Asarinin attenuates bleomycin-induced pulmonary fibrosis by activating PPARγ.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease that lacks effective treatment modalities. Once patients are diagnosed with IPF, their median survival is approximately 3-5 years. PPARγ is an important target for the prevention and treatment of pulmonary fibrosis. Asarinin is a lignan compound that can be extracted from food plant Asarum heterotropoides. In this study, we investigated the therapeutic effects of asarinin in a pulmonary fibrosis model constructed using bleomycin in mice and explored the underlying mechanisms. Intraperitoneal administration of asarinin to mice with pulmonary fibrosis showed that asarinin effectively attenuated pulmonary fibrosis, and this effect was significantly inhibited by the PPARγ inhibitor GW9662. Asarinin inhibited TGF-ß1-induced fibroblast-to-myofibroblast transition in vitro, while GW9662 and PPARγ gene silencing significantly inhibited this effect. In addition, asarinin inhibited not only the canonical Smad pathway of TGF-ß but also the non-canonical AKT and MAPK pathways by activating PPARγ. Our study demonstrates that asarinin can be used as a therapeutic agent for pulmonary fibrosis, and that PPARγ is its key target.

Molecular cloning and function analysis of insulin-like growth factor-binding protein 1a in blunt snout bream (Megalobrama amblycephala).

Insulin-like growth factor-binding protein 1 (IGFBP-1), a hypoxia-induced protein, is a member of the IGFBP family that regulates vertebrate growth and development. In this study, full-length IGFBP-1a cDNA was cloned from a hypoxia-sensitive Cyprinidae fish species, the blunt snout bream (Megalobrama amblycephala). IGFBP-1a was expressed in various organs of adult blunt snout bream, including strongly in the liver and weakly in the gonads. Under hypoxia, IGFBP-1a mRNA levels increased sharply in the skin, liver, kidney, spleen, intestine and heart tissues of juvenile blunt snout bream, but recovered to normal levels after 24-hour exposure to normal dissolved oxygen. In blunt snout bream embryos, IGFBP-1a mRNA was expressed at very low levels at both four and eight hours post-fertilization, and strongly at later stages. Embryonic growth and development rates decreased significantly in embryos injected with IGFBP-1a mRNA. The average body length of IGFBP-1a-overexpressed embryos was 82.4% of that of the control group, and somite numbers decreased to 85.2%. These findings suggest that hypoxia-induced IGFBP-1a may inhibit growth in this species under hypoxic conditions.

The goldfish hAT-family transposon Tgf2 is capable of autonomous excision in zebrafish embryos.

The goldfish (Carassius auratus) Tgf2 transposon is a vertebrate DNA transposon that belongs to the hAT transposon family. In this study, we constructed plasmids containing either the full-length Tgf2 transposon (pTgf2 plasmid) or a partially-deleted Tgf2 transposon (ΔpTgf2 plasmid), and microinjected these plasmids into fertilized zebrafish (Danio rerio) eggs at the one- to two-cell stage. DNA extracted from the embryos was analyzed by PCR to assess transient excision, if any, of the exogenous plasmid and to verify whether Tgf2 is an autonomous transposon. The results showed that excision-specific bands were not detected in embryos injected with the ΔpTgf2 plasmid, while bands of 300-500bp were detected in embryos injected with pTgf2, which indicated that the full-length Tgf2-containing plasmid could undergo autonomous excision in zebrafish embryos. DNA cloned from 24 embryos injected with pTgf2 was sequenced, and the results suggested that Tgf2 underwent self-excision in zebrafish embryos. Cloning and PCR analysis of DNA extracted from embryos co-injected with ΔpTgf2 and in vitro-transcribed transposase mRNA indicated that partially-deleted-Tgf2-containing ΔpTgf2 plasmid also underwent excision, in the presence of functional transposase mRNA. DNA cloned from 25 embryos co-injected with ΔpTgf2 and transposase mRNA was sequenced, and the results suggested that partially-deleted Tgf2 transposons plasmids were excised. These results demonstrated that excisions of Tgf2 transposons were mediated by the Tgf2 transposase, which in turn confirmed that Tgf2 is an autonomous transposon.

Goldfish transposase Tgf2 presumably from recent horizontal transfer is active.

Hobo/Activator/Tam3 (hAT) superfamily transposons occur in plants and animals and play a role in genomic evolution. Certain hAT transposons are active and have been developed as incisive genetic tools. Active vertebrate elements are rarely discovered; however, Tgf2 transposon was recently discovered in goldfish (Carassius auratus). Here, we found that the endogenous Tgf2 element can transpose in goldfish genome. Seven different goldfish mRNA transcripts, encoding three lengths of Tgf2 transposase, were identified. Tgf2 transposase mRNA was detected in goldfish embryos, mainly in epithelial cells; levels were high in ovaries and mature eggs and in all adult tissues tested. Endogenous Tgf2 transposase mRNA is active in mature eggs and can mediate high rates of transposition (>30%) when injected with donor plasmids harboring a Tgf2 cis-element. When donor plasmid was coinjected with capped Tgf2 transposase mRNA, the insertion rate reached >90% at 1 yr. Nonautonomous copies of the Tgf2 transposon with large-fragment deletions and low levels of point mutations were also detected in common goldfish. Phylogenetic analysis indicates the taxonomic distribution of Tgf2 in goldfish is not due to vertical inheritance. We propose that the goldfish Tgf2 transposon originated by recent horizontal transfer and maintains a highly native activity.

Effect of histamine on regional cerebral blood flow of the parietal lobe in rats.

Histamine is a powerful modulator that regulates blood vessels and blood flow. The effect of histamine on the extracortical vessels has been well described, while much less is known about the effect of histamine on intracortical vessels. In this study, we investigated the effect of histamine on regional cerebral blood flow in rat parietal lobe with laser Doppler flowmetry. The pharmacological characteristics of distinct ways (intracerebroventricular injection, intraperitoneal injection, and cranial window infusion) in applying histamine to the brain were also obtained and compared. Histamine applied in three ways all produced a decrease of rCBF in parietal lobe in a concentration-dependent manner. Cranial window infusion was the most effective way and intraperitoneal injection of L-histidine was the most ineffective, although it is a simple and applied way. To determine which type of receptor takes part in the vessel contraction induced by histamine, H1 receptor antagonist, diphenhydramine, and H2 receptor antagonist, cimetidine, were applied, respectively, before histamine administration. When the injection of cimetidine was conducted in advance, histamine still resulted in a decrease of infusion amount; while the injection of diphenhydramine was conducted in advance, the infusion of blood amount wasn't changed. These findings indicated that histamine could result in a reduction of rCBF in the rat parietal lobe and this effect of histamine may attribute partly to its combination with H1 receptor.

[Effect of glutamate on vascular endothelial growth factor mRNA and protein expressions in hypoxic rat astrocytes in vitro].

OBJECTIVE: To study the effect of glutamate on the expression of vascular endothelial growth factor (VEGF) mRNA and protein in cultured rat astrocytes under hypoxia. METHODS: Cultured rat astrocytes were randomly divided control group, glutamate group, hypoxia group and hypoxia+glutamate group. The cells in the control and glutamate groups were cultured under nomoxic condition (95% air and 5% CO(2)), and those in the other two groups under hypoxic condition (94% N(2), 5% CO(2) and 1% O(2)). The total RNA was extracted from the cells at different time points of hypoxic exposure for real-time FQ-PCR and ELISA to detect the expression of VEGF mRNA and protein in cultured astrocytes, respectively. RESULTS: The expressions of VEGF mRNA and protein underwent no significant changes in the control glutamate groups, but increased obviously in both hypoxia and hypoxia+glutamate groups at 2, 4, 6, 8 and 12 h of hypoxic exposure. At these time points, VEGF expressions at both the mRNA and protein levels were significantly higher in hypoxia+glutamate group than in hypoxia group. CONCLUSION: Glutamate at 1 micromol/L can further increase the expression of VEGF mRNA and protein in astrocytes exposed to hypoxia, which may result from the adaptive changes of glutamate receptors in hypoxic astrocytes.

[Comparison and correlative analysis of trace elements in five kinds of radix curcumae].

Traditional Chinese medicine materials (TCM) contained abundant trace elements needed in human body. Mixed acid (HNO3:HClO4) was added into samples treated by microwave digestion, and a systematic study was carried out by selection of the most appropriate working conditions and optimization of the sample mass. Some inorganic elements in 5 kinds of radix curcumae (Curcuma wenyujin Y. H. Chen et C. Ling, Curcuma longa L., Curcuma kwangsiensis S. G. Lee et C. F. Liang, Curcuma phaeocaulis Val. and Curcuma chuanyujin C. K Hsieh et H. Zhang) necessary to lives were determined by flames atomic absorption spectrometry (AAS). Elements, such as K, Ca, Na, Mg, Zn, Fe, Cu and Mn were detected. The results show that there are similar elements in the five kinds of radix curcumae, while differences only exist in a few elements contents. The correlative analysis of trace elements in samples was carried out by cluster analysis of the 4 kinds of radix curcumae (Curcuma wenyujin Y. H. Chen et C. Ling, Curcuma longa L., Curcuma kwangsiensis S. G. Lee et C. F. Liang and Curcuma phaeocaulis Val. ) in codex populations in a cluster, different from the unofficial sample (Curcuma chuanyujin C. K. Hsieh et H. Zhang). The cluster analysis results are consistent with the morphological characteristics of the traditional taxonomy results. The results showed that the analytic method is advisable. This paper provides scientific basis for deeply studing the relation between trace elements and drug effect of radix curcumae.

[A simple method for assessment of RNA integrity in laser capture microdissection samples].

OBJECTIVE: To develop a simple method for assessment of RNA integrity in laser capture microdissection (LCM) samples. METHODS: The total RNA were isolated from the LCM samples and the sections before and after microdissection and examined by agarose gel electrophoresis. Real-time PCR was employed to assess the RNA from LCM samples, and the quantity of RNA was theoretically estimated according to the average total RNA product in mammalian cells (10 ng/1000 cells). RESULTS: When the total RNA from the sections before and after microdissection was intact, the RNA from LCM samples also had good quality, and the 28S and 18S rRNAs were visualized by ethidium bromide staining. Real-time PCR also showed good RNA quality in the LCM samples. CONCLUSION: A simple method for quantitative and qualitative assessment of the RNA from LCM samples is established, which can also be applied to assessment of DNA or proteins in LCM samples.

[Neurons in the corpus callosum of rats: expression of Cav2.2 and their connection].

OBJECTIVE: To prove the existence neurons in the rat corpus callosum, the potential function of these neurons and their connection. METHODS: Immunohistochemistry was used performed to examine the expressions of NeuN, a mature neuron marker,and N-type voltage-dependent valcium channel alpha1-subunit (Cav2.2)in the section of the rat corpus callosum. Horseradish peroxidase (HRP) normal sodium solution (30%), the retrograde tracer,was injected under the frontal forceps of corpus callousm and HRP absorbed by the process of neurons in the brain slices was stained with tetramethyl benzidine. RESULTS: There were some NeuN positive cells in the rat corpus callosum and Cav2.2 was detected in some of these NeuN positive cells.Neurons with positive HRP were found in the rat corpus callosum and some of these neurons connected to the cortex or corpus striatum. CONCLUSION: There are a few neurons in the corpus callosum of adult rats and some of them express Cav2.2. Neurons in the corpus callosum have connections with the brain cortex or corpora striatum.

[Construction and identification of a plasmid with EGFP reporter gene for enhancer function analysis].

OBJECTIVE: To construct a plasmid vector with EGFP reporter gene for functional analysis of enhancers. METHODS: EGFP DNA was amplified by PCR from plasmid pEGFP-N1 DNA and subcloned into plasmid PGL3-promoter backbone without luc(+) gene to construct the enhancer-identifying vector pEGFP-enhancer. Different copies of hypoxia response element (HRE) sequence were synthetized and subcloned into the multiple cloning site of the plasmid pEGFP-enhancer. Using Lipofectamine 2000, the recombined pEGFP-HRE and pEGFP-5HRE plasmids were transfected into the Hela cells respectively. After hypoxic or normoxic cell culture, EGFP expression in the cells was detected by flow cytometry and fluorescence microscopy. RESULTS: After hypoxic exposure, the fluorescence intensity of EGFP in the Hela cells transfected with the plasmid increased with the enhancer HRE copies, while the fluorescence intensity underwent no significant changes after normoxic cell culture. CONCLUSION: we have successfully constructed the enhancer expression vector plasmid pEGFP-enhancer, which can identify the activity of the enhancers through EGFP expression.

[Effect of ligustrazine on cell proliferation in subventricular zone in rat brain with focal cerebral ischemia-reperfusion injury].

OBJECTIVE: To observe the effect of ligustrazine on cell proliferation in subventricular zone (SVZ) in rat brain with focal cerebral ischemia reperfusion injury. METHODS: Male SD rats were randomly divided into a normal group,a sham operation group,a ligustrazine treatment group, and a control group. The ligustrazine treatment group and the control group were further divided into 5 subgroups: 1d, 3d, 7d, 14d, and 21d reperfusion after 2h middle cerebral artery occlusion (MCAO). The focal cerebral ischemia-reperfusion model was made by MCAO. S phase cells were labelled with BrdU. Immunohistochemistry method was conducted to detect the BrdU positive cells. The total number of BrdU positive cells in the SVZ was measured. The expression of neuro nitric oxide synthase (nNOS) was detected with Western blot method. RESULTS: There was a significant increase of BrdU positive cells in SVZ of ligustrazine treatment in the 1d and 3d group compared with that of the control group (P<0.01). The total number of BrdU positive cells reached a peak in 7d group and declined afterwards. Cells proliferated also in SVZ on the contralateral side, and peaked at 7d. The nNOS expression of ligustrazine administration after the focal cerebral ischemia-reperfusion decreased at 1d and 3d after the reperfusion compared with that of the control group (P<0.05), and increased at 7d, but with no significant difference compared with that of the control group. CONCLUSION: Ligustrazine may promote the cell proliferation in SVZ of adult rats with ischemia-reperfusion injury by decreasing the nNOS expression.

[Effect of ligustrazine on nNOS expression and neuranagenesis in adult rats after cerebral ischemia-reperfusion injury].

OBJECTIVE: To observe the effect of ligustrazine on cell proliferation in the subventricular zone (SVZ) and dentate gyrus (DG) and nNOS expression in rat brain after cerebral ischemia-reperfusion injury. METHODS: Male SD rats were randomly divided into normal control group, sham operation group, model group and ligustrazine treatment group. The latter two groups were further divided into 5 subgroups for observation at 1, 3, 7, 14 and 21 days after reperfusion following a 2-hour middle cerebral artery occlusion (MCAO). The cells in S phase were labeled with BrdU, and immunohistochemistry was employed to detect BrdU- and nNOS-positive cells. The numbers of BrdU-positive cells in the SVZ and DG were measured. The expression of nNOS was detected by Western blotting. RESULTS: nNOS expression increased significantly in the model group as compared to the sham operation group (P<0.05), and ligustrazine treatment significantly lowered the expression level in comparison with the model group (P<0.05). Compared with the model group, a significant increase in BrdU-positive cells occurred in the SVZ of rats 1 and 3 days after igustrazine treatment (P<0.05), along with an increase of DG BrdU-positive cells. CONCLUSION: Ligustrazine significantly restrains ischemia-reperfusion injury-induced nNOS activity enhancement and promotes cell proliferation in the SVZ and DG of adult rats after ischemia-reperfusion injury.