Effects of Bushen-Yizhi formula on age-related inflammation and oxidative stress in senescence-accelerated mice.

The present study aimed to investigate the possible effects and underlying molecular mechanism of Bushen­Yizhi formula (BSYZ), a traditional Chinese medicine, on age­related degeneration of brain physiology in senescence­accelerated mouse prone 8 (SAMP8) mice. SAMP8 mice (age, 6 months) were administered BSYZ (1.46, 2.92 and 5.84 g/kg/day) for 30 days. Morris water maze and step­down tests demonstrated that BSYZ significantly improved memory impairments in SAMP8 mice. In addition, BSYZ significantly enhanced the expression levels of peroxisome proliferator­activated receptor­Î³ and B­cell lymphoma extra­large, and downregulated the expression levels of inflammatory mediators, glial fibrillary acidic protein, cyclooxygenase­2, nuclear factor­κB and interleukin­1ß in the brain compared with untreated SAMP8 mice. Furthermore, BSYZ reversed disordered superoxide dismutase activity, malondialdehyde content and glutathione peroxidase activity, and ameliorated apoptosis and histological alterations. The present study indicated that BSYZ may attenuate cognitive impairment in SAMP8 mice, and modulate inflammation, oxidative stress and neuronal apoptosis. These results suggested that BSYZ may have the potential to be further developed into a therapeutic agent for protection against age­related neurodegenerative diseases.

Atractylenolide I stimulates intestinal epithelial repair through polyamine-mediated Ca2+ signaling pathway.

BACKGROUND: An impairment of the integrity of the mucosal epithelial barrier can be observed in the course of various gastrointestinal diseases. The migration and proliferation of the intestinal epithelial (IEC-6) cells are essential repair modalities to the healing of mucosal ulcers and wounds. Atractylenolide I (AT-I), one of the major bioactive components in the rhizome of Atractylodes macrocephala Koidz. (AMR), possesses multiple pharmacological activities. This study was designed to investigate the therapeutic effects and the underlying molecular mechanisms of AT-I on gastrointestinal mucosal injury. METHODS: Scratch method with a gel-loading microtip was used to detect IEC-6 cell migration. The real-time cell analyzer (RTCA) system was adopted to evaluate IEC-6 cell proliferation. Intracellular polyamines content was determined using high performance liquid chromatography (HPLC). Flow cytometry was used to measure cytosolic free Ca2+ concentration ([Ca2+]c). mRNA and protein expression of TRPC1 and PLC-γ1 were determined by real-time PCR and Western blotting assay respectively. RESULTS: Treatment of IEC-6 cells with AT-I promoted cell migration and proliferation, increased polyamines content, raised cytosolic free Ca2+ concentration ([Ca2+]c), and enhanced TRPC1 and PLC-γ1 mRNA and protein expression. Depletion of cellular polyamines by DL-a-difluoromethylornithine (DFMO, an inhibitor of polyamine synthesis) suppressed cell migration and proliferation, decreased polyamines content, and reduced [Ca2+]c, which was paralleled by a decrease in TRPC1 and PLC-γ1 mRNA and protein expression in IEC-6 cells. AT-I reversed the effects of DFMO on polyamines content, [Ca2+]c, TRPC1 and PLC-γ1 mRNA and protein expression, and restored IEC-6 cell migration and proliferation to near normal levels. CONCLUSION: Our data demonstrate that AT-I stimulates intestinal epithelial cell migration and proliferation via the polyamine-mediated Ca2+ signaling pathway. Therefore, AT-I may have the potential to be further developed as a promising therapeutic agent to treat diseases associated with gastrointestinal mucosal injury, such as inflammatory bowel disease and peptic ulcer.

[Sijunzi Decoction Promote the Repair of Intestinal Epithelial Cell Injury via Activating TLR-2/My D88 Signaling Pathway].

Objective: To study the effect and mechanism of Sijunzi decoction( SJZD) containing serum on the repairing of gastrointestinal mucosal damage. Methods: SJZD containing serum was prepared by serum pharmacological method. Cell migration model was established by tips scratch method, Real-time-cell-analyzer( RTCA) was used to mensurate IEC-6 cell proliferation, the mRNA and protein expression of TLR-2 and My D88 mRNA were detected by qRT-PCR and Western blot analysis,respectively. Results: Medium dose( 10%) and high dose( 20%) of SJZD containing serum stimulated IEC-6 cell migration at 8 h after cell damage; medium dose( 10%)and high dose( 20%) of SJZD containing serum increased IEC-6 cell proliferation at 12 h after cell damage; low dose( 5%),medium dose( 10%) and high dose( 20%) of SJZD containing serum enhanced IEC-6 proliferation both at 24 h and 36 h after cell damage. Medium dose( 10%) and high dose( 20%) of SJZD containing serum upregulated TLR-2 and My D88 mRNA and protein expression,respectively. Conclusion: Sijunzi decoction can repair the injury of gastrointestinal mucosal barrier,the mechanism may be related to its effect on activating TLR-2 / My D88 signaling pathway,and promoting IEC-6 cell migration and proliferation.

[Effects of Methanol Extracts from Atractylodes macrocephalae Rhizoma on Small Intestinal Epi- thelial Cell Proliferation and Migration, and Expression of Phospholipase C-γl].

OBJECTIVE: To observe the effects of methanol extracts from Atractylodes macro- cephalae Rhizoma (AMR) on the proliferation and migration of IEC-6 cell (small intestinal epithelial cells) and the expression of phospholipase C-γ1 (PLC-γ1) , and to explore the mechanism of AMR (a Chinese herb capable of invigorating Pi replenishing qi) for promoting repair of gastrointestinal mucosal injury. METHODS: IEC-6 cells were divided into the blank group, the positive control (spermidine, SPD; 5 µmol/L) group, AMR extracts groups (50, 100, and 200 mg/L). The alpha-difluoromethylornithine (DFMO, polyamines synthesis inhibitor) group, the SPD +DFMO group, AMR extracts (50, 100, and 200 mg/L) +DF- MO groups were set up in stress test. IEC-6 cells were cultured by adherence for 24 h,and then treated with AMR extracts for appropriate periods of time. Effects of IEC-6 cell proliferation after action of AMR extracts were detected by Real-time Cell Analyzer (RTCA). The effect of AMR extracts on IEC-6 cell migration number was detected using scratch method. mRNA and protein expressions of PLC-γ1 levels were detected by fluorescent quantitative polymerase chain reaction ( RT-qPCR) and Western blot respectively. RESULTS: Compared with the blank group, AMR extracts showed no obvious effect on IEC-6 cell proliferation (P >0. 05). But SPD and AMR extracts (100 and 200 mg/L) not only promoted IEC-6 cell migration (P <0. 01), but also improved mRNA and protein expressions of PLC-γl in the process of cell migration (P <0. 01). Compared with the DFMO group, SPD and AMR extracts (100 and 200 mg/L) could reverse inhibitory effects of DFMO on cell migration, and mRNA and protein expressions of PLC-γl (all P <0. 01). CONCLUSION: AMR extracts played roles in repairing gastrointestinal mucosal injury possibly by promoting polyamine mediated intestinal epithelial cell migration, and its effect on intestinal epithelial cell proliferation was not main potentcy.

Phenotypic characterization of type II collagen-induced arthritis in Wistar rats.

The aim of the present study was to determine a more specific, efficient and simple method for the induction of collagen-induced arthritis (CIA) in rats. Different strains of rats were injected at the base of the tail with bovine type II collagen (CII) emulsified in incomplete Freund's adjuvant (IFA). The onset and severity of arthritis were evaluated by clinical assessment. The established CIA model was analyzed using a comprehensive examination of clinical, hematological, histological and radiological parameters. The results demonstrated that Wistar rats were the most susceptible strain to CIA followed by Wistar Furth rats, with Sprague Dawley rats being the least susceptible. Following primary and booster immunization, female Wistar rats developed severe arthritis, with an incidence of >83% and low variability in clinical signs. The development of arthritis was accompanied by a significantly elevated erythrocyte sedimentation rate compared with that in the control rats. The radiographic examination revealed bone matrix resorption, considerable soft tissue swelling, periosteal new bone formation and bone erosion in the arthritic joints of the CIA rats. Histopathologically, the synovial joints of CIA rats were characterized by synovial hyperplasia, pannus formation, marked cellular infiltration, bone and cartilage erosion and narrowing of the joint space. The administration of an intradermal injection of only 200 µg bovine CII emulsified in IFA at the base of the tail therefore leads to the successful development of a CIA rat model. This well-characterized CIA rat model could be specifically used to study the pathophysiology of human rheumatoid arthritis as well as to test and develop anti-arthritic agents for humans.

Atractylodes macrocephala Koidz stimulates intestinal epithelial cell migration through a polyamine dependent mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodes macrocephala Koidz (AMK), a valuable traditional Chinese herbal medicine, has been widely used in clinical practice for treating patients with disorders of the digestive system. AMK has shown noteworthy promoting effect on improving gastrointestinal function and immunity, which might represent a promising candidate for the treatment of intestinal mucosa injury. The aim of this study was to investigate the efficacy of AMK on intestinal mucosal restitution and the underlying mechanisms via intestinal epithelial (IEC-6) cell migration model. MATERIALS AND METHODS: A cell migration model of IEC-6 cells was induced by a single-edge razor blade along the diameter of the cell layers in six-well polystyrene plates. After wounding, the cells were grown in control cultures and in cultures containing spermidine (5µM, SPD, reference drug), alpha-difluoromethylornithine (2.5mM, DFMO, polyamine inhibitor), AMK (50, 100, and 200mg/L), DFMO plus SPD and DFMO plus AMK for 12h. The polyamines content was detected by high-performance liquid chromatography (HPLC) with pre-column derivatization. The Rho mRNAs expression levels were assessed by Q-RT-PCR. The Rho and non-muscle myosin II proteins expression levels were analyzed by Western blot. The formation and distribution of non-muscle myosin II stress fibers were monitored with immunostaining techniques using specific antibodies and observed by confocal microscopy. Cell migration assay was carried out using inverted microscope and the Image-Pro Plus software. All of these indexes were used to evaluate the effectiveness of AMK. RESULTS: (1) Treatment with AMK caused significant increases in cellular polyamines content and Rho mRNAs and proteins expression levels, as compared to control group. Furthermore, AMK exposure increased non-muscle myosin II protein expression levels and formation of non-muscle myosin II stress fibers, and resulted in an acceleration of cell migration in IEC-6 cells. (2) Depletion of cellular polyamines by DFMO resulted in a decrease of cellular polyamines levels, Rho mRNAs and proteins expression, non-muscle myosin II protein formation and distribution, thereby inhibiting IEC-6 cell migration. AMK not only reversed the inhibitory effects of DFMO on the polyamines content, Rho mRNAs and proteins expression, non-muscle myosin II protein formation and distribution, but also restored cell migration to control levels. CONCLUSIONS: The results obtained from this study revealed that AMK significantly stimulates the migration of IEC-6 cells through a polyamine dependent mechanism, which could accelerate the healing of intestinal injury. These findings suggest the potential value of AMK in curing intestinal diseases characterized by injury and ineffective repair of the intestinal mucosa in clinical practice.

Atractylodes macrocephala Koidz promotes intestinal epithelial restitution via the polyamine--voltage-gated K+ channel pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodes macrocephala Koidz (AMK) has been used widely as a digestive and tonic in traditional Chinese medicine. AMK has shown noteworthy promoting effect on intestinal epithelial cell migration, which might represent a promising candidate for the treatment of intestinal mucosa injury. The aim of this study was to investigate the efficacy of AMK on intestinal mucosal restitution and the underlying mechanisms via IEC-6 cell migration model. MATERIALS AND METHODS: A wounding model of IEC-6 cells was induced by a single-edge razor blade along the diameter of six-well polystyrene plates. The cells were grown in control cultures and in cultures containing spermidine (5 µmol/L, SPD, reference drug), alpha-difluoromethylornithine (2.5 mmol/L, DFMO, polyamine inhibitor), AMK (50, 100, and 200 µg/mL), DFMO plus SPD and DFMO plus AMK for 24h. The membrane potential (MP) and cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) were detected by flow cytometry, and polyamines content was determined via high-performance liquid chromatography (HPLC). The expression of Kv1.1 mRNA and protein levels were assessed by RT-qPCR and Western blot analysis, respectively. Cell migration assay was carried out using the Image-Pro Plus software. All of these indexes were used to evaluate the effectiveness of AMK. RESULTS: (1) Treatment with AMK caused significant increases in cellular polyamines content, membrane hyperpolarization, an elevation of [Ca(2+)]cyt and an acceleration of cell migration in IEC-6 cells, as compared to control group. (2) AMK not only reversed the inhibitory effects of DFMO on the polyamines content, MP, and [Ca(2+)]cyt but also restored IEC-6 cell migration to control levels. (3) The Kv1.1 mRNA and protein expression were significantly increased by AMK treatment in control and polyamine-deficient IEC-6 cells. CONCLUSIONS: The results of our current studies revealed that treatment with AMK significantly stimulates the migration of intestinal epithelial cells through polyamine-Kv1.1 channel signaling pathway, which could promote the healing of intestinal injury. These results suggest the potential usefulness of AMK to cure intestinal disorders characterized by injury and ineffective repair of the intestinal mucosa.

Sweet tea leaves extract improves leptin resistance in diet-induced obese rats.

AIM OF THE STUDY: Dietary obesity is usually characterized by leptin resistance and abnormal lipid metabolism. Lithocarpus polystachyus Rehd.(Sweet Tea) leaf is a kind of Chinese folkloric medicine, and it has been widely used for obesity, diabetes, and hypertension in South China. The present study is aimed at investigating the pharmacological mechanism of the anti-hyperleptinaemia effects of Sweet Tea leaves extract in high fat diet-induced obese rats. MATERIALS AND METHODS: We induced high fat diet obesity for 14 weeks to test the corrective effects of three ST doses (75, 150 and 300 mg/kg per day) for 8 weeks. At the end of the experiment, body weight, fasting blood glucose and serum lipids, superoxide dismutase (SOD), malondialdehyde (MDA), fasting serum insulin and leptin, C-reactive protein, adiponectin and resistin levels were measured, Homeostasis Model Assessment for Insulin Resistance (HOMA-IR) was also calculated. mRNA gene expression of PPARγ (peroxisome proliferator-activated receptor γ) and C/EBPα(CCAAT/enhancer-binding protein α) in epididymal adipose tissue of DIO control and experimental groups were evaluated. RESULTS: Sweet Tea leaves extract could significantly decrease the levels of serum lipids, attenuate body weight gain and lower circulating leptin and insulin levels, ameliorate the state of oxidative stress, raise serum adiponectin, reduce circulating CRP and resistin levels, and depress the expression of PPARγ and C/EBPα in epididymal adipose tissue of obese rats. CONCLUSION: The present findings suggest that ST can effectively attenuate the leptin resistance at least through anti-hyperlipidemic activity and thus has the therapeutic potential in treating hyperlipidemia and hyperleptinaemia related to dietary obesity.