreatic peptide, also has significant pulmonary vasodilator activity.

Comparison of responses to pituitary adenylate cyclase activating peptides 38 and 27 in the pulmonary vascular bed of the cat.

Responses to pituitary adenylate cyclase activating polypeptide (PACAP)-38 were investigated and compared with responses to PACAP-27 and vasoactive intestinal polypeptide (VIP) in the pulmonary vascular bed of the intact-chest cat under constant flow conditions. Under low resting tone baseline conditions, injections of PACAP-38 had little or no effect on lobar arterial pressure; however, when tone in the pulmonary vascular bed was raised to a high steady level (35-40 mm Hg) with U46619, intralobar injections of PACAP-38 caused dose-related decreases in lobar arterial pressure without altering left atrial pressure. The peptide induced biphasic changes in systemic arterial pressure. PACAP-38 was more potent than VIP in decreasing lobar arterial pressure, and both peptides were significantly less potent than PACAP-27 in dilating the pulmonary vascular bed. The present data show that PACAP-38 has significant vasodilator activity in the pulmonary vascular bed of the cat, and that the 27 amino acid form of the peptide is approximately 3-fold more potent than PACAP-38.

Effects of 17 beta-estradiol on endothelium-dependent relaxation induced by acetylcholine in female rat aorta.

Estrogens have been postulated to play an important role in modulation of vascular responses to endogenous reactive substances. The effects of chronic in vivo treatment with 17 beta-estradiol on relaxant responses to acetylcholine were investigated in the rat aorta isolated from prepubertal female rats. The selectivity of effects of 17 beta-estradiol on acetylcholine-induced relaxation was evaluated using histamine, another endothelium-dependent relaxant in the rat aorta. 17 beta-Estradiol significantly enhanced endothelium-dependent relaxation induced by acetylcholine, but did not alter the vascular responses to acetylcholine in endothelium-denuded aortic rings isolated from prepubertal female rats. In contrast, 17 beta-estradiol did not change endothelium-dependent relaxation induced by histamine in endothelium-intact aortic rings. The results of the present study demonstrate that 17 beta-estradiol selectively enhances acetylcholine-induced endothelium-dependent relaxation in the rat aorta.

Synthetic human adrenomedullin and ADM15-52 have potent short-lasting vasodilator activity in the pulmonary vascular bed of the cat.

Responses to synthetic human adrenomedullin, a novel hypotensive peptide localized in several organ systems, including the lung, and the carboxy terminal 15-52 amino acid fragment of adrenomedullin (ADM15-52) were investigated in the pulmonary vascular bed of the intact-chest cat. Under constant flow conditions when baseline tone in the pulmonary vascular bed was raised to a high steady level, injections of adrenomedullin and ADM15-52 into the perfused lobar artery in doses of 0.1-1 nmol, caused significant dose-related decreases in lobar arterial pressure. Since left atrial pressure was unchanged, the decreases in lobar arterial pressure reflect decreases in pulmonary lobar vascular resistance. Adrenomedullin and ADM15-52 exhibited similar vasodilator activity and were approximately 3-fold more potent than bradykinin in the pulmonary vascular bed of the cat. Pulmonary vasodilator responses to adrenomedullin and ADM15-52 were rapid in onset and lasted for 150-200 sec, depending on the dose of the peptide injected. The present results demonstrate that synthetic human adrenomedullin and ADM15-52 possess potent, short-lasting vasodilator activity in the pulmonary vascular bed of the cat and suggest that amino acids 15-52 in the peptide are important for the expression of vasodilator activity in the pulmonary vascular bed of the cat.

Comparative effects of adrenomedullin, an adrenomedullin analog, and CGRP in the pulmonary vascular bed of the cat and rat.

Responses to synthetic human adrenomedullin (ADM), a novel hypotensive peptide initially isolated from human pheochromocytoma cells, an ADM analog (ADM15-52), and a structurally related peptide, calcitonin gene-related peptide (CGRP), were compared in the pulmonary vascular bed of the cat and rat under constant flow conditions. When tone was increased with U46619, intraarterial injections of ADM (0.03-0.3 nmol), ADM15-52 (0.03-0.3 nmol), and of CGRP (0.03-0.3 nmol) caused dose-related decreases in pulmonary arterial perfusion pressure. When the relative vasodilator activity of the peptides was compared on a nmol basis, ADM was approximately 10-fold more potent in the cat than in the rat, whereas vasodilator responses to CGRP were very similar in both species. CGRP was slightly more potent than ADM in the rat, whereas ADM was slightly more potent than CGRP in the cat. ADM and ADM15-52 had similar pulmonary vasodilator activity in the cat, whereas the full sequence peptide was slightly more potent than ADM15-52 in the rat. The present data demonstrate that ADM has significant vasodilator activity in the pulmonary vascular beds of the cat and of the rat, and that the relative potency of the vasodilator effects of ADM and ADM15-52 are different in the two species.

[Analytical study on processing of Rheum palmatum L. by HPLC].

This paper deals with the effect of processing on Rheum palmatum. A HPLC method for the determination of rhein in the drug processed five different ways has been established. The method is simple, specific and accurate. The recovery is 98.52% and relative standard deviation.