To investigate the protective effects of eplerenone on adriamycin-induced renal injury and the possible mechanisms involved, 36 male Sprague-Dawley rats were randomly divided into control group, adriamycin nephropathy (AN) group and eplerenone-treated group (100 mg·kg(-1)·d(-1) eplerenone). Blood pressure, 24-h urinary protein, serum potassium, sodium and creatinine were measured 28 days after adriamycin injection (a single tail intravenous injection of 6.5 mg/kg adriamycin). The morphological changes of renal tissues were observed by light and electron microscopy. Immunohistochemistry and Western blotting were performed to examine the expression of TGF-β(1) and desmin in renal cortex. The results showed that 28 days after adriamycin injection, there were no significant changes in the level of serum potassium, sodium, creatinine concentrations and blood pressure values in the rats of the three groups. Meanwhile, the 24-h proteinuria excretion in the AN group was significantly higher than that in the control group (P<0.01), but that in the eplerenone-treated group was substantially reduced when compared with that in the AN group (P<0.05). Mild mesangial cell proliferation and matrix expansion, diffuse deformation and confluence of foot processes in podocytes were found in the AN group. By contrast, rats in the eplerenone-treated group exhibited obvious attenuation of these morphological lesions. The protein expression of TGF-β(1) and desmin in the AN group was markedly up-regulated in contrast to that in the control group (P<0.01), whereas that in the eplerenone-treated group was much lower than in the AN group (P<0.05). It was concluded that eplerenone may ameliorate the proteinuria and the development of pathological alteration in adriamycin-induced nephropathy presumably via the inhibition of cytokine release, and restore the morphology of podocytes independent of its blood pressure-lowing effects.

This study was aimed to observe the analgesia of curing-injury Cataplasma and discuss the Nav1 . 7 expression in dorsal root ganglion ( DRG ) in model rats with formaldehyde-induced inflammatory pain . A total of 36 Sprague-Dawley rats were divided into three groups, which were the blank control group (n = 12), model group ( n = 12 ) , and treatment group ( n = 12 ) . The blank control group was without any treatment . The model group was injected with 0 . 1 mL 5% saline formalin on the left rear foot . The treatment group was applied with curing-injury Cataplasma on the left rear foot 24 h before the injection of 0 . 1 mL 5% saline formalin in the establishment of animal model . The behavior reactions to pain of model rats were observed . Dubuisson score was recorded and compared . Meanwhile , L3-6 DRG was collected from rats in each group . The expres-sion of Nav1 . 7 was detected by real-time quantitative PCR and western blot . The results showed that the pain reaction integral in the treatment group was lower than the model group ( P < 0 . 05 ) . Results from the real-time quantitative PCR showed that the relative expression of Nav1 . 7 mRNA in the model group was more than the treatment group . And the relative expression of Nav1 . 7 mRNA in the treatment group was more than the blank control group . There was significant difference among three groups ( P < 0 . 05 ) . There was no statistical difference at the three time points within three groups. Results from the western blot showed that the relative expression of Nav1 . 7 in the model group was more than the treatment group . And the expression of Nav1 . 7 in the treatment group was more than the blank control group . There was significant difference among three groups (P < 0.05). There was no statistical difference at the three time points within three groups. It was concluded that the curing-injury Cataplasma can alleviate inflammatory pain response in rats, and have certain analgesia effect . Meanwhile , it can influence Nav1 . 7 expression in DRG in model rats with formaldehyde-induced inflam-matory pain .

This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA (shRNA) targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside (PAN)-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein (GFP)-contained plasmids (pGC) to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein expression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN+TRPC6 shRNA transfected group and PAN+scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.

This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA (shRNA) targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside (PAN)-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein (GFP)-contained plasmids (pGC) to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein expression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN+TRPC6 shRNA transfected group and PAN+scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.
Animals ; Cell Membrane Permeability ; drug effects ; physiology ; Cell Survival ; drug effects ; physiology ; Cells, Cultured ; Gene Knockdown Techniques ; Mice ; Mice, Knockout ; Podocytes ; drug effects ; physiology ; Puromycin Aminonucleoside ; pharmacology ; TRPC Cation Channels ; genetics ; metabolism

To investigate the protective effects of eplerenone on adriamycin-induced renal injury and the possible mechanisms involved,36 male Sprague-Dawley rats were randomly divided into control group,adriamycin nephropathy (AN) group and eplerenone-treated group (100 mg·kg-1·d-1 eplerenone).Blood pressure,24-h urinary protein,serum potassium,sodium and creatinine were measured 28 days after adriamycin injection (a single tail intravenous injection of 6.5 mg/kg adriamycin).The morphological changes of renal tissues were observed by light and electron microscopy.lmmunohistochemistry and Western blotting were performed to examine the expression of TGF-β1 and desmin in renal cortex.The results showed that 28 days after adriamycin injection,there were no significant changes in the level of serum potassium,sodium,creatinine concentrations and blood pressure values in the rats of the three groups.Meanwhile,the 24-h proteinuria excretion in the AN group was significantly higher than that in the control group (P＜0.01),but that in the eplerenone-treated group was substantially reduced when compared with that in the AN group (P＜0.05).Mild mesangial cell proliferation and matrix expansion,diffuse deformation and confluence of foot processes in podocytes were found in the AN group.By contrast,rats in the eplerenone-treated group exhibited obvious attenuation of these morphological lesions.The protein expression of TGF-β1 and desmin in the AN group was markedly up-regulated in contrast to that in the control group (P＜0.01),whereas that in the eplerenone-treated group was much lower than in the AN group (P＜0.05).It was concluded that eplerenone may ameliorate the proteinuria and the development of pathological alteration in adriamycin-induced nephropathy presumably via the inhibition of cytokine release,and restore the morphology of podocytes independent of its blood pressure-lowing effects.

ObjectiveTo study the anti-inflammatory effects of Blumea balsamifera （L.） DC oil （BBO） based on nuclear factor kappa-B （NF-κB） nonclassical and arachidonic acid （AA） pathway. MethodsEffects of BBO on the production of slow reacting substance of anaphylaxis （SRS-A） were detected by the ileal smooth muscle method. The contents of prostaglandin E₂ （PGE₂） and leukotriene B₄ （LTB₄） in lipopolysaccharides （LPS） -induced macrophages were detected by ELISA kit. The expression of COX-2， 5-LOX， FLAP and RelB were detected by qRT-PCR. Western blot was performed to detect the effects of BBO on the level of NF-κB nonclassical pathway proteins TNF receptor associated factor 3 （TRAF3）， TNF receptor associated factor 2 （TRAF2）， NF-κB-inducing kinase （NIK）， p100 and RelB. ResultsThe contractile tension of guinea pig ileum was reduced （P<0.001）， and the SRS-A production inhibition rate reached 65.34% at 1mg·mL^-1 BBO concentration. Compared with LPS group， BBO reduced the concentrations of PGE₂ （P<0.05） and LTB₄ （P<0.05）_， and decreased the expressions of COX-2 （P<0.05）， 5-LOX （P<0.05） and FLAP （P<0.05） in AA pathway at concentrations of 40-80 μg·mL^-1. Moreover， 40-80 μg·mL^-1 BBO decreased the concentrations of TRAF3 （P<0.05）， TRAF2 （P<0.05）， and NIK （P<0.05）， and further inhibited the phosphorylation of p100 （P<0.05）， as well as the level of the transcription factor RelB in genes （P<0.05） and proteins （P<0.05） in nonclassical NF-κB pathway， whereas BBO did not cause such changes. ConclusionBBO may potentially exert its anti-inflammatory effects by suppressing the regulatory proteins TRAF3 and TRAF2 and the transcription factor RelB in NF-κB nonclassical pathway. The inhibitory action extending to the induction kinase function of NIK， further hindering the phosphorylation of p100 and its binding with the transcription factor RelB. Consequently， downstream elements in the AA pathway， including the pivotal rate-limiting enzymes COX-2， 5-LOX and FLAP， were altered. This modulation influences the levels of inflammatory mediators such as PGE₂ and LTB₄.

OBJECTIVE To optimize the extraction process of blumeatin from Blumea balsamifera and to evaluate its antibacterial and anti-inflammatory activity. METHODS The content of blumeatin in the extract of B. balsamifera was determined by HPLC. On the basis of the single factor experiment， the extraction technology of blumeatin was optimized by the Box-Behnken response surface method with the volume fraction of ethanol， liquid-solid ratio and extraction time as the factors， using the yield of blumeatin as index. Microdilution method was used to determine the antibacterial activity of blumeatin against Streptococcus pyogenes， Staphylococcus aureus， Streptococcus agalactiae， Streptococcus mutans， Bacillus subtilis and Micrococcus luteus. The anti-inflammatory activity of blumeatin was evaluated by ear swelling test and capillary permeability test in mice. RESULTS The optimal extraction technology was as follows： ethanol concentration of 90%， liquid-material ratio of 15∶1， extraction time of 2 h at 80 ℃； the yield of blumeatin using this extraction process was 1.97 mg/g. The minimum inhibitory concentrations of blumeatin for S. pyogenes， S. aureus， S. agalactiae， S. mutans， B. subtilis and M. luteus were 50.00， 200.00， 400.00， 400.00， 800.00 and 1 600.00 μg/mL， respectively； the minimum bactericidal concentrations of blumeatin for S. pyogenes and S. aureus were 400.00 and 1 600.00 μg/mL， respectively. Blumeatin could significantly inhibit the ear swelling induced by xylene and capillary permeability induced by acetic acid in mice（P＜0.05 or P＜0.01）. CONCLUSIONS The optimized extraction technology of blumeatin is stable and feasible. The extracted blumeatin has a certain antibacterial effect against S. pyogenes and a good anti-inflammatory activity.