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.

Abstract: To explore the inflammatory changes and the changes of β-amyloid in the brain of rats with experimental periodontitis induced by ligation. Methods: Eighteen Sprague Dawley rats were randomly divided into 3 groups(n=6): the negative control group, chronic periodontitis group and chronic periodontitis treated with intraperitoneal injection of TAK-242 group. The experimental periodontitis model was established by ligation of the necks of bilateral maxillary first molar and inoculation ofPorphyromonas gingivalis(P.gingivalis). At the end of the second month after the successful modeling, the samples were collected from the rats. The damage of the alveolar bone was analyzed by Micro-CT. The mRNA expression levels of interleukin(IL)-6, IL-1β and tumor necrosis factor-α(TNF-α) in gingival tissue and hippocampal tissue, the mRNA expression level of Toll like receptors-4(TLR4), leukocyte differentiation antigen 14(CD14) and NF-κB in hippocampal tissue of rats were detected by qPCR. The protein expression levels of IL-6, IL-1β, TNF-α, myloid-beta protein-40(Aβ40) and Aβ42in hippocampal tissue of rats were evaluated by ELISA. Results: Experimental periodontitis model of rats could be successfully established by ligation of the neck of the rat's bilateral maxillary first molars and inoculation with porphyromonas gingivalis. The results of qPCR and ELISA showed that experimental periodontitis up-regulated the expression levels of inflammatory factors(IL-6, IL-1β and TNF-α) in hippocampus of rats and the result of ELISA showed that the level of Aβ42in hippocampus of experimental periodontitis rats increased. But the pretreatment with TAK-242 intraperitoneal injection could reduce the up-regulated the expression of inflammatory factors and Aβ42by down-regulating the TLR4/NF-κB signaling pathway. Conclusion Experimental periodontitis in rats induced by ligation combined with inoculation of porphyromonas gingivalis can result in inflammation in the brain and promote the accumulation of Aβ42in the brain, and it is reasonable to speculate that inflammation may play an important role in the correlation between periodontitis and systemic diseases such as Alzheimer's disease.

Potassium 2-(1-hydroxypentyl)-benzoate (d,l-PHPB), a new drug candidate for ischemic stroke at the phase II clinic trial, has been shown to protect neurons by inhibiting oxidative injury and reducing neuron apoptosis in previous studies. But the mechanisms of d,l-PHPB remain to be studied. In this study, a neuron-astrocytes co-culture system was used to elucidate the roles of astrocytes in neuroprotection of d,l-PHPB under oxygen-glucose deprivation/reoxygenation (OGD/R) condition. Our data showed that d,l-PHPB reduced neuronal apoptosis in mono-culture system and this effect was enhanced in neuron-astrocyte co-culture system under the OGD/R condition. Meanwhile, d,l-PHPB obviously increased the levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), which were mainly secreted from astrocytes, in the co-culture system after OGD/R. The PI3K/AKT and ERK signaling pathways as well as the p-TRKA/B receptors were involved in the process. In addition, the levels of TNF-and IL-1secreted from astrocytes after OGD/R were markedly reduced after d,l-PHPB treatment, which was mainly due to the suppression of phosphorylated p38. In conclusion, the present study demonstrates that the neuroprotective effects of d,l-PHPB were improved by astrocytes, mainly mediated by increasing the release of BDNF/NGF and attenuating inflammatory cytokines.

As a most effective monomer composition from bark of Pacific Yew,paclitaxel and its derivatives are used in clinical practice as broad spectrum anticancer drugs.Since its discovery in the 1970 s,many researches had been carried out,mainly focusing on the modification,structure-activity relationship and pharmacological activity.The great successes pressed ahead the development of a series of taxol-like drugs,including taxol,docetaxel,cabazitaxel,larotaxel.Nowadays,studies of taxol are still the hotpots,which concentrated on the new source such as cultivation of tissue,fungus culture and new dosage forms.As the representative of drugs research from natural source,taxol is worth to be summarized of its history and ongoing development for looking forward to bring new innovation mentality in new drugs.

Objective To study the value of high frequency ultrasound in predicting spontaneous closure of patent duct arteriosus (PDA) in premature neonates. Methods One hundred and seventy premature neonates aged less than 37 weeks of gestation were enrolled. They were all admitted in the neonatal intensive care unit of Jinhua People′ s Hospital within 24 hours after birth. Echocardiographic examinations was conducted using GE′s Vivid I Portable color Doppler ultrasonography with routine cardiac probe (frequency 1.7-3.4 MHz) and high-frequency transducer (frequency 7.5 MHz). Up to the 7th day, the echocardiography was carried out every day after birth until the closure of PDA .They were divided to two groups according to whether spontaneous closure of PDA took place with in 7 days after birth (the control group) or not (the PDA group). There were 16 premature neonates in PDA group, including 6 male and 10 female. The mean gestational week at birth was (35.3±1.4) weeks. The mean birth weight was (2 330.9±325.5) g. Thirty-two premature neonates were included incontrol group, including 13 male and 19 female. The mean gestational week at birth was (35.0±1.3) weeks. The mean birth weight was (2 337.5±287.8) g. The clinical characteristics and echocardiographic parameters were compared between the two groups. Results The detection rates of high-frequency ultrasound and routine echocardiography were all 100%. The spontaneous closure rate of PDA in preterm infants at 24 hours, 48 hours, 72 hours were 18.8%(32/170), 61.2%(104/170), 78.8%(134/170) respectively. Within 7 days after birth, the closure rate was 90.6%(154/170), therefore the incidence of PDA in premature neonates was about 9.4%(16/170) in our study. The display rate of entire duct arteriosus using high frequency ultrasound was signiifcantly higher than that using routine ultrasound [82%(39/48) vs 46%(22/48), P<0.001, 100%(48/48) vs 77.1%(37/48), P<0.01]. The diameters of duct arteriosus in PDA group were signiifcantly larger than that in control group [(2.08±0.4) mm vs (1.09±0.22) mm, P<0.001]. However, the remaining echocardiographic parameters showed no signiifcant difference between the two groups (P>0.05). To predict the spontaneous closure of PDA in preterm neonates within 7 days after birth by high frequency ultrasound, the sensitivity, specificity and accuracy were 87.5%, 90.6% and 66.5% respectively using the cutoff point of 1.55 mm. While the sensitivity, speciifcity and accuracy were 75%, 93.7%, 44.15%by routine echocardiography with the cutoff point of 2.36 mm. Conclusions High-frequency ultrasound can conifrm the diagnosis of PDA in preterm infants and it is better than routine echocardiography in depicting the structure of arterial duct. The larger PDA diameter was suggestive of the lower possibility of spontaneous close.

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 .

OBJECTIVE: To observe the effect of curing-injury cataplasma on the expression of aquaporin protein 3 (AQP-3) in skeletal muscle of rat model with acute injury in soft tissues. METHODS: A total of 54 SD rats were randomly divided into 3 groups, and by using 10% sodium sulfide the depilating treatment was made in the thigh lateral of each left hind leg 1 day before modeling. The depilatory area in the control group was merely marked with striking range, not attacked for modeling. In the depilatory area of the modeling group, the blowing apparatus was used to attack the marked range to establish the model of soft tissue swelling with acute injury, to which none medication was given. In the drug treatment group, immediately after establishing the model of soft tissue swelling with acute injury, curing-injury cataplasma was scattered on the stricken area, and fixed with bandage. After the modeling, the rats were killed at 1 h, 6 h, 1 d, 3 d, 5 d, and 7 d, 3 rats in each group at each time point. In the marked area some tissue was taken, and the dry/wet proportion method was used to detect the water content in the skeletal muscle. Western blot and qPCR method were used for the AQP-3 protein and the level of gene expression. RESULTS: At the six time points, for the modeling and drug treatment groups, the water content of skeletal muscle was higher than that of the control group (P<0.05). At 3 d, 5 d and 7 d, the water content in the drug treatment group was lower than that of the modeling group (P<0.01); for the modeling and drug treatment groups, AQP-3 protein and the level of gene expression were higher than those of the control group. There was significant difference between the drug treatment group and the modeling group (P<0.01). CONCLUSION Curing-injury cataplasma can relieve soft tissue swelling with acute injury, and accelerate the repair process after the injury.
Animals ; Aquaporin 3 ; metabolism ; Drugs, Chinese Herbal ; therapeutic use ; Hindlimb ; injuries ; Male ; Muscle, Skeletal ; metabolism ; Rats ; Rats, Sprague-Dawley ; Soft Tissue Injuries ; drug therapy ; metabolism

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

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.