Electroacupuncture alleviates PTSD-like behaviors by modulating hippocampal synaptic plasticity via Wnt/ß-catenin signaling pathway.

Abnormalities in hippocampal synaptic plasticity contribute to the pathogenesis of post-traumatic stress disorder (PTSD). The Wnt/ß-catenin signaling pathway is critical for the regulation of synaptic plasticity. PTSD symptoms can be alleviated by correcting impaired neural plasticity in the hippocampus (Hipp). Electroacupuncture (EA) has a therapeutic effect by relieving PTSD-like behaviors. However, little is known about whether the Wnt/ß-catenin pathway is involved in EA-mediated improvements of PTSD symptoms. In this study, we found that enhanced single prolonged stress (ESPS)-induced PTSD led to abnormal neural plasticity, characterized by the decline of dendritic spines, the expression of postsynaptic density 95 (PSD95), and synaptophysin (Syn) in the stressed Hipp along with the reduction of Wnt3a and ß-catenin, and increased GSK-3ß. EA significantly alleviated PTSD-like behaviors, as assessed by the open field test, elevated platform maze test and conditioning fear test. This was paralleled by correcting abnormal neural plasticity by promoting the expression of PSD95 and Syn, as well as the number of dendritic spines in the Hipp. Importantly, EA exerted anti-PTSD effects by augmenting the expression levels of Wnt3a and ß-catenin, and decreasing that of GSK-3ß. The effects mediated by EA were abolished by XAV939, an inhibitor of the Wnt/ß-catenin pathway. This suggests that EA relieved ESPS-induced PTSD-like behaviors, which can largely be ascribed to impaired neural plasticity in the Hipp. These findings provide new insights into possible mechanisms linking neural plasticity in the Hipp as potential novel targets for PTSD treatment in EA therapy.

Antinociceptive C19-diterpenoid alkaloids from the roots of Aconitum nagarum.

Three new C19-diterpenoid alkaloids, nagarumines A-C (1-3), together two known alkaloids, deoxyaconitine (4) and N-deethyldeoxyaconitine (5), were isolated from the roots of Aconitum nagarum. The structures of the new compounds were elucidated by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy, as well as high resolution mass spectrometry. The in vivo pharmacological studies revealed that nagarumine C (3) possessed comparable antinociceptive activity (ED50 = 76.0 µmol/kg) with the positive control drugs aspirin and acetaminophen.

Accurate 16S Absolute Quantification Sequencing Revealed Vaginal Microecological Composition and Dynamics During Mixed Vaginitis Treatment With Fufang FuRong Effervescent Suppository.

Background: The diagnosis and treatment of mixed vaginitis are more complicated than single pathogenic infections, and there may be adverse reactions and several contraindications to conventional antibiotic therapy. Therefore, this study aimed to evaluate the preliminary effects of Fufang Furong Effervescent Suppository for the management of aerobic vaginitis (AV) mixed with bacterial vaginosis (BV) using Accurate 16S absolute quantification sequencing (Accu16S). Methods: In the present randomized, blind, multi-center clinical trial, women (20 to 55 years) who had received a diagnosis of AV+BV were randomly assigned into clindamycin positive control (n = 41) and Fufang Furong Effervescent Suppository (n = 39) groups. The follow-up occurred in three time periods (V1: -2~0 days; V2: 15-17 days; V3: 40 ± 3 days). At each visit, two vaginal swabs, one for clinical evaluation and one for laboratory examination, were taken from each patient. The Nugent score, Donders' score, drug-related complications, recurrence rates, and microecological changes of vaginal swabs were assessed in the time three periods. Results: At baseline, the two groups were similar in frequency of presentation with vaginal burning, odor, abnormal discharge, and itching. No meaningful differences in Nugent and Donders' scores were detected between the two groups at stage V2 (Nugent: p = 0.67; Donders': p = 0.85) and V3 (Nugent: p = 0.97; Donders: p = 0.55). The Furong group presented fewer complications compared to the Clindamycin group. However, this difference was not statistically significant (p = 0.15). Additionally, Accu16S indicated that the total abundance of bacteria in both groups sharply decreased in stage V2, but slightly increased in V3. In stage V3, the absolute abundance of Lactobacillus in the Furong group was considerably higher compared to untreated samples (p < 0.05). On the other hand, no momentous increase was detected in the Clindamycin group (p > 0.05). Conclusion: Fufang Furong Effervescent Suppository can be as effective as clindamycin cream in the management of AV+BV while may restore the vagina microecosystem better.

Assay establishment and validation of a high-throughput organoid-based drug screening platform.

BACKGROUND: Organoids are three-dimensional structures that closely recapitulate tissue architecture and cellular composition, thereby holding great promise for organoid-based drug screening. Although growing in three-dimensional provides the possibility for organoids to recapitulate main features of corresponding tissues, it makes it incommodious for imaging organoids in two-dimensional and identifying surviving organoids from surrounding dead cells after organoids being treated by irradiation or chemotherapy. Therefore, significant work remains to establish high-quality controls to standardize organoid analyses and make organoid models more reproducible. METHODS: In this study, the Z-stack imaging technique was used for the imaging of three-dimensional organoids to gather all the organoids' maximum cross sections in one imaging. The combination of live cell staining fluorescent dye Calcein-AM and ImageJ assessment was used to analyze the survival of organoids treated by irradiation or chemotherapy. RESULTS: We have established a novel quantitative high-throughput imaging assay that harnesses the scalability of organoid cultures. Using this assay, we can capture organoid growth over time, measure multiple whole-well organoid readouts, and show the different responses to drug treatments. CONCLUSIONS: In summary, combining the Z-stack imaging technique and fluorescent labeling methods, we established an assay for the imaging and analysis of three-dimensional organoids. Our data demonstrated the feasibility of using organoid-based platforms for high-throughput drug screening assays.

Effects of Two Polysaccharides From Traditional Chinese Medicines on Rat Immune Function.

The aim is to study the immune function effect of two polysaccharides extracted from traditional Chinese herbs on rats. Ultrasonic-assisted extraction was used to extract the polysaccharide from traditional Chinese medicines. MTT assay was used to determine the effects of two polysaccharides on the conversion of pig peripheral T lymphocytes. For this, 24 Sprague-Dawley rats were selected for the clinical trial and divided into groups B (blank), CK (cyclophosphamide inhibitory control), AP (angelica polysaccharide), and RIP (radix isatidis polysaccharide). Except for group B, other groups can induce the immunodeficiency by using cyclophosphamide. Rats of the AP and RIP groups were given gavage of 1 mL of AP and RIP. The blood was sampled from the eyeball on days 0, 7, 14, 21, 28, and 35, respectively, to determine immune cells, IgG and IgM of immunoglobulin, body weight, and spleen index. Results: The average content of AP and RIP was 51.27 and 14.8%, and the extraction rate was 75.23 and 60.94%. The maximum stimulation index was 1.407 when the concentration of AP was 8,000 µg mL-1 and 1.5 when the concentration of RIP was 125 µg mL-1. Both kinds of polysaccharides can alleviate the decline of white blood cells, lymphocytes, monocytes, neutrophils, and serum IgG and IgM caused by cyclophosphamide. The two polysaccharides can regulate the rapid recovery of weight in immunosuppressed rats and increase the spleen index of immunosuppressed SD rats. The polysaccharides from the two traditional Chinese medicines can alleviate the immunosuppression caused by cyclophosphamide and promote the immune function of the body, which can be used as raw material resources of new veterinary medicine.

Immediate Effect of Local Vibration Therapy for Sport-induced Fatigue Based on Traditional Chinese Medicine's Holistic Theory.

INTRODUCTION: Vibration therapy has been widely used and published in alleviating muscle fatigue. However, reports on applying vibration therapy based on the holisitic theory of traditional Chinese medicine (TCM) remains limited. This study is to evaluate the immediate effect of vibration therapy on exercise-induced muscle fatigue. METHODS: For this retrospective parallel controlled study, all data were from a previously approved and completed clinical trial. Participants (n=40) in the clinical trial included local Greco-Roman wrestling and Judo athletes in south China. The participants were equally randomly divided into the intervention group (n=20) and control group (n=20). The intervention group received a seven-week vibration intervention-based TCM holistic theory combined with conventional therapy, such as stretching, massage, and flapping, while the control group only received the conventional therapy. Surface electromyography (sEMG) of the lumbar segment of erector spinae was measured for each participant pre- and postintervention, and the two-point discrimination thresholds of the data were differentiated and compared with panel data analysis. RESULTS: For the control group, the pre- and postintervention sEMG measure showed no significant difference (p=0.333), whereas significant difference (p=0.004) was observed for the intervention group. Further, the pre- and postintervention two-point discrimination test also showed a significant difference (p=0.016) for the intervention group. DISCUSSION: The application of vibration therapy based on TCM holistic theory may have an immediate effect in reducing sport-induced muscle fatigue from intensive training. Future larger sample size and robust designed clinical trial is warranted to evaluate the long-term effect of the intervention.

Oxyresveratrol induces apoptosis and inhibits cell viability via inhibition of the STAT3 signaling pathway in Saos­2 cells.

Oxyresveratrol (ORES) is a natural phenolic compound with multiple biological functions including antioxidation, anti­inflammation and neuroprotection; however, the inhibitory effect of ORES on osteosarcoma remains largely unknown. The present study aimed to determine the effects of ORES on osteosarcoma cell Saos­2. Cell Counting Kit­8 assay was performed to detect Soas­2 cell viability. Annexin­FITC/PI staining and JC­1 staining were used to measure cell apoptosis and the change of mitochondrial membrane potential. In addition, western blotting was conducted to determine the expression levels of apoptotic proteins and the phosphorylation of STAT3. It was found that ORES inhibited cell viability and induced apoptosis of osteosarcoma Saos­2 cells in a concentration­dependent manner. In addition, ORES increased the expression levels of apoptotic proteases caspase­9 and caspase­3 and reduced mitochondrial membrane potential. In response to ORES treatment, the expression levels of pro­apoptotic proteins, Bad and Bax, were enhanced, whereas those of anti­apoptotic proteins, Bcl­2 and Bcl­xL, were reduced. In addition, the phosphorylation of STAT3 was attenuated in Saos­2 cells after treatment with ORES. Inhibition of cell viability and apoptosis induction by ORES were rescued by enhancement of STAT3 activation upon treatment with IL­6. Collectively, the present study indicated that ORES induced apoptosis and inhibited cell viability, which may be associated with the inhibition of STAT3 activation; thus, ORES represents a promising agent for treating osteosarcoma.

Using RNA-Seq to Explore the Repair Mechanism of the Three Methods and Three-Acupoint Technique on DRGs in Sciatic Nerve Injured Rats.

Objective: To study the effects of the three methods and three-acupoint technique on DRG gene expression in SNI model rats and to elucidate the molecular mechanism of the three methods and three-acupoint technique on promoting recovery in peripheral nerve injury. Methods: 27 male SD rats were randomly divided into three groups: a Sham group, the SNI group, and the Tuina group. The Tuina group was treated with a tuina manipulation simulator to simulate massage on points, controlling for both quality and quantity. Point-pressing, plucking, and kneading methods were administered quantitatively at Yinmen (BL37), Chengshan (BL57), and Yanglingquan (GB34) points on the affected side once a day, beginning 7 days after modeling. Intervention was applied once a day for 10 days, then 1 day of rest, followed by 10 more days of intervention, totally equaling 20 times of intervention. The effect of the three methods and three-point technique on the recovery of injured rats was evaluated using behavior analysis. RNA sequencing (RNA-Seq) analysis of differentially expressed genes in DRGs of the three groups of rats was also performed. GO and KEGG enrichment was analyzed and verified using real-time PCR. Results: RNA-Seq combined with database information showed that the number of differentially expressed genes in DRG was the largest in the Tuina group compared with the SNI group, totaling 226. GO function is enriched in the positive regulation of cell processes, ion binding, protein binding, neuron, response to pressure, response to metal ions, neuron projection, and other biological processes. GO function is also enriched in the Wnt, IL-17, and MAPK signaling pathways in the KEGG database. PCR results were consistent with those of RNA sequencing, suggesting that the results of transcriptome sequencing were reliable. Conclusion: The three methods and three-acupoint technique can promote the recovery of SNI model rats by altering the gene sequence in DRGs.

Blood homocysteine and folic acid levels may provide reference value for the treatment of sudden total frequency deafness.

BACKGROUND: The cause of sudden sensorineural hearing loss (SSNHL) is still unknown. Literature has indicated that there is a statistically significant correlation between hyperhomocysteinemia and SSNHL, yet there is lack of study in the relationship concerning total frequency deafness subtype of SSNHL. This study investigated the relationship between plasma concentration of homocysteine (Hcy), serum concentration of folic acid and occurrence and treatment responding in total frequency deafness adult patients, and explored whether targeted early intervention was associated with improved clinical outcome in this subgroup. METHODS: A total of 54 consecutive adult patients with diagnosis of sudden total frequency deafness in a single institution was enrolled into the study group. Two control groups were established. Control group 1 was derived from inpatients with normal listening comprehension. Control group 2 included 52 patients with sudden total frequency deafness treated in a parallel hospital. Blood concentration of folic acid and Hcy was investigated. Treatment included Ginkgo biloba extract, dexamethasone, hyperbaric oxygen, folic acid, vitamin B6, and optional vitamin B12. All data was statistically analyzed. Blood level of Hcy and folic acid was compared between study group and control group 1. RESULTS: Although there was no clear evidence for the divergence trend of Hcy and folic acid levels individually, the results showed that the study group had higher blood level of Hcy and lower blood level of folic acid, than control group. In the study group, 24 patients (44.44%) demonstrated treatment effectiveness after the 2-week treatment course. Patients without vertigo had higher effective rate than patients with vertigo (P<0.05). CONCLUSIONS: Effective rate of study group was higher than control group 2 which had no folic acid and vitamin B6/B12 supplement. High blood Hcy and low blood folic acid were closely associated in patients with sudden total frequency deafness. The currently accepted concept of treatment for sudden total frequency deafness is not essentially satisfactory. Testing of plasma Hcy and serum folic acid may provide referential value for its treatment and prognosis evaluation.

Anti-proliferative and anti-migratory effects of Scutellaria strigillosa Hemsley extracts against vascular smooth muscle cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The abnormal increase in vascular smooth muscle cell (VSMC) proliferation and migration are critical events in the pathogenesis of cardiovascular diseases (CVDs) including restenosis and atherosclerosis. The dried roots of Scutellaria baicalensis Georgi (common name: Huangqin in China) have been confirmed to possess beneficial effects on CVD by clinical and modern pharmacological studies. Flavonoids in Huangqin exert anti-proliferative and anti-migratory effects. Similar to Huangqin, Scutellaria strigillosa Hemsley (SSH) has been used to clear heat and damp and is especially rich in flavonoids including wogonin, wogonoside, baicalein, and baicalin. However, there have been few of reports about pharmacological activities of SSH. AIM OF THE STUDY: To investigate the anti-proliferative and anti-migratory properties of Scutellaria strigillosa Hemsley extract (SSHE) in vitro and in vivo and explore its possible mechanism of action. MATERIALS AND METHODS: The chemical constituents of SSHE were analyzed by ultra-high performance liquid chromatography coupled with triple time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS). Cell proliferation and migration were investigated using BrdU incorporation assay and cell scratch test, respectively. The protein expression was determined by western blotting. In vivo, we established an artery ligation model of C57BL/6 mice and orally administered them with 50 or 100 mg/kg/day of SSHE. The carotid arteries were harvested and the intima-media thickness was examined 28 days post-ligation. RESULTS: Twelve compounds were identified and tentatively characterized. SSHE significantly inhibited the VSMC proliferation and migration stimulated by PDGF-BB and decreased the relative protein expression of regulatory signaling intermediates. Furthermore, the expression of SM22α was significantly elevated in SSHE-pretreated VSMCs, whereas knockdown of SM22α impaired the PDGF-BB-induced proliferation and migration arrest. Meanwhile, both ROS generation and the phosphorylation of ERK decreased in SSHE-pretreated VSMCs. In carotid artery ligation mice model, SSHE treatment significantly inhibited neointimal hyperplasia. CONCLUSIONS: SSHE significantly inhibited the PDGF-BB-induced VSMC proliferation, migration, and neointimal hyperplasia of carotid artery caused by ligation. Upregulation of SM22α expression, inhibition of ROS generation and ERK phosphorylation were, at least, partly responsible for the effects of SSHE on VSMCs.

Simultaneous detection of four flavonoids and two alkaloids in rat plasma by LC-MS/MS and its application to a comparative study of the pharmacokinetics between Abri Herba and Abri mollis Herba extract after oral administration.

Abri Herba and Abri mollis Herba both were important members of the Leguminosae family in southwestern China. Abri mollis Herba was often used as Abri Herba due to their proximity, but there are few studies on pharmacokinetics to compare their main identical active compositions. A sensitive and selective high-performance liquid chromatography with tandem mass spectrometry method in the positive/negative electrospray ionization switching mode was developed and validated for the simultaneous analysis of four flavonoids and two alkaloids in rat plasma. The chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of methanol and 0.5% acetic acid. The detection of the target compounds was conducted in multiple-reaction monitoring mode with a hybrid triple quadrupole linear ion trap mass spectrometer equipped with positive/negative ion-switching electrospray ion source. The differences in pharmacokinetics were discovered, which indicated that the substitution between them is some degree of irrationality. The validated method was successfully applied to pharmacokinetic study of the six components in male rat plasma after oral administration of Abri Herba and Abri mollis Herba extract and the results in the study would provide a useful guide for the clinical application of Abri Herba with those in Abri mollis Herba.

A chemometric-assisted LC-MS/MS method for the simultaneous determination of 17 limonoids from different parts of Xylocarpus granatum fruit.

The marine mangrove Xylocarpus granatum is used as a folk medicine and is rich in bioactive limonoids. The quantitative determination of the chemical composition and distribution of limonoids in different parts of X. granatum fruit (fruit peel, seed coat, seed kernels, seed, and fruit) is significant for authentication and quality control purposes. However, the quantitative determination of limonoids in X. granatum has not yet been reported. In this study, a chemometric-assisted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of 17 limonoids to reveal the chemical composition and distribution in different parts of X. granatum fruit. Ultrasonic-assisted extraction, optimized by response surface methodology (RSM), was more accurate than the general one-variable-at-a-time method. The overall distribution of 17 limonoids in different parts of X. granatum fruit had the following order: seed kernels > seed > fruit, and 13 limonoids showed a rank order of seed kernels > seed > fruit > fruit peel > seed coat. Furthermore, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to analyze the LC-MS/MS data and provide a chemometric model for easy visualization and interpretation to classify the different parts of X. granatum fruit. In addition, the study indicated that the chemometric-assisted strategy, consisting of RSM, PCA, and OPLS-DA for the development, optimization, and data analysis of multicomponent quantitation by LC-MS/MS, is effective and feasible. This study provided the chemical composition and distribution evidence for the authentication and quality control of X. granatum fruit.

Sucrose esters improve the colloidal stability of nanoethosomal suspensions of (-)-epigallocatechin gallate for enhancing the effectiveness against UVB-induced skin damage.

Nanoethosomal suspensions, composed of phospholipids, ethanol, and water, are novel lipid carriers. These suspensions have been reported to enhance the permeation of drugs into the skin as a result of the interdigitation effect of ethanol on the lipid bilayer of liposomes and by increasing the fluidity of lipids in the stratum corneum. The physical stability of the nanoethosomal suspension is still a critical research problem until now. This study investigated the commercial palm sucrose esters to improve the colloidal stability of nanoethosomal suspensions. The results indicated that palm sucrose esters (PSE) were effective for stabilizing nanoethosomal suspension of (-)-epigallocatechin gallate (EGCG) from green tea. A PSE concentration of 0.15% was optimal for a nanoethosomal suspension which gave mean diameter 75.5 ± 3.5 nm, zeta potential -30.8 ± 3.2 mV and polydispersity index 0.207 ± 0.017. Moreover, the effectiveness of stabilization was influenced by the degree of esterification of the sucrose esters: the sucrose polyesters could prolong the stability of nanoethosomes loaded with EGCG to a year, but the sucrose monoesters only provided less than 6 months of stabilization. EGCG nanoethosomal suspension stabilized by sucrose polyesters shows better inhibition effectiveness against UVB-induced skin damage than native EGCG. The nanoethosomal suspension has the potential for its utilization as skin care and other products. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2416-2425, 2017.

"Three Methods and Three Points" regulates p38 mitogen-activated protein kinase in the dorsal horn of the spinal cord in a rat model of sciatic nerve injury.

Tuina is a traditional Chinese treatment for sensory disturbances caused by peripheral nerve injury and related diseases. Our previous studies showed that tuina regulates relevant regions and indices of the spinal dorsal horn using the Dian, Bo, and Rou method in Yinmen (BL37), Yanglingquan (GB34), and Weizhong (BL40). Treatment prevents muscle atrophy, protects spinal cord neurons, and promotes sciatic nerve repair. The mechanisms of action of tuina for treating peripheral nerve injury remain poorly understood. This study established rat models of sciatic nerve injury using the crushing method. Rats received Chinese tuina in accordance with the principle of "Three Methods and Three Points," once daily for 20 days. Tuina intervention reduced paw withdrawal latency and improved wet weight of the gastrocnemius muscle, as well as promoting morphological recovery of sciatic nerve fibers, Schwann cells, and axons. The protein expression levels of phospho-p38 mitogen-activated protein kinase, tumor necrosis factor-α, and interleukin-1ß also decreased. These findings indicate that "Three Methods and Three Points" promoted morphological recovery and improved behavior of rats with peripheral nerve injury.

Protective effect of panaxatriol saponins extracted from Panax notoginseng against MPTP-induced neurotoxicity in vivo.

AIM OF THE STUDY: Panaxatriol saponins (PTS), the main constituents extracted from Panax notoginseng, a Chinese herbal medicine, has been shown to be an effective agent on various diseases. Our previous study has demonstrated that PTS is an inducer of thioredoxin-1 (Trx-1) and has a possible potential as a therapeutic agent for Parkinson's disease (PD). However, the effect of PTS on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in vivo is unknown. MATERIALS AND METHODS: Using locomotor activity test and traction test, we detected the effect of PTS on MPTP-induced behavioral impairment. Tyrosine hydroxylase, Trx-1, cyclooxygenase-2, pro-caspase-9, pro-caspase-12 and caspase-3 expressions in the anatomical region of substantia nigra pars compacta (SNc) were tested by Western blot. RESULTS: PTS provided neuroprotection against the loss of dopaminergic neurons and behavioral impairment caused by MPTP. MPTP-induced neuronal death in the SNc was suppressed by PTS through increasing Trx-1 expression, suppressing cyclooxygenase-2 over-expression and inhibiting mitochondria-mediated apoptosis. CONCLUSIONS: PTS, an inducer of Trx-1, has pluripharmacological properties in the protection against PD including enhancing antioxidant activity, acting as neurotrophic factor, modulating inflammation and inhibiting mitochondria-mediated apoptosis.

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 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.