Ultraviolet light C in promoting rapid healing of delayed wound union: A case report.

INTRODUCTION AND IMPORTANCE: The purpose of this case is to present a useful phototherapy for rapid healing of recalcitrant postoperative wound disunion due to implantation of allogeneic bone and metal. CASE PRESENTATION: In this case, a middle-aged man was diagnosed as complex fracture of the left tibial plateau which caused movement loss, swelling and pain of the left knee. The patient's wound disunion was still present after been surgery for 26 days, with yellowish liquid persistently oozing out of the wound after several wound dressing. We treated the postoperative wound with ultraviolet light C (UVC) to promote wound healing. CLINICAL DISCUSSION: Incision disunion is a common complication of surgery, which increases patients' hospital days. Prevention and treatment of wound disunion is one of the key considerations for perioperative management. CONCLUSION: During 3-day consecutive UVC treatment, the yellowish wound exudate gradually reduced and disappeared. Subsequently, the non-healing postoperative wound scared over and healed rapidly. As a convenient, non-invasive, non-polluting and effectively physical therapy, UVC can promote rapid healing of recalcitrant wound disunion caused by implants in complex tibial plateau fracture.

Is There a Regular Pattern in the Recovery of Parathyroid Function After Thyroid Cancer Surgery?

PURPOSE: To investigate whether there is a pattern of recovery of parathyroid function after thyroid cancer surgery. PATIENTS AND METHODS: The study included 183 patients with papillary thyroid cancer (PTC) who underwent "total thyroidectomy (TT)" plus "unilateral central lymph node dissection (UCLND)" or "bilateral central lymph node dissection (BCLND)". The intact parathyroid hormone (iPTH) and serum calcium (sCa) were analyzed several times within 1 month after surgery to explore the recovery pattern of parathyroid gland function. Then, these 183 cases were divided into group A (97 cases) with UCLND and group B (86 cases) with BCLND to analyze whether the impairment and recovery of parathyroid function were different between the two subgroups. RESULTS: Postoperative hypoparathyroidism was seen in 115 out of 183 cases. iPTH values decreased significantly on postoperative day (POD) 1 compared with preoperative values, dropped to the lowest point on POD 3, showed an increasing trend on POD 5 and 14, and increased to 85.0% of preoperative values at POD30, whereas changes in sCa differ from changes in iPTH, which showed the lowest sCa value on POD1, and rebounded on the POD3 with the intervention of calcium supplementation, and continued to rise on the POD5 and POD14, and the sCa value reached 96.6% of the preoperative level at POD30. Subgroup analysis showed that temporary hypoparathyroidism was more pronounced in group B than in group A. SCa and iPTH levels in both subgroups showed the same trend of first decrease and then increase. CONCLUSION: The recovery of hypocalcemia and hypo-iPTHemia in the first month after thyroid cancer surgery shows a trend of decreasing and then increasing, and knowing the recovery of parathyroid function at different time points is of great value to surgeons and patients alike.

Clinical evidence-guided network pharmacology analysis reveals a critical contribution of ß1-adrenoreceptor upregulation to bradycardia alleviation by Shenxian-Shengmai.

BACKGROUND: Shenxian-Shengmai (SXSM) Oral Liquid is a CFDA-approved patent Chinese Herbal medicine, which has been clinically used for the treatment of bradycardia. However, its active components and action mechanism remain to be established. The present study aimed to evaluate the efficacy of SXSM on bradycardia and to identify the possible active components and their pharmacological targets for this action. METHODS: A literature-based meta-analysis was performed to evaluate the clinical efficacy of SXSM on bradycardia, which was confirmed by a rat ex vivo cardiac model. Network pharmacology analysis was then conducted to reveal the potential targets of SXSM active components and their anti-arrhythmia mechanisms. Finally, the identified drug-target interaction was confirmed by immunofluorescence assay in cardiomyocyte. RESULTS: Meta-analysis of the available clinical study data shows that Shenxian-Shengmai Oral Liquid has a favorable effect for bradycardia. In an ex vivo bradycardia model of rat heart, SXSM restored heart rate by affecting Heart rate variability (HRV) which is associated with autonomic nervous system activity. A drug-target-pathway network analysis connecting SXSM components with arrhythmia suggested that a prominent anti-arrhythmia mechanisms of SXSM was via ß1-adrenergic signaling pathway, which was subsequently validated by immunofluorescence assay showing that SXSM indeed increased the expression of ADRB1 in cultured cardiomyocytes. CONCLUSION: By combining approaches of clinical evidence mining, experimental model confirmation, network pharmacology analyses and molecular mechanistic validation, we show that SXSM is an effective treatment for bradycardia and it involves multiple component interacting via multiple pathways, among which is the critical ß1-adrenergic receptor upregulation. Our integrative approach could be applied to other multi-component traditional Chinese medicine investigation where ample clinical data are accumulated but advanced mechanistic studies are lacking.

Inhibition of Autophagy Attenuated Intestinal Injury After Intestinal I/R via mTOR Signaling.

BACKGROUND: Intestinal ischemia/reperfusion (I/R) is a grave condition related to high morbidity and mortality. Autophagy, which can induce a new cell death named type II programmed cell death, has been reported in some intestinal diseases, but little is known in I/R-induced intestinal injury. In this study, we aimed to explore the role of autophagy in intestinal injury induced by I/R and its potential mechanisms. MATERIALS AND METHODS: The rats pretreated with rapamycin or 3-methyladenine had intestinal I/R injury. After reperfusion, intestinal injury was measured by Chiu's score, intestinal mucosal wet-to-dry ratio, and lactic acid level. Intestinal mucosal oxidative stress level was measured by malondialdehyde and superoxide dismutase. Autophagosome, LC3, and p62 were detected to evaluate autophagy level. Mammalian target of rapamycin (mTOR) was detected to explore potential mechanism. RESULTS: Chiu's score, intestinal mucosal wet-to-dry ratio, lactic acid level, malondialdehyde level, autophagosomes, and LC3-II/LC3-I were significantly increased, and superoxide dismutase level and expression of p62 were significantly decreased in intestinal mucosa after intestinal ischemia/reperfusion. Pretreatment with rapamycin significantly aggravated intestinal injury evidenced by increased Chiu's score, intestinal mucosal wet-to-dry ratio and lactic acid level, increased autophagy level evidenced by increased autophagosomes and LC3-II/LC3-I and decreased expression of p62, and downregulated expression of p-mTOR/mTOR. On the contrary, pretreatment with 3-methyladenine significantly attenuated intestinal injury and autophagy level and upregulated expression of p-mTOR/mTOR. CONCLUSIONS: In summary, autophagy was significantly enhanced in intestinal mucosa after intestinal ischemia/reperfusion, and inhibition of autophagy attenuated intestinal injury induced by I/R through activating mTOR signaling.

Novel Competitive Chemiluminescence DNA Assay Based on Fe3O4@SiO2@Au-Functionalized Magnetic Nanoparticles for Sensitive Detection of p53 Tumor Suppressor Gene.

A simple, rapid response time and ultrahigh sensitive chemiluminescence (CL) DNA assay based on Fe3O4@SiO2@Au-functionalized magnetic nanoparticles (Au-MNPs) was developed for detection of p53 tumor suppressor gene. In this study, 2',6'-dimethylcarbonylphenyl-10-sulfopropyl acridinium-9-carboxylate 4'-NHS ester (NSP-DMAE-NHS), as a new kind of highly efficient luminescence reagent, was immobilized on the complementary sequence of the wild-type p53 (ssDNA) to improve the detection sensitivity. The optimal concentration of ssDNA-(NSP-DMAE-NHS) conjugates mixed with the wild-type p53 (wtp53) samples respectively. Then, the wtp53-Au-MNPs conjugates were added to continue the competitive reaction in the above solution. Subsequently, the Au-MNPs separated under magnetic field, measured by a homemade luminescent measurement system. Under optimal conditions, the method exhibited ultrasensitive sensitivity with a detection limit of 0.001 ng mL-1 (0.16 pM), a wide range of liner response from 0.001 ng mL-1~6.6 µg mL-1. Therefore, the immunomagnetic nanocomposites-based detection strategy was rapid, low-cost, and highly sensitive that can be easily extended to the early diagnosis of cancer development and monitoring of patient therapy.

MOF based fluorescent assay of xanthine oxidase for rapid inhibitor screening with real-time kinetics monitoring.

The activity assay of xanthine oxidase (XO) is of great application value in clinical diagnosis because the abnormal level of this enzyme is related to a series of pathological states. In this work, a Zr based metal-organic framework (BTB-MOF) with stable photoluminescence in pure water and buffer solution was synthesized. The examination about the fluorescent responses of this material to xanthine and its oxidation product, uric acid, showed that, although both of them affected the emission of BTB-MOF in quenching form, the efficiencies presented much difference. Taking advantage of this feature, a fluorescent method was developed for the activity assay of XO, that is, BTB-MOF was added to the enzymatic oxidation system as a sensor to transduce the proceeding of the reaction real-timely to the signal of fluorescent intensity change. Our method can work under the interference of normal biologically related species, and precisely reflect XO activity in the range of 0.2-40 U L-1 (detection limit = 0.004 U L-1). With consecutive fluorescence intensity scan, this assay could be applied as a high speed screening method of XO inhibitors with the testing time of 1 min. This work shows the wide potential application of MOFs in enzyme analysis.

An integrated anti-arrhythmic target network of compound Chinese medicine Wenxin Keli revealed by combined machine learning and molecular pathway analysis [corrected].

Wenxin Keli (WK), a Chinese patent medicine, is known to be effective against cardiac arrhythmias and heart failure. Although a number of electrophysiological findings regarding its therapeutic effect have been reported, the active components and system-level characterizations of the component-target interactions of WK have yet to be elucidated. In the current study, we present the first report of a new protective effect of WK on suppressing anti-arrhythmic-agent-induced arrhythmias. In a model of isolated guinea pig hearts, rapid perfusion of quinidine altered the heart rate and prolonged the Q-T interval. Pretreatment with WK significantly prevented quinidine-induced arrhythmias. To explain the therapeutic and protective effects of WK, we constructed an integrated multi-target pharmacological mechanism prediction workflow in combination with machine learning and molecular pathway analysis. This workflow had the ability to predict and rank the probability of each compound interacting with 1715 target proteins simultaneously. The ROC value statistics showed that 97.786% of the values for target prediction were larger than 0.8. We applied this model to carry out target prediction and network analysis for the identified components of 5 herbs in WK. Using the 124 potential anti-arrhythmic components and the 30 corresponding protein targets obtained, an integrative anti-arrhythmic molecular mechanism of WK was proposed. Emerging drug/target networks suggested ion channel and intracellular calcium and autonomic nervous and hormonal regulation had critical roles in WK-mediated anti-arrhythmic activity. A validation of the proposed mechanisms was achieved by demonstrating that calaxin, one of the WK components from Gansong, dose-dependently blocked its predicted target CaV1.2 channel in an electrophysiological assay.

A new azadirachta from the crude extracts of neem (Azadirachta Indica A. Juss) seeds.

One new compound, 2H, 3H-cyclopent[b] furo [2',3':4,5] naphtho [2,4-d] heptlactone-[3,7] furan-6aceticacid, 3-(acetyloxy)-8-(3-furyl)-2a, 4a, 4b, 4c,5,5a, 6, 6a, 8, 9,9a, 10a,10b-13 hydrogen-2a,5a,6a,5-tetramethyl-3-[[(2E)-2-methyl-1-oxo-2-butenyl]oxy]-methyl ester, named azadirachta R (1), along with 10 known ones, Azadirachta A, AZ-B, AZ-D, AZ-H, AZ-I, nimbin, deacetylnimbin, salannin, deacetylsalannin and azadiradione were isolated from the crude extracts of neem (Azadirachta indica A. Juss) seeds, which were determined by UV, IR, HR-ESI-MS and NMR data analyses. According to the in vitro antibacterial activity experimental results, this compound showed good antibacterial activity to two bacteria, the minimum inhibitory concentration and the minimum bactericidal concentration of compound 1 to two kinds of bacteria are 50 and 25 mg/L, respectively, which were determined by resazurin colour-micro-dilution method. The experimental results provide a theoretical basis for the comprehensive utilisation of azadirachtin compounds in the future.

[Species-associated differences in foliage-root coupling soil-reinforcement and anti-erosion].

This paper took four kinds of common soil and water conservation plants of the study area, Caragana microphylla, Salix psammophila, Artemisia sphaerocephala and Hippophae rhamnides at ages of 4 as the research object. Thirteen indicators, i.e., single shrub to reduce wind velocity ration, shelterbelt reducing wind velocity ration, community reducing wind velocity ration, taproot tensile strength, representative root constitutive properties, representative root elasticity modulus, lateral root branch tensile strength, accumulative surface area, root-soil interface sheer strength, interface friction coefficient, accumulative root length, root-soil composite cohesive, root-soil composite equivalent friction angle, reflecting the characteristics of windbreak and roots, were chose to evaluate the differences of foliage-root coupling soil-reinforcement and anti-erosion among four kinds of plants by analytic hierarchy process (AHP) under the condition of spring gale and summer rainstorm, respectively. The results showed the anti-erosion index of foliage-root coupling was in the sequence of S. psammophila (0.841) > C. microphylla (0.454) > A. sphaerocephala (-0.466) > H. rhamnides (-0.829) in spring gale, and C. microphylla (0.841) > S. psammophila (0. 474) > A. sphaerocephala (-0.470) > H. rhamnides (-0.844) in summer rainstorm. S. psammophila could be regarded as one of the most important windbreak and anti-erosion species, while C. microphylla could be the most valuable soil and water conservation plant for the study area.

Self-Replenishable Anti-Waxing Organogel Materials.

Solid deposition, such as the formation of ice on outdoor facilities, the deposition of scale in water reservoirs, the sedimentation of fat, oil, and grease (FOG) in sewer systems, and the precipitation of wax in petroleum pipelines, cause a serious waste of resources and irreversible environmental pollution. Inspired by fish and pitcher plants, we present a self-replenishable organogel material which shows ultra-low adhesion to solidified paraffin wax and crude oil by absorption of low-molar-mass oil from its crude-oil environment. Adhesion of wax on the organogel surface was over 500 times lower than adhesion to conventional material surfaces and the wax was found to slide off under the force of gravity. This design concept of a gel with decreased adhesion to wax and oil can be extended to deal with other solid deposition problems.

Glycogen synthase kinase-3ß is involved in electroacupuncture pretreatment via the cannabinoid CB1 receptor in ischemic stroke.

We have previously shown that electroacupuncture (EA) pretreatment produces neuroprotective effects, which were mediated through an endocannabinoid signal transduction mechanism. Herein, we have studied the possible contribution of the phosphorylated form of glycogen synthase kinase-3ß (GSK-3ß) in EA pretreatment-induced neuroprotection via the cannabinoid CB1 receptor (CB1R). Focal transient cerebral ischemia was induced by middle cerebral artery occlusion in rats. Phosphorylation of GSK-3ß at Ser-9 [p-GSK-3ß (Ser-9)] was evaluated in the penumbra tissue following reperfusion. Infarct size and neurological score were assessed in the presence of either PI3K inhibitors or a GSK-3ß inhibitor 72 h after reperfusion. Cellular apoptosis was evidenced by TUNEL staining and determination of the Bax/Bcl-2 ratio 24 h after reperfusion. The present study showed that EA pretreatment increased p-GSK-3ß(Ser-9) 2 h after reperfusion in the ipsilateral penumbra. Augmented phosphorylation of GSK-3ß induced similar neuroprotective effects as did EA pretreatment. By contrast, inhibition of PI3K dampened the levels of p-GSK-3ß(Ser-9), and reversed not only the neuroprotective effect but also the anti-apoptotic effect following EA pretreatment. Regulation of GSK-3ß by EA pretreatment was abolished following treatment with a CB1R antagonist and CB1R knockdown, whereas two CB1R agonists enhanced the phosphorylation of GSK-3ß. Therefore we conclude that EA pretreatment protects against cerebral ischemia/reperfusion injury through CB1R-mediated phosphorylation of GSK-3ß.

Hypocholesterolemia of Rhizoma Coptidis alkaloids is related to the bile acid by up-regulated CYP7A1 in hyperlipidemic rats.

This study is to investigate the cholesterol-lowering effect and the new mode of action of coptis alkaloids on high lipid diet-induced hyperlipidemic rats. Coptis alkaloids extract (CAE) was prepared by alcohol extraction from Rhizoma Coptidis that have been quality-controlled according to the protocol. The cholesterol-lowering effect of CAE was evaluated on SD rats fed with high-lipid diet. Serum level of lipid, Bile acid and cholesterol in the liver and feces of the rats were measured using colorimetric assay kit. RT-PCR and Western blot were used to analyze the mRNA and protein expression of cholesterol metabolism-related genes including cholesterol 7α-hydroxylase (CYP7A1), peroxisome proliferator-activated receptor-alpha (PPARα) and farnesoid X receptor (FXR) in the livers of the rats. A HPLC analysis was used to assess the activity of CYP7A1. The results showed that CAE reduced the levels of serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C). CYP7A1 gene expression and its activity was up-regulated dose-dependently accompanying with the increased level of bile acid and the reduced cholesterol level in the livers of the CAE treated hyperlipidemic rats. Meanwhile, the mRNA expression of PPARα was also up-regulated in dose-dependent way accompanying the down-modulation of the FXR mRNA expression in the livers of the CAE treated hyperlipidemic rats. The results indicate that the cholesterol-lowering effect of coptis alkaloid extract is at least partly attributed to its promoting the cholesterol conversion into bile acids by up-regulating the gene expression of CYP7A1 and thus increasing its activity in the liver of the hyperlipidemic rats, which might related to the positive regulation of PPARα and the negative modulation of FXR.

[Detection of antioxidative capacity of bamboo leaf extract by scavenging hydroxyl free radical].

By studying on absorption spectrum of red compound coming from the reaction system of Fe2+ and 1, 10-phenanthroline and the capacity of antioxidant TBHQ and bamboo leaf extract for scavenging hydroxyl free radical, some results were drawn as follows: the determining wavelength of bamboo leaf extract for scavenging hydroxyl free radical by spectrophotometric method is 509.1 nm, and IC50 (the value of antioxidant concentration at scavenging half of hydroxyl free radical)was used as the index to evaluate scavenging capacity. The determined IC50 values were TBHQ (0.040), M20 (0.378), M40 (0.323), M60 (0.334), and bamboo leaf extract could be used as natural antioxidant.

[Detection of antioxidative capacity of bamboo leaf extract by scavenging superoxide anion free radical].

By studying absorption spectrum, oxidation velocity of pyrogallic acid reaction system, the effect of pyrogallic acid concentration and buffer pH value on the scavenging rate for superoxide anion free radical coming from reaction system was investigated, and some results were obtained as follows, the wavelength used for the pyrogallic acid reaction system was determined by spectrophotometric method: 319.5 nm. Some parameters of the reaction system were determined as follows: reaction volume: 10 mL, reaction time: 9 min, volume of pyrogallic acid (3 mmol x L(-1)): 0.3 mL, pH value of buffer: 8.2 and oxidation velocity: 0.035. The relation between scavenging rate for superoxide anion free radical and concentration of TBHQ and bamboo leaf extract samples was studied by our method, and IC50 (the value of antioxidant concentration at scavenging half of superoxide anion free radical) was used as the index to evaluate scavenging capacity. IC50 value of TBHQ and most effective samples were as follows: TBHQ (95.01 mg x L(-1)) and M40 (298.69 mg x L(-1)). M40 and other bamboo leaf extract could be used as natural antioxidant.

[Detection of antioxidative capacity of bamboo leaf extract by scavenging organic free radical DPPH].

By studying on absorption spectrum of DPPH solution and DPPH reaction system, the conclusion was drawn as follows, the wavelength for the determination of DPPH reaction system by spectrophotometric method was chosen to be 518.4 nm, DPPH reaction system was 4.00 mL of 257.7 mg x L(-1) DPPH+1 mL of antioxidant solution with the reaction time of DPPH reaction system of 40 min. The relation between DPPH radical scavenging rate and the concentration of TBHQ and BHT was studied, and IC50 (the value of antioxidant concentration for scavenging half of DPPH radical) was used as the index to evaluate scavenging capacity. The determining IC50 values were as follows: TBHQ (21.14 mg x L(-1)), BHT(42.09 mg x L(-1)) and M40 (108.40 mg x L(-1)). Bamboo leaf extract could be used as natural antioxidant.

[The role of apoptosis and the related genes in non-alcoholic steatohepatitis].

OBJECTIVE: To study the role of apoptosis and the expression of apoptosis-related genes Fas ligand (FasL), Fas, caspase-3 and caspase-8 in an animal model of non-alcoholic steatohepatitis (NASH). METHODS: An experimental progressive NASH model was established by feeding male C57BL6/J mice with a high fat, methionine-choline deficient (MCD-) diet for two days, five days, ten days, three weeks and eight weeks. Control mice were fed methionine-choline supplemented (MCD+) diet. Hepatic steatosis, inflammation and fibrosis were graded by examining their H and E stained liver sections. Hepatocyte apoptosis was detected by TUNEL assay. Expressions of mRNA and protein of FasL, Fas and caspase-8 were performed by quantitative real time RT-PCR and Western blot. Caspase-3 activity assay was conducted using ApoAlert caspase-3 assay kit. RESULTS: In MCD- mice, minimal hepatic steatosis was observed at day 5, and by day 10, mild steatosis with inflammatory infiltration was found. Severe steatohepatitis was noted at week 3, and fibrosis at week 8. TUNEL assay showed that apoptotic index in MCD- group was higher than that in MCD+ group at week 3 (15.59%+/-4.87% vs 5.17%+/-3.19%, P less than 0.05) and at week 8 (11.29%+/-3.22% vs 5.41%+/-1.54%, P less than 0.05). Compared to MCD+ group, the expression of FasL was dramatically increased on day 10 and in week 3 in MCD- mice both at the mRNA and protein levels (P less than 0.05 and P less than 0.01). Expression of Fas mRNA was up-regulated in weeks 3 and 8 (P less than 0.01), and expression of Fas in protein level was higher at week 8 (P less than 0.01) in MCD- group. Expression of caspase-8 significantly increased at the mRNA level at week 3 and week 8 (P less than 0.01 and P less than 0.05 respectively) and at the protein level at week 8 (P less than 0.05) in MCD- group. In all of the time points except for day 5, caspase-3 activities were significantly more enhanced in MCD- group than that in MCD+ group (P less than 0.05). CONCLUSIONS: In our experimental NASH model, hepatic apoptosis was frequently detected. Increased apoptosis was probably attributable to up-regulation of apoptosis-related genes, such as FasL/Fas system, and activation of the caspase pathway. These changes may provoke hepatic apoptosis and the development of inflammation and fibrosis.

Comparison of protocatechuic aldchyde in Radix Salvia miltiorrhiza and corresponding pharmacological sera from normal and fibrotic rats by high performance liquid chromatography.

AIM: To observe the effect of protocatechuic aldchyde on the proliferation of hepatic stellate cells (HSCs). METHODS: Liver fibrosis was induced in rats by carbon tetrachloride (CCl4). Then normal and fibrotic drug sera were extracted from rats. The effects of protocatechuic aldchyde, raw Radix Salvia miltiorrhiza and drug sera of Salvia miltiorrhiza on HSC growth were determined by CCK-8. The protocatechuic aldchyde was separated by high performance liquid chromatography (HPLC) in a Alltima C18 column (250 mm mult 4.6 mm, 5 microm) with a mobile phase of acetonitrile-4% glacial acetic acid solution (gradient elution) at the wavelength of 281 nm. RESULTS: Protocatechuic aldchyde, raw Radix Salvia miltiorrhiza and drug sera of Salvia miltiorrhiza were found to have inhibitory effects on proliferation of rat HSCs. Raw Radix Salvia miltiorrhiza had a stronger inhibitory effect than the drug sera. The fibrotic drug sera showed a higher suppressive effect than the normal drug sera (P < 0.05). Protocatechuic aldchyde was found in crude materials of both Radix Salvia miltiorrhiza and its corresponding drug sera. The average recovery (n = 6) was 110.5% for raw Salvia miltiorrhiza Bge, 102% for normal drug sera and 105.2% for fibrotic drug sera. The relative standard devitation (RSD) was 0.37%, 1.96% and 1.51%, respectively (n = 6). The contents of protocatechuic aldchyde were 0.22%, 0.15% and 0.19%, respectively (n = 6) (P < 0.05). The RSD was 0.33%, 0.75% and 1.24% (n = 6) for raw material of Radix Salvia miltiorrhiza, normal drug sera and fibrotic drug sera, respectively. The samples were stable for 6 d. CONCLUSION: Protocatechuic aldchyde can inhibit the growth of HSCs. HPLC is suitable for the determination of virtual bioactive components of Chinese herbal medicines in vitro.

[Effects of Radix Salviae Miltiorrhizae on Ca2+ in hepatic stellate cells].

OBJECTIVE: To investigate the effects of Radix Salviae Miltiorrhizae (RSM) on intracellular free calcium in hepatic stellate cells (HSCs). METHODS: After the model of hepatic fibrosis was established in SD rats, RSM [20 ml/(kg x d)] was given via gastrogavage to the rats of treatment groups while the same volume of 0.9% NaCl was given to the rats of control groups twice a day for 6 consecutive days. Then the blood sample was drawn from the inferior vena cava, and the serum was extracted for pharmacological studies. After 24 h incubation with 10% drug serum, HSCs were loaded with Fluo-3/AM, a Ca2+ marker, and were observed with laser scanning confocal microscopy (LSCM). RESULTS: By comparison with controls, both RSM pharmacological serums decreased [Ca2+]i in HSCs significantly in the condition of using Ang II or not (P<0.05). CONCLUSION: RSM decreased [Ca2+]i in activated HSCs, which may be one of important ways to block liver fibrosis.

Effects of pharmacological serum from normal and liver fibrotic rats on HSCs.

AIM: To make drug sera of Salvia miltiorrhiza and Yigankang, both of which are Chinese herbs that activate bleeding and eliminate stasis, in normal rats and those with liver fibrosis, respectively. To investigate and compare the effects of the two different drug sera on the proliferation and activation of hepatic stellate cells (HSCs). METHODS: Some rats were induced with liver fibrosis: 40% carbon tetrachloride (CCl4) subcutaneous injection, twice a week for 9 wk. Salvia miltiorrhiza, Yigankang, colchicines and normal saline were administered into the stomachs of normal rats and those with liver fibrosis. Drug sera were extracted 5 d later. HSCs in vitro were cultivated in different drug sera for 24 h. The rates of proliferation and expression of alpha-smooth muscle actin (alpha-SMA) were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and immunocytochemistry stain, respectively. RESULTS: The drug sera from normal and liver fibrotic rats could be used to cultivate HSCs and to observe the effects of the corresponding components of herbs on HSCs. Salvia miltiorrhiza and Yigankang had better inhibitory effects on HSCs than colchicines (MTT: normal drug serum: Salvia miltiorrhiza 0.42+/-0.08, Yigankang 0.32+/-0.10 vs colchicines 0.45+/-0.12 pathological drug serum: Salvia miltiorrhiza 0.33+/-0.02, Yigankang 0.26+/-0.01 vs colchicines 0.41+/-0.09. P<0.05). The drug sera of Salvia miltiorrhiza, Yigankang from liver fibrotic rats had a stronger inhibitory effect than the same ones from normal rats (MTT: Salvia miltiorrhiza: normal drug serum 0.42+/-0.08 vs pathological drug serum 0.33+/-0.02. Yigankang: normal drug serum 0.32+/-0.10 vs pathological drug serum 0.26+/-0.01. P<0.05). CONCLUSION: Salvia miltiorrhiza and Yigankang could inhibit the expression of alpha-SMA and the proliferation of HSCs. The drug sera from normal and liver fibrotic rats had different effects on HSCs, probably due to different metabolic processes, effective components and different quantities of drug contents in drug sera from rats with different states of liver.

Efficacy of Chinese medicine Yi-gan-kang granule in prophylaxis and treatment of liver fibrosis in rats.

AIM: To investigate the efficacy of a Chinese medicine, Yi-gan-kang granule (granules for benefiting the liver), in prophylaxis and treatment of liver fibrosis in rats and its possible mechanism. METHODS: One hundred and forty Sprague-Dawley rats were randomly divided into seven groups (20 each): group 1, blank control group without any interference during the study; group 2, CCl4-induced liver fibrosis group; group 3, pig serum-induced liver fibrosis group; group 4, prophylaxis group of CCl4-induced liver fibrosis by Yi-gan-kang; group 5, prophylaxis group of pig serum-induced liver fibrosis by Yi-gan-kang; group 6, treatment group of CCl4-induced liver fibrosis by Yi-gan-kang; group 7, treatment group of CCl4-induced liver fibrosis by Yi-gan-kang. At wk 6, 10, 14 and 20 (baseline for CCl4 or big serum induction), five rats in each group were anesthetized and their livers were removed for pathological studies including immunohistochemical studies for alpha-SMA, type I collagen and in situ hybridization of tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA of hepatic stellate cells (HSCs). Anti-lipid peroxidation in isolated mitochondria and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay for proliferation and terminal deoxynucleotidyl transferase-medicated dUTP-biotin nick end-labeling (TUNEL), flow cytometry and electron microscopy for apoptosis in isolated HSCs were also studied. RESULTS: The mean number of pseudolobuli at wk 10, 14 and 20 in the prophylaxis group was significantly less than that in the control group (P<0.05 or 0.01). The effect of prophylaxis at wk 14 in CCl4 rats and at wk 10 in pig serum-induced rats was much better than that of treatment group (P<0.01). The thickness (in microm) of fibers both in pig serum-induced prophylaxis and in treatment groups at wk 14 and 20 was significantly less than that in control group (P<0.05). The number of fibers both in prophylaxis and in treatment groups from wk 10 or 14 to 20 was significantly less than that in control group (P<0.05 or P<0.01). The tissue HSC positive rates of type I collagen, alpha-SMA and TIMP-1 mRNA, which represented the active phenotype of HSCs in tissues, remained very high from wk 6 to the end of model making in control group. While in prophylaxis group, they were at a relatively low level. In treatment group, there was a gradual decreasing trend. Time- and dose-dependent effects of anti-lipid peroxidation on isolated mitochondria, cell proliferation and apoptosis in cultured HSCs were also observed during the study. CONCLUSION: Yi-gan-kang can effectively inhibit or inverse the course of liver fibrogenesis in CCl4- and pig serum-induced rat models.