High-Survival Rate After Microinjection of Mouse Oocytes and Early Embryos With mRNA by Combining a Tip Pipette and Piezoelectric-Assisted Micromanipulator.

Utilizing microinjection to introduce biological molecules such as DNA, mRNA, siRNA, and proteins into the cell is well established to study oocyte maturation and early embryo development in vitro. However, microinjection is an empirical technology. The cellular survival after microinjection is mainly dependent on the operator, and an experienced operator should be trained for a long time, from several months to years. Optimizing the microinjection to be highly efficient and quickly learned should be helpful for new operators and some newly established laboratories. Here, we combined the tip pipette and piezo-assisted micromanipulator to microinject the oocyte and early embryos at different stages of mouse. The results showed that the survival rate after microinjection was more than 85% for cumulus-oocyte complex, germinal vesicle oocyte, two-cell, and four-cell embryos, and close to 100% for MII oocyte and zygotes. The high-rate survival of microinjection can save many experimental samples. Thus, it should be helpful in studying some rare animal models such as aging and conditional gene knockout mice. Furthermore, our protocol is much easier to learn for new operators, who can usually master the method proficiently after several training times. Therefore, we would like to publicly share this experience, which will help some novices master microinjection skillfully and save many laboratory animals.

Chinese Medicine Involving Triple Rehabilitation Therapy for Knee Osteoarthritis in 696 Outpatients: A Multi-Center, Randomized Controlled Trial.

OBJECTIVE: To determine the effects of Chinese medicine (CM) involving triple rehabilitation therapy on the progression of knee osteoarthritis (KOA). METHODS: A total of 722 patients recruited from 38 community health service centers located in China from March 2013 to March 2017 were randomly divided into treatment and control groups equally, using a cluster randomization design. Health education combined with CM involving triple rehabilitation therapy for KOA (electro-acupuncture, Chinese medicinal herb fumigating-washing, and traditional exercises) was administered in the treatment group while conventional rehabilitation therapy (physical factor therapy, joint movement training, and muscle strength training) was administered in the control group. Patients with a visual analog scale (VAS) scores ≽4 were treated with dispersible meloxicam tablets (7.5 mg, once daily). The Lequesne index scores, VAS scores, range of motion (ROM), lower limb muscle strength, knee joint circumference, quantitative scores of KOA symptoms, and the short-form 36 item health survey questionnaire (SF-36) scores were measured for each patient at 5 checkpoints (before treatment, at the 2nd week and the 4th week during the 4-week treatment period, at 1 month and 3 months after end of treatment), and adverse reactions were observed also. RESULTS: A total of 696 patients completed the entire process, with 351 in the treatment group and 345 in the control group. At all treatment checkpoints, the treatment group demonstrated better outcomes than the control group with regard to the total Lequesne index scores, effective rate and improvement rate of the total Lequesne index scores, VAS scores, lower limb muscle strength, knee circumference, quantitative scores of KOA symptoms, and SF-36 scores as well (P<0.05 or P<0.01). No adverse reactions were encountered in this study. CONCLUSIONS: CM involving triple rehabilitation therapy can alleviate KOA-related pain and swelling, improve lower limb muscle strength, promote flexion and activity of the knee and improve the quality of life in patients undergoing KOA. It is suitable for patients with early or mid-stage KOA. (Registration No. ChiCTR-TRC-12002538).

MIR22HG Regulates the Proliferation, Epithelial-Mesenchymal Transition, and Apoptosis in Colorectal Carcinoma.

Background: Recent investigations have suggested that long noncoding RNA (lncRNA) MIR22HG is commonly dysregulated in multiple types of malignancies. Nevertheless, the role of these MIR22HG in human colorectal carcinoma (CRC) are not well explored. Materials and Methods: Quantitative real-time polymerase chain reaction (qPCR) and in situ hybridization (ISH) assay were used to measure the expression of MIR22HG. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and migration, as well as invasion assays, were utilized to determine the roles of MIR22HG on growth, apoptosis, migration, and invasiveness of CRC cell. The expression of E-cadherin and N-cadherin was measured using Western blotting and immunohistochemistry staining assay. CRC cell growth in vivo was analyzed using nude mice xenograft. Results: The qPCR and ISH assay revealed that MIR22HG was downregulated in CRC sample compared with in normal tissue. MIR22HG was also significantly downexpressed in CRC cells compared with that in normal colonic epithelial cell line. Overexpression of MIR22HG inhibited the growth, migration ability, and invasiveness of CRC cell in vitro. In addition, MIR22HG suppressed the epithelial-mesenchymal transition (EMT) and induced the apoptosis of human CRC cell. Moreover, the authors demonstrated that MIR22HG inhibited the tumor growth of CRC cell and regulated the expression of EMT markers (E-cadherin and N-cadherin) in vivo. Conclusion: Altogether, these results imply that lncRNA MIR22HG restrained the aggressive phenotypes of CRC cell.

Quercetin Covalently Linked Lipid Nanoparticles: Multifaceted Killing Effect on Tumor Cells.

The encapsulation of hydrophobic drugs is a problem that many researchers are working on. The goal of this study is to achieve the delivery of hydrophobic drugs by means of prodrugs and nanoformulations for a stronger tumor cell-killing effect and explore related killing mechanisms. Lipophilic quercetin (Qu) was covalently linked to glyceryl caprylate-caprate (Gcc) via disulfide bonds-containing 3,3'-dithiodipropionic acid (DTPA) to synthesize novel lipid Qu-SS-Gcc. Qu-SS-Gcc lipid nanoparticles (Qu-SS-Gcc LNPs) were fabricated using the solvent diffusion technique. The intracellular release of Qu by cleavage of nanocarriers was determined by liquid chromatography and compared with the uptake of free Qu. Detection methods, such as fluorescent quantitation, flow cytometry, and western blot were applied to explore the action mechanism induced by Qu. It was revealed that Qu-SS-Gcc LNPs could be cleaved by the high concentrations of reduction molecules in MCF-7/ADR (human multidrug-resistant breast cancer) cells, followed by the release of Qu. The intracellular Qu content produced by dissociation of Qu-SS-Gcc LNPs was higher than that produced by internalization of free Qu. The resulting release of Qu exerted superior cell-killing effects on MCF-7/ADR cells, such as P-gp inhibition by binding to P-gp binding sites, blocking the cell cycle in the G2 phase, and causing cell apoptosis and autophagy. Moreover, it was revealed autophagy triggered by a low concentration of Qu-SS-Gcc LNPs was beneficial to cell survival, while at a higher concentration, it acted as a cell killer. Qu-SS-Gcc LNPs can realize massive accumulation of Qu in tumor cells and exert a multifaceted killing effect on tumor cells, which is a reference for the delivery of hydrophobic drugs.

Chitosan-modified lipid nanodrug delivery system for the targeted and responsive treatment of ulcerative colitis.

Targeted and sensitive drug release at the colitis site is critical for the effective therapy of ulcerative colitis and reduction of side effects from the drug. Herein, we used 3,3'-dithiodipropionic acid (DTPA) to covalently link quercetin (Qu) and glyceryl caprylate-caprate (Gcc) via ester bonds to prepare Qu-SS-Gcc lipid nanoparticles (Qu-SS-Gcc LNPs). Dexamethasone (Dex) was used as a model drug, and chitosan (CSO) was modified on the surface of Qu-SS-Gcc LNPs to obtain CSO-modified Dex-loaded Qu-SS-Gcc LNPs (CSO/Dex/LNPs). The encapsulation efficiency and drug loading of CSO/Dex/LNPs were 93.1 % and 8.1 %, respectively. The in vitro release results showed that CSO/Dex/LNPs had esterase-responsive characteristics and could release the drug rapidly in esterase-containing artificial intestinal fluid. A human colorectal adenocarcinoma cell (Caco-2) monolayer was used as the intestinal cell barrier model. Transmembrane resistance measurements and permeation experiments showed that CSO/Dex/LNPs had a protective effect on the lipopolysaccharide (LPS)-stimulated Caco-2 cell monolayer and increased the expression of E-cadherin in LPS-stimulated Caco-2 cells. Moreover, CSO/Dex/LNPs could significantly reduce the expression of the inflammatory factors TNF-α, IL-6 and NO in LPS-stimulated RAW 264.7 cells. The ulcerative colitis mouse model was constructed by using C57BL/6 mice. The in vivo distribution results showed that CSO/Dex/LNPs had colon-targeting effects and strong retention ability in the colons of mice with colitis. The results also showed that CSO/Dex/LNPs had better anti-inflammatory effects than free Dex, which could reduce colonic atrophy, reduce histomorphological changes and increase the expression of E-cadherin in the colon. Furthermore, the expression levels of TNF-α, IL-6 and NO in the CSO/Dex/LNP-treated group were 37.4 %, 35.5 % and 33.2 % of those in mice with colitis, respectively.

Different spinal subtypes with varying characteristics of lumbar disc degeneration at specific level with age: a study based on an asymptomatic population.

BACKGROUND: The relationship between spinal sagittal subtypes and lumbar disc degeneration is unclear. Thus, we aimed to investigate the relationship between lumbar intervertebral disc degeneration and age in asymptomatic healthy individuals with different sagittal alignments. METHODS: In this cross-sectional observational study, we examined 209 asymptomatic young and middle-aged volunteers (123 women and 86 men) who were divided into the following three groups according to age: groups A (20-30 years), B (31-40 years), and C (41-50 years). The volunteers underwent full-spine standing lateral radiography and magnetic resonance imaging (MRI, 3.0 T) of the lumbar spine. Based on panoramic radiography, two observers measured the spinopelvic parameters and classified the spine into Roussouly subtypes. The degree of disc degeneration was assessed based on T2-weighted images according to the Pfirrmann classification. RESULTS: There was a statistically significant difference in the degree of degeneration of type I spine between groups B and C at L4-L5 (P < 0.03) and L5-S1 (P < 0.01) and between groups A and C at L1-L2 (P < 0.04) and L4-L5 (P < 0.01). The degeneration degree of type II spine at all levels were significantly different between groups A and C. No statistically significant difference was found between groups A and B in all subtypes except for type II spine at L1-L2 (P < 0.04). A significant difference was found at four levels between groups B and C in type III spine (P < 0.05) and between groups A and C. For type IV spine, there was a significant difference in the degree of degeneration at L4-L5 (P < 0.02) between groups A and C. Moreover, almost all single parameters were not strongly correlated with the degree of disc degeneration. CONCLUSION: The different spinal subtypes have characteristics of lumbar disc degeneration at specific levels with age. We considered that spinal classification could be used as a predictor of lumbar disc degeneration. Our data may be helpful to increase awareness of the relationship between spinal subtypes and lumbar disc degeneration. LEVEL OF EVIDENCE: 3.

A novel biocompatible, simvastatin-loaded, bone-targeting lipid nanocarrier for treating osteoporosis more effectively.

An insufficient drug concentration at the target site and drug efflux resulting in poor efficacy are recognized as important obstacles in osteoporosis treatment. Simvastatin (SIM), which can treat osteoporosis by promoting osteoblast differentiation and mineralization through the bone morphogenetic proteins (BMP)-Smad signaling pathway, has lower bioavailability, and less bone tissue distribution. Herein, novel lipid nanoparticles (LNPs) delivering SIM (SIM/LNPs) for osteoporosis therapy were developed with aspartic oligopeptide (ASP n , here ASP6)-based bone-targeting moieties grafted to the nanoparticles (SIM/ASP6-LNPs) in an attempt to increase the concentration of SIM in bones with a relatively low dose to minimize adverse effects. In vivo experiments indicated that the ASP6-LNPs exhibited ideal bone-targeting characteristics, and in vitro cell evaluation experiments showed LNPs have good biocompatibility with MC3T3-E1 cells. The cell mineralization experiment revealed that the SIM-loaded LNPs induced osteoblast differentiation and the formation of mineralized nodules in MC3T3-E1 cells, achieving the same efficacy as that of SIM. Pharmacodynamic experiments revealed that SIM/ASP6-LNPs improved the efficacy of SIM on the recovery of bone mineral density when compared to SIM/LNPs or to SIM alone. Therefore, SIM/ASP6-LNPs may represent a potential bone-targeting drug delivery system (DDS) that contributes to the development of a novel osteoporosis treatment.

Correction: A novel biocompatible, simvastatin-loaded, bone-targeting lipid nanocarrier for treating osteoporosis more effectively.

[This corrects the article DOI: 10.1039/D0RA00685H.].

[The relationship between myocardial remodeling and endoplasmic reticulum stress and autophagy in rats with ischemic cardiomyopathy].

OBJECTIVE: To study the relationship between myocardial remodeling and endoplasmic reticulum stress and autophagy in rats with ischemic cardiomyopathy. METHODS: Thirty-six male SD rats were divided into normal control group, sham-operated group and ischemic cardiomyopathy group (n=12). Echocardiography was performed before operation in three groups. Rats in sham-operated group closed their thoracic cavity without ligation of coronary artery after thoracotomy. The rats in ischemic cardiomyopathy group were closed their thoracic cavity after ligating of coronary artery for 20 minutes and recovered reperfusion. After operation for 4 weeks, rats in three groups were killed after taking echocardiography. The myocardial tissues were taken for HE staining and Masson staining to observe the pathological changes of myocardium and the expressions of glucose-regulated protein 78 (GRP78), microtubule-associated protein 1 light chain 3- I (LC3-I), microtubule-associated protein 1 light chain 3- II (LC3-II), Bcl-2 interacting protein (Beclin-I) and the ratio of LC3-II/LC3-I were detected by Western blot. RESULTS: Compared with normal group and sham-operated group, ischemic cardiomyopathy rats had significant differences in echocardiography and myocardial pathology; the myocardial array was disordered, myocardial fibrosis was increased, mitochondrial vacuolation was serious. Mean while, the expressions of GRP78, LC3-I, LC3-II, Beclin-I and LC3-II/LC3-I ratio had significant changes. CONCLUSION: Autophagy and endoplasmic reticulum stress may play important roles in myocardial remodeling in rats with ischemic cardiomyopathy.

Factors Associated with Cervical Spine Alignment in an Asymptomatic Population: A Preliminary Analysis.

BACKGROUND: Cervical sagittal imbalance compromises health-related quality of life and can lead to myriad incapacitating symptoms through compression of the spinal cord. Questions regarding which parameters play primary roles in the progression of cervical sagittal imbalance and which might be compensatory factors remain unanswered. METHODS: This study enrolled 246 asymptomatic volunteers from July 2016 to June 2018. After demographic and radiologic parameters were measured, the data were analyzed using correlation coefficient test and multiple regression analysis. A predictive equation was assessed with variance analysis, residual analysis, collinearity analysis, and a paired t test. RESULTS: Average values are as follows: orbital tilt, 64 ± 6°; orbital slope (OS), 15 ± 6°; C0-C2 lordosis (C0C2), 28 ± 8°; cervical lordosis (CL), 5 ± 11°; C2-C7 sagittal vertical axis (C2C7SVA), 15 ± 8 mm; T1 slope (TS), 17 ± 6°; thoracic inlet angle, 69 ± 8°; thoracic kyphosis, 34 ± 9°; lumbar lordosis, 50 ± 10°; sacral slope, 38 ± 7°; pelvic index, 48 ± 9°; sagittal vertical axis, 10 ± 19 mm. Correlations of C2C7SVA were observed with body mass index (BMI), OS, C0C2, CL, and TS. The validated predictive equation was: C2C7SVA = 0.38 × BMI - 0.73 × OS + 0.73 × C0C2 + 0.15 × CL + 0.18 × TS - 6.53. CONCLUSIONS: BMI, OS, C0C2, CL, and TS were primary influencers in the progression of cervical sagittal imbalance and established a predictive equation of asymptomatic population, which can provide clinical advice and remind surgeons of the primary influencers of reconstructive surgery for better prognoses.

Acceptable Chin-Brow Vertical Angle for Neutral Position Radiography: Preliminary Analyses Based on Parameters of the Whole Sagittal Spine of an Asymptomatic Chinese Population.

OBJECTIVE: We sought to acquire the whole sagittal spine parameters and investigated the acceptable chin-brow vertical angle (CBVA) for neutral position radiography in an asymptomatic Chinese population. METHODS: The parameters measured in 257 asymptomatic volunteers included CBVA, occipital slope, orbital tilt, occipital incidence, C0-C2 Cobb angle, C2-C7 Cobb angle, C1-C7 Cobb angle, C2-C7 sagittal vertical axis and absolute rotation angle, cervical tilt, cranial tilt, T1 slope, and thoracic kyphosis, and others. We used Pearson correlation analyses to find relationships between CBVA and other variables. The subjects were divided into 5 groups according to the CBVA percentile: group A, 0%-20% CBVA; group B, 20%-40% CBVA; group C, 40%-60% CBVA; group D, 60%-80% CBVA; and group E, 80%-100% CBVA. We used analysis of variance to analyze differences among the 5 groups. RESULTS: Orbital tilt, Occipital incidence, C1-C7 Cobb angle, C2-C7 sagittal vertical axis, and cranial tilt all increased with increasing CBVA (P < 0.001). The occipital slope, C2-C7 Cobb angle, C2-C7 absolute rotation angle, cervical tilt, T1 slope, and thoracic kyphosis decreased with decreasing CBVA (P < 0.05). No correlations between other sagittal parameters and the CBVA were found. A slight deviation was found in groups B-D, with a greater deviation in groups A, C, and E. CONCLUSIONS: An acceptable range of -1.5° to 5.8° is recommended for the CBVA for cervical radiography in the neutral position. When spinal surgeons evaluate the cervical plane, the effects of the CBVA deviation on cervical curvature must be considered.

Rational Design of Redox-Responsive and P-gp-Inhibitory Lipid Nanoparticles with High Entrapment of Paclitaxel for Tumor Therapy.

An insufficient drug concentration at the target site and drug efflux resulting in poor efficacy is recognized as important obstacles in tumor treatment. Herein, novel lipid nanoparticles (LNPs) with redox-responsive properties based on disulfide bond-contained, quercetin (Qu)-grafted glyceryl caprylate-caprate (Gcc) are introduced (Qu-SS-Gcc LNPs). Qu-SS-Gcc LNPs show good entrapment of paclitaxel (PTX) due to π-π stacking between the aromatic rings of Qu and PTX. In vitro experiments indicate that Qu-SS-Gcc LNPs can selectively respond to high levels of reducing substances by breakdown of disulfide bonds, thus achieving rapid and efficient drug release, and only dissociate rapidly in tumor cells rather than in normal cells. Meanwhile, the Qu released concomitantly with the breakdown of disulfide bonds combines with P-gp and inhibits the drug efflux triggered by P-gp. Using an orthotopic 4T1 mouse mammary tumor model in BALB/c mice, PTX/Qu-SS-Gcc LNPs exhibit superior antitumor efficacy compared to Taxol, in addition better biosafety and inhibition of chemotherapy-triggered P-gp overexpression are achieved. Taken together, this work designs and implements redox-responsive drug release and drug efflux inhibition in tumor cells via modified LNPs, which not only leads to efficient drug release but also solves the problem of drug efflux that exists in stimulus-responsive systems.

Rate of presence of 11 thoracic vertebrae and 6 lumbar vertebrae in asymptomatic Chinese adult volunteers.

BACKGROUND: An increasing number of studies on spinal morphology in asymptomatic Asian and Western patients have been reported. Variation in spinal anatomy among patients is considered as the cause of wrong-level surgery in up to 40% of cases. The present study examined the rate of presence of 11 thoracic vertebrae and 6 lumbar vertebrae in 293 asymptomatic Chinese adult volunteers. METHODS: From May 27, 2016, to November 11, 2017, a cohort of 325 asymptomatic Chinese adults meeting the study exclusion criteria was recruited. The radiographs were examined by a spine surgeon and a radiologist to assess the number of thoracic and lumbar vertebrae. RESULTS: In total, 293 volunteers were included in this study: 17 (5.8%) had 11 thoracic vertebrae, and 16 (5.5%) had 6 lumbar vertebrae. Among all volunteers, 12 (4.1%) had 7 cervical vertebrae (C), 11 thoracic vertebrae (T), and 5 lumbar vertebrae (L); 5 (1.7%) had 7C, 11T, and 6L; and 11 (3.8%) had 7C, 12T, and 6L. There was no difference between the findings of the spine surgeon and the radiologist. CONCLUSIONS: For the first time, this study describes the rate of presence of 11 thoracic vertebrae and 6 lumbar vertebrae in 293 asymptomatic Chinese adult volunteers. Variations in the number of thoracic and lumbar vertebrae tend to be ignored by spine surgeons. We encourage spinal surgeons and researchers to be aware of such variations when performing thoracic- and lumbar-level surgery and assessing spinal alignment and parameters.

Transport Mechanisms of Solid Lipid Nanoparticles across Caco-2 Cell Monolayers and their Related Cytotoxicology.

Solid lipid nanoparticles (SLNs) have been extensively investigated and demonstrated to be a potential nanocarriers for improving oral bioavailability of many drugs. However, the molecular mechanisms related to this discovery are not yet understood. Here, the molecular transport mechanisms of the SLNs crossing simulative intestinal epithelial cell monolayers (Caco-2 cell monolayers) were studied. The cytotoxicology results of the SLNs in Caco-2 cells demonstrated that the nanoparticles had low cytotoxicity, had no effect on the integrity of the cell membrane, did not induce oxidative stress, and could significantly reduce cell membrane fluidity. The endocytosis of the SLNs was time-dependent, and their delivery was energy-dependent. For the first time, the transport of the SLNs was directly verified to be a vesicle-mediated process. The internalization of the SLNs was mediated by macropinocytosis pathway and clathrin- and caveolae (or lipid raft)-related routes. Transferrin-related endosomes, lysosomes, endoplasmic reticulum (ER), and Golgi apparatus were confirmed to be the main destinations of the SLNs in Caco-2 cells. As for the transport of the SLNs in Caco-2 cell monolayers, the results demonstrated that the SLNs transported to the basolateral side were intact, and the transport of the nanoparticles did not destroy the structure of tight junctions. The transcytosis of the SLNs across the Caco-2 cell monolayer was demonstrated to be mediated by the same routes as that in the endocytosis study. The ER, Golgi apparatus, and microtubules were confirmed to be important for the transport of the SLNs to both the basolateral and apical membrane sides. This study provides a more thoroughly understand of SLNs transportation crossing intestinal epithelial cell monolayers and could be beneficial for the fabrication of SLNs.

[Effects of Mongolian new medicine-â ¡ on cardiac functions, endoplasmic reticulum stress and apoptosis in congestive heart failure rats with dilated cardiomyopathy].

OBJECTIVE: To investigate the effects of Mongolian new medicine-Ⅱ oncardiac functions, myocardial pathology, endoplasmic reticulum stress and myocardial apoptosis in congestive heart failure with dilated cardiomyopathy in rats. METHODS: Thirty SD male rats were randomly dividedinto 3 groups(n=10):control group, dilated cardiomyopathy group (intraperitoneal injection of adriamycin 2 mg/kg body weight, 1 time/week, 4 weeks after treatment were observed for 4 weeks), Mongolian new medicine-Ⅱ group(intraperitoneal injection of adriamycin 2 mg/kg body weight, 1 time/week, 4 weeks after treatment, 30 mg/(kg·d)was given Mongolian new medicine-Ⅱ orally for 4 weeks). During the experiment, general conditions of rats were observed. After 8 weeks, these rats were killed after measurement of the cardiac function indexes by high frequency echocardiography. The morphological changes of myocardial tissues were observed by using HEstaining, VG staining and electron microscopic. The myocardial apoptosis was detected by TUNEL method and the expressions of endoplasmic reticulum chaperone GRP78, GRP94, pro-apoptotic factor CHOP and caspase-3 were monitored by Western blot. RESULTS: ① Compared with dilated cardiomyopathy group, the cardiac systolic and diastolic functions were significantly increased in Mongolian new medicine-Ⅱ group, which were reflected in that left ventricular contraction diameter(LVIDs) and left ventricular end-diastolic diameter(LVIDd) were decreased, and left ventricular shortening fraction(FS) and ejection fraction(EF) were increased. The hemodynamic parameters of rats were improved significantly in Mongolian new medicine-Ⅱ group. ②Compared with dilated cardiomyopathy group, the myocardial lesion score was decreased and fibrosis of tissue space was relieved in Mongolian new medicine-Ⅱ group. ③Compared with dilated cardiomyopathy group, the apoptosis of myocardial cells was decreased. ④The expressions of endoplasmic reticulum chaperone GRP78, GRP94, pro-apoptotic factor chop and caspase-3 were decreased in Mongolian new medicine-Ⅱ group. CONCLUSIONS: Mongolian new medicine-Ⅱ could improve the pathologic alterations of cardiac cells and cardiac functions, decrease endoplasmic reticulum stress, the degree of fibrosis and myocardial apoptosis. The experimental results may be one of the mechanisms of treatment function of Mongolian new medicine-Ⅱ on dilated cardiomyopathy.

Protective effects of paeoniflorin against FasL-induced apoptosis of intervertebral disc annulus fibrosus cells via Fas-FasL signalling pathway.

In the present study, we demonstrate that the degeneration of intervertebral discs is caused by ageing and apoptosis of matrix cells. Apoptosis is as essential as the function of proteoglycan synthesis in assessing the possible degeneration of intervertebral discs; paeoniflorin (PF) induces cytoprotective effects on various types of cells. In this study, the function of PF in inhibiting Fas ligand (FasL)-induced apoptosis in annulus fibrosus cells was assessed, and the correlation between apoptosis and the Fas-FasL pathway was determined. Annulus fibrosus cells were derived from the intervertebral discs of 1-month-old Sprague Dawley rats; the cells were characterised by toluidine blue staining and subjected to apoptosis with FasL. PF was diluted to various concentrations and added to annulus fibrosus cells at various times. The impact of PF and FasL on cell apoptosis of annulus fibrosus cells was determined by flow cytometry. Western blot analysis was performed to determine the protein expression levels of Fas and caspase-3. The percentages of apoptotic annulus fibrosus cells as well as the expression levels of caspase-3 and Fas were significantly reduced following treatment with 208, 20.8 or 2.08 µM PF. PF inhibits the activation of the Fas-FasL signal pathway and decreases FasL-induced apoptosis of annulus fibrosus cells.

[Effect of guizhi plus gegen decoction on ultrastructural changes of intervertebral disc annulus fibrosus cells].

OBJECTIVE: To observe the effect of Guizhi plus Gegen Decoction (GGD) on ultrastructural changes of intervertebral disc annulus fibrosus cells. METHODS: Rats' intervertebral disc annulus fibrosus cells were isolated and cultured using adherence wall screening method. After annulus fibrosus cells were intervened by GGD, the microstructure and ultrastructural features of untreated annulus fibrosus cells and annulus fibrosus cells treated by GGD containing serum at different concentrations were observed under optical microscope and electron microscope. RESULTS: Under optical microscope, most annulus fibrosus cells showed irregular polygons and few in star shape with rich superficial ecphyma. The nuclei were oval, large and complete. Under electron microscope, most cells in the blank group were oval after intervened by GGD containing serum at different concentrations. The nucleus was large, deviated, and irregular, the heterochromatin scattered diffusely, partial mitochondria vacuolized, and rough endoplasmic reticulum dilated. In the low dose GGD group, increased mitochondria and condensed density could be seen. The rough endoplasmic reticulum were expanded, lipid drops or glycogen could be occasionally seen. In the middle dose GGD group, increased endoplasmic reticulum expansion and condensed density could be seen. More medium density protein sediment could be seen. Increased mitochondria with condensed density could be seen, showing irregular cystic form with various sizes nucleus. In the high dose GGD group, increased rough endoplasmic reticulum with obvious expansion could be seen. More high density protein sediment could be seen. The nuclei were deviated. More mitochondria could be seen with secretory granules in them. CONCLUSIONS: After intervened by GGD containing serum at different concentrations, the ultrastructure of annulus fibrosus cells were manifested as follows: (1) The endoplasmic reticulum increased more in the middle and high dose GGD groups than in the blank group and the low dose GGD group. Greater density protein sediment occurred, especially in the high dose GGD group. (2) GGD played an important role in preventing ultrastructural changes induced by the degeneration of annulus fibrosus cells.