[Bushen Huoxue Recipe Inhibited Vascular Calcification in Chronic Renal Failure Rats by Regulating BMP-2/Runx2/Osterix Signal Pathway].

OBJECTIVE: To observe the effect of Bushen Huoxue Recipe (BHR) on inhibiting vascular calcification (VC) in chronic renal failure (CRF) rats by regulating BMP-2/Runx2/Osterix signal pathway, and to explore its possible mechanism. METHODS: Thirty SD rats were randomly divided into the normal group, the model group, and the BHR group, 10 in each group. Rats in the model group and the BHR group were administered with 250 mg/kg adenine suspension by gastroagavage and fed with 1.8% high phosphorus forage, once per day in the first 4 weeks, and then gastric administration of adenine suspension was changed to once per two days in the following 5-8 weeks. Rats in the BHR group were administered with BHR at the daily dose of 55 g/kg by gastrogavage in the first 8 weeks, once per day. Equal volume of normal saline was given to rats in the normal group by gastrogavage for 8 weeks. Histological changes in renal tissue and aorta VC were observed by HE staining and alizarin red staining respectively. Levels of calcium (Ca), phosphorus (P), serum creatinine (Cr), blood urea nitrogen (BUN), and intact parathyroid hormone (iPTH) in serum were detected. Protein expression levels of bone morphogenetic protein (BMP-2), Runt related transcription factor (Runx2) , and Osterix were detected by Western blot. RESULTS: HE staining showed that compared with the normal group, disordered glomerular structure, tubular ectasia and dropsy, intracavitary inflammatory cell infiltration, dark brown crystal deposition in kidney tubules, renal interstitial fibrosis, and decreased number of renal blood vessels in the model group. Compared with the model group, normal glomerular numbers increased more, reduced degree of tubular ectasia, decreased number of inflammatory cells, and reduced adenine crystal deposition in the BHR group. Alizarin red staining showed that compared with the normal group, calcified nodes could be found in the model group, with extensive deposition of red particle in aorta. Compared with the model group, calcified nodes were reduced in the BHR group. Compared with normal group, serum levels of P, SCr, BUN, and iPTH significantly increased, serum Ca level significantly decreased, protein expressions of BMP-2, Runx2, Osterix also increased in the model group (P < 0.05, P < 0.01). Compared with the model group, serum levels of P, SCr, BUN, and iPTH levels significantly decreased, serum Ca level significantly increased, protein expressions of BMP-2, Runx2, Osterix also decreased in the BHD group (P < 0.05, P < 0.01). CONCLUSION: BHD could improve renal function, Ca-P metabolism, and renal histological changes in CHF rats, down-regulate the expression level of BMP-2/Runx2/Osterix signal pathway in vascular calcification of CRF, which might be one of the mechanisms for inhibiting VC in CHF.

[Effect of Mild Moxibustion with Different Terms at Different Acupoints on Renal Functions and Renal Pathological Changes in Focal Segmental Glomerulosclerosis Rats].

OBJECTIVE: To observe changes of urinary microprotein, and serum creatinine, urea nitrogen and uric acid levels in focal segmental glomerulosclerosis (FSGS) rats treated by mild moxibustion, so as to explore the mechanism of moxibustion underlying improvement of FSGS. METHODS: SD rats were randomized into normal control (normal), sham operation (sham), model, medication (Losartan), moxibustion-Shenshu (BL 23) and moxibustion-Geshu (BL 17) groups. The latter two moxibustion groups were further divided into 10 min, 20 min and 30 min subgroups (n=6 in each group/subgroups). The FSGS model was established by unilateral nephrectomy combined with injection of Losartan into the tail vein twice. Mild moxibustion was applied to bilateral BL 17 and BL 23 for 10, 20 and 30 min, respectively, once every other day, for 12 weeks. The contents of urinary microglobulin α 1, micro-albumin, transferring and IgG were assayed using enzyme linked immunosorbent assay (ELISA), and serum creatinine, urea nitrogen and uric acid contents (indexes of renal function) determined using an automatic biochemical analyzer. The pathological changes of the kidney tissue was observed by using a microscope after periodic acid schiff (PAS) staining. RESULTS: No significant differences were found between the normal control and sham groups in the levels of all the urinary and serum indexes (P>0.05) and pathological changes of the renal tissues. Compared with the normal control group, the contents of urinary microglobulin α 1, micro-albumin, transferrin and IgG, and serum creatinine, urea nitrogen and uric acid were significantly increased in the model group (P<0.01). Following medication and moxibustion, the contents of the aforementioned 7 indexes in the Losartan group, and urinary microglobulin α 1, micro-albumin, transferrin and IgG, and serum creatinine levels in the moxibustion BL 23-20 min and 30 min groups, and the micro-albumin and transferrin contents in the BL-17 10 min group and IgG level in the BL-23 10 min group, the serum creatinine and urea nitrogen levels at the 3 time-points of both moxibustion BL 23 and BL 17 groups, and serum uric acid in the moxibustion BL 23 30 min, and BL17 20 and 30 min groups were all considerably down-regulated (P<0.05, P<0.01). The therapeutic effects of moxibustion 30 min were notably better than moxibustion 10 min in reducing urinary microglobulin α 1, micro-albumin, transferring and IgG levels (P<0.05, P<0.01). Results of PAS staining showed that the injury of the renal tissue as the endothelial and mesangial cellular proliferation, collagen proteinosis, interstitial fibrosis, etc were relatively milder in the Losartan and moxibustion 20 and 30 min groups. CONCLUSIONS: Mild moxibustion may reduce proteinuria, and improve the kidney function and pathological changes in FSGS rats, and longer duration of moxibustion is better in achieving therapeutic effect.

Variation in Phenolics, Flavanoids, Antioxidant and Tyrosinase Inhibitory Activity of Peach Blossoms at Different Developmental Stages.

Peach blossoms were harvested and classified into six developmental stages: (I) bud emerging stage; (II) middle bud stage; (III) large bud stage; (IV) initial-flowering stage; (V) full-flowering stage; and (VI) end-flowering stage. The contents of total phenolics, flavanoids, individual phenolic compounds as well as antioxidant and tyrosinase inhibitory activity of peach blossoms at different developmental stages were investigated. The total phenolic contents varied from 149.80 to 74.80 mg chlorogenic acid equivalents/g dry weight (DW), and the total flavanoid contents ranged from 93.03 to 44.06 mg rutin equivalents/g DW. Both the contents of total phenolics and flavanoids decreased during blossom development. Chlorogenic acid was the predominant component, accounting for 62.08%-71.09% of the total amount of identified phenolic compounds in peach blossom. The antioxidant capacities determined by different assays and tyrosinase inhibitory activity also showed descending patterns during blossom development. Significant correlations were observed between antioxidant capacities with contents of total phenolics and total flavanoids as well as chlorogenic acid, cinnamic acid and kaempferol-3-O-galactoside, while the tyrosinase inhibitory activity had lower correlations with total phenolics and total flavanoids as well as chlorogenic acid, quercetin-3-O-rhamnoside, kaempferol-3-O-galactoside and cinnamic acid. The antioxidant activities of peach blossom seemed to be more dependent on the phenolic compounds than tyrosinase inhibitory activity.

Soft tissue contusion repairing effects of Hong Yao with different penetration enhancers.

ETHNOPHARMACOLOGICAL RELEVANCE: Hong Yao aerosol (HYA) is a new dosage form developed from Hong Yao, a traditional Chinese medicine preparation, which has the efficacy of promoting soft tissue contusion repair, anti-inflammation, and analgesia. AIM: To evaluate the soft tissue contusion repairing, anti-inflammatory and analgesic effects of HYA formulations with different penetration enhancers (PE) and to quantify the transdermal absorption of HYA component. MATERIALS AND METHODS: Three preparations of HYA with different PEs were made: DMSO preparation (5% DMSO as additional PE), Azone preparation (3% azone as additional PE), and NAPE preparation (no additional PE). Four in vivo rodent models were employed to evaluate the pharmacodynamic effects of the HYAs: mouse soft tissue contusion model, rat paw edema model, mouse ear swelling model, and mouse analgesia model of electric-stimulated foot. In vitro skin penetration experiment was applied to evaluate the transdermal absorption of HYA components. RESULTS: All HYA preparations showed effects on soft tissue contusion repairing, anti-inflammation, and analgesia compared with the blank control (p<0.05). There was no significant difference among the three preparations. Pathological variation of mice skin and the pain response time (PRT) reduction phenomena indicated that DMSO preparation induced skin irritation. In vitro skin penetration experiment showed no significant difference between DMSO and NAPE on absorption enhancement of ferulic acid from HYA. CONCLUSIONS: NAPE preparation was considered as best and Menthol/borneol (6.3%, W/W, 4:1.75) in HYA might be a good PE combination.

[Effects of high-dose N-acetylcysteine on the lung tissues of rats exposed to silica].

OBJECTIVE: To explore the effects of high-dose N-acetylcysteine on the lung tissues of rats exposed to silica. METHODS: Ninety-six Wistar rats were randomly divided into model group, intervention group and control group (32 rats for each group). The rats of model group and intervention group were exposed to silica by intratracheal infusion of silica dust suspension. The rats in the intervention group were orally given high dose N-acetylcysteine. In 3, 7, 14, 28 days after exposure, eight rats in each group were sacrificed, respectively and the lung samples were collected. The pathological changes of lung were evaluated by HE and Masson staining methods. The levels of TNF-alpha and IL-8 in the BALF were detected by ELISA. RESULTS: Compared with the control group, the alveolitis and pulmonary fibrosis in the intervention group were significantly reduced. In 3, 7, 14, 28 days after exposure, the lung/body coefficients in the intervention group were 9.30 +/- 0.78, 6.29 +/- 0.74, 7.63 +/- 0.88, 6.06 +/- 1.16 respectively, which were significantly lower than those (13.84 +/- 1.61, 9.23 +/- 0.87, 11.23 +/- 1.25, 9.56 +/- 0.76, P < 0.01 ) in the model group (P < 0.01). At the different time points, the levels of TNF-alpha and IL-8 in the BALF in the intervention group were significantly higher than those in the control group (P < 0.01), but were significantly lower than those in the model group (P < 0.01). CONCLUSION: The intervention with high dose N-acetylcysteine can significantly reduce the alveolitis and the TNF-alpha and IL-8 levels in the BALF, therefore, inhibit and delay the development of pulmonary fibrosis of rats exposed to silicon dioxide.

[Effects of two controlled-release fertilizers with different proportions of N, P and K on the nutrient uptake and growth of Chrysanthemum morifolium Ramat].

A pot experiment was conducted to study the effects of two controlled-release fertilizers CRFA (4% resin-coated, N: P2O5: K2O = 14: 14: 14) and CRFB (4% resin-coated, N: P2O5: K2O = 20: 8:10) on the nutrient uptake and growth of Chrysanthemum morifolium, with common compound fertilizer CCF (N: P2O5: K2O = 15: 15: 15) as the control. Six treatments were installed, i. e., CCF1 (CCF, 6 g N x pot(-1)), CCF2 (CCF, 3 g N x pot(-1)), CRFA1 (CRFA, 6 g x pot(-1)), CRFA2 (CRFA, 3 g x pot(-1)), CRFB1 (CRFB, 6 g x pot(-1)), and CRFB (CRFB, 3 g x pot(-1)). On the 30th day of applying common compound fertilizer CCF1 and CCF2, soil available N, P and K contents were 163.29 and 145.26 mg x kg(-1), 180.39 and 163.13 mg x kg(-1), and 300.08 and 213.15 mg x kg(-1), respectively, and decreased rapidly since then. In treatments CRFA1, CRFB1, CRFA2, and CRFB, soil available N content increased slowly, and reached the peak on the 60th day after fertilizing, being 129.51, 138.65, 118.36, and 126.31 mg x kg(-1), respectively. Soil available P content had the same variation trend. Its maximum concentration was 169.54 and 133.46 mg x kg(-1) in treatments CRFA1 and CRFA2 on the 30th day after fertilizing, and 137.13 and 84.68 mg x kg(-1) in treatments CRFB1 and CRFB2 on the 60th day after fertilizing, and decreased slowly then. The agronomic traits such as leaf area, leaf area index, branch number, flowering rate, flower number, and flower diameter, etc., in treatments CRFA and CRFB were obviously better than those of the control, and CRFB was better than CRFA, suggesting that CRFB more matched the nutrient demand of C. morifolium. Under the conditions of present experiment, applying CRFB2 obtained the highest yield.

Abdominal acupuncture for insomnia in women: a randomized controlled clinical trial.

A randomized single-blind trial was conducted to evaluate the efficacy of short-term abdominal acupuncture as a novel treatment for insomnia in Chinese women. Forty-four patients between the ages of 22 and 56 were randomly assigned to an acupuncture (n = 23) and a medication group (n = 21). The acupuncture group received abdominal acupuncture once a day for the first three days and once every three days for the remaining 11 days. In addition, every subject in acupuncture group also received a placebo pill once daily. Abdominal acupuncture was administered according to a standardized protocol involving four master and four adjunctive acupoints: Zhongwan (CV 12), Xiawan (CV 10), Guanyuan (CV 4), and Qihai (CV 6); bilateral Shangqu (KI 17), Huaroumen (ST 24), Xiafengshidian, and Qipang. Subjects in the medication group were treated with sham acupuncture at the same time as the acupuncture group and received estazolam once a day. The outcome measure was the Leeds Sleep Evaluation Questionnaire (LSEQ), administered before and after the trial. Subjects who received abdominal acupuncture lowered their LSEQ scores by an average of 26.32 points (95% CI: 37.34, 15.30). After controlling for potential confounding factors, the effect of abdominal acupuncture in relieving insomnia was still statistically significant. Results indicate that short-term abdominal acupuncture is more effective than pharmacological treatment for relieving insomnia in adult women and has few adverse effects.

[Pharmacokinetic and tissue distribution study of scutellarin in rats].

OBJECTIVE: To investigate the pharmacokinetic and distribution character of scutellarin in plasma and tissues in rats, in order to provide some references for rational drug use in the clinic. METHOD: The solution of scutellarin was administered to rats (80 mg x kg(-1)) by oral gavage. A high performance liquid chromatography method determinated the scutellarin concentration in rat plasma and tissue. The plasma samples were performed by solid phase extraction method. The other biological samples were extracted by ethyl acetate. RESULT: The range of scutellarin in plasma and tissue in rats were 10-1280 ng x mL(-1) (R2 > 0.99), 40-1280 ng x g(-1) (R2 > 0.99), respectively. The lowest detection of scutellarin were 10 ng x mL(-1) and 40 ng x g(-1), the precision were less than 8%. The main pharmacokinetic parameters of scutellarin were as follows: tmax, Cmax, AUC and MRT being (7.7 +/- 0.9) h, (288.0 +/- 75.2) microg x L(-1), (5.6 +/- 1.6) microg x mL(-1) x h(-1), (17.5 +/- 1.4) h(-1), respectively. CONCLUSION: These methods applied the study of pharmacokinetics of scutellarin. After oral the scutellarin in rats, the concentration-time course doesn't obey any compartment model. The concentration-time curve is the double peaks.

[Comparison between the qualitative similarity and the quantitative similarity of chromatographic fingerprints of traditional Chinese medicines].

To explore the differences between the qualitative similarity and the quantitative similarity of chromatographic fingerprints of traditional Chinese medicines, the quantitative similarity calculated by vector shadow C%, apparent quantitative similarity R%, quantitative similarity P%, etc. were firstly proposed to disclose the quantitative information characteristics of traditional Chinese medicines fingerprints. The HPLC fingerprints of both the standard Fructus gardeniae and the ten batches of Fructus gardeniae produced in different places were evaluated by the new parameters to obtain good results. The contrasted fingerprint contained 35 peaks while geniposide was selected as the reference peak. The HPLC fingerprint had good precision and reproducibility with the RSD of the relative retention time less than 1.5% and the RSD of the relative peak area within 5%. The qualitative similarity and quantitative similarity between each crude drug and the contrasted fingerprint were quantitatively calculated, the values of C%, P%, etc., were applied in the quality control practice, which had less errors. What is more, this method could be used for the overall quality control of Fructus gardeniae and especially suits for qualitative and quantitative evaluations of the chromatographic fingerprints both in chemical constituent distribution and in contents. The quantitative parameters such as C% and P% can be used to objectively, authentically and thoroughly display the content information characteristics. When they combined with the qualitative similarity, it will be the good method to evaluate the chromatographic fingerprints of traditional Chinese medicines.

[Experimental study on the theraputic effect of crescent Euphorbia on lewis lung cancer in mice].

OBJECTIVE: To observe the inhibitory effects of crescent euphorbia on tumor growth, immunoregulation, the side effect on the mice blood system, cell cycle and to investigate its effect on apoptosis of tumor cells. METHOD: Crescent euphorbia was administered gastrally to C57BL/6J mouse implanted with Lewis lung cancer for 12 days. Inhibition on tumor growth, immunoregulation,the side effect on the mice blood system and cell cycle were observed and its effect on apoptosis of lung cancer cells were investigated. RESULT: (1) Inhibitory rates of crescent euphorbia at doses of 7.5, 15, 30 g x kg(-1) and 60 g x kg(-1) were 0.61%, 16.93%, 32.81% and 58.26% respectively, significant differenel with those of controls at doses of 30 g x kg(-1) and 60 g x kg(-1)(P < 0.05). (2) Although no effect on thymus index in normal mouse at all dose of crescent euphorbia was found, spleen index in tumor-bear mice was reduced significantly at dose of 60 g x kg(-1)(P < 0.05). (3) No side effect on the miceblood system was found. (4) The percentage of S phase of cell cycle was increased in the group of crescent euphorbia at dose of 30 g x kg(-1), 60 g x kg(-1) and the apoptotic rate was (16.43 +/- 18.69)% and (24.37 +/- 15.48)% by flow eytometry (P < 0.05). The apoptotic index (AI)of these two groups were (4.00 +/- 7.50)% and (5.93 +/- 5.96)% by TUNEL, however apoptosis was found in the group at doses of 60 g kg(-1) (P < 0.01). CONCLUSION: Crescent euphorbia can inhibit the growth of Lewis lung cancer in mice, no side effection for the mice blood system and the effect might be associated with induction of apoptosis on cancer cells.

Oridonin induces a caspase-independent but mitochondria- and MAPK-dependent cell death in the murine fibrosarcoma cell line L929.

Oridonin, an active component isolated from Rabdosia rubescences, has been reported to exhibit antitumor effects, but little is known about its molecular mechanisms of action. In this study, the growth-inhibitory activity of oridonin for L929 cells is in time- and dose-dependent manner. After treatment with various concentrations of oridonin for 12 h, the majority of L929 cells underwent apoptosis as measured by an LDH activity-based assay. Although apoptotic bodies were observed in oridonin-treated L929 cells, DNA fragmentation as a hallmark of apoptosis was not found. The pan-caspase inhibitor, z-VAD, and caspase-3 inhibitor, z-DEVD, sensitized L929 cells to oridonin, however, a PARP inhibitor (DPQ) effectively blocked oridonin-induced cell death. After 12 h treatment, PARP proenzyme was significantly cleaved. This result indicated that oridonin-induced L929 cell death required PARP degradation in a caspase-independent manner. In addition, an MEK/ERK inhibitor (PD98059) markedly blocked oridonin-induced cell death, whereas a p38 inhibitor (SB203580) and JNK inhibitor (SP600125) weakly protected the cells against death. Treatment with 41.2 microM oridonin for 12 h induced significant and persistent ERK activation and p38 inactivation in L929 cells without evident changes in the protein levels. The responsiveness of ERK and p38 to oridonin suggests the involvement of these kinases in this apoptotic process. Moreover, oridonin increased the ratio of Bax/Bcl-2 protein expression, whereas it had no effect on the expression of Bcl-xL. These results indicate that regulation of the Bcl-2 and MAPK families maybe the effector mechanisms of oridonin-induced L929 cell death, independent of the caspase pathway.

Oridonin induces apoptosis of HeLa cells via altering expression of Bcl-2/Bax and activating caspase-3/ICAD pathway.

AIM: To study the mechanisms by which oridonin inhibited HeLa cell growth in vitro. METHODS: Viability of oridonin-induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. Caspase activity was assayed using fluorometric protease assay. ICAD, Bcl-2, and Bax proteins expression were detected by Western blot analysis. RESULTS: Oridonin induced oligonucleosomal fragmentation of DNA and increased caspase-3 activity, on the other hand, reduced the expression of inhibitor of caspase-3-activated DNase (ICAD), a caspase-3 substrate, at 12 h in HeLa cells. Oridonin-induced DNA fragmentation, caspase-3 activation and down-regulation of ICAD expression were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk (z-Asp-Glu-Val-Asp-fmk). However, pretreatment with an inhibitor of poly (ADP-ribose) polymerase (PARP), 3, 4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ), did not suppress oridonin-induced HeLa cell death. In addition, oridonin-induced apoptosis was associated with an increase in the expression of the apoptosis inducer Bax, and a significant reduction in expression of the apoptosis suppressor Bcl-2 in mitochondria. CONCLUSION: Oridonin induces HeLa cells apoptosis by altering balance of Bcl-2 and Bax protein expression and activation of caspase-3/ICAD pathway.