Chaihu Longgu Mulitang Relieves Generalized Anxiety Disorder in Rats via p38 MAPK/NF-κB Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo study whether Chaihu Longgu Mulitang can inhibit hypothalamic inflammation， mitigate anxiety-like behavior， and alleviate anxiety symptoms by regulating the p38 mitogen-activated protein kinase/nuclear factor-κB （p38 MAPK/NF-κB） signaling pathway in the rat model of generalized anxiety disorder （GAD）. MethodTwelve out of 74 Wistar rats were randomly selected as the blank group， and the remaining rats were subjected to chronic restraint stress for the modeling of GAD. The open field test （OFT） and elevated Porteus maze test （PMT） were conducted 14 days after modeling to detect the anxiety-like behaviors. Sixty successfully modeled rats were selected and randomized into model， low-， medium-， and high-dose （6， 12， and 24 g·kg-1， respectively） Chaihu Longgu Mulitang， and diazepam （1 mg·kg-1） groups （n=12） and administrated with corresponding drugs for 14 consecutive days. OFT and PMT were then carried out to examine the anxiety-like behaviors of the rats. The levels of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and interleukin-1β （IL-1β） in the hypothalamus and serum of rats were determined by the enzyme-linked immunosorbent assay （ELISA）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）was conducted to determine the mRNA levels of p38 MAPK， NF-κB p65， nuclear factor κB inhibitor α （IκBα）， and ionized calcium binding adaptor molecule 1 （Iba-1）. The protein levels of p38 MAPK， phosphorylated （p）-p38 MAPK， NF-κB p65， p-NF-κB p65， and IκBα in the hypothalamus of rats were determined by Western blot. The expression of Iba-1 in the hypothalamic microglia was detected by immunofluorescence assay. ResultCompared with the blank group， the model group had decreased body weight， scattered dark yellow fur， increased irritability， and preference to hibernation in the corner. In addition， the modeled rats showed increased edge movement distance and time in OFT （P<0.01） and decreased movement distance and time and the number of entries in the open arm in PMT （P<0.01）. The modeling increased the fluorescence intensity of Iba-1 in paraventricular nucleus of hypothalamus （P<0.01）， elevated the levels of IL-1β， IL-6， and TNF-α in the serum and hypothalamus （P<0.01）， up-regulated the protein and mRNA levels of p38 MAPK， NF-κB p65， p-p38 MAPK， p-NF-κB p65， and Iba-1 （P<0.05， P<0.01）， and down-regulated the protein and mRNA levels of IκBα （P<0.01） in the hypothalamus. Compared with the model group， medium- and high-dose Chaihu Longgu Mulitang and diazepam increased the body weight， improved the fur and behaviors， decreased the edge movement distance and time in OFT （P<0.05， P<0.01）， and increased the movement distance and time in the open arm in PMT （P<0.05， P<0.01）. Furthermore， they decreased the fluorescence intensity of Iba-1 in hypothalamic microglia （P<0.05， P<0.01）， lowered the levels of IL-1β， IL-6， and TNF-α in the serum and hypothalamic tissue （P<0.05， P<0.01）， down-regulated the mRNA and protein levels of p38 MAPK， NF-κB p65， p-p38 MAPK， p-NF-κB p65， and Iba-1 （P<0.05， P<0.01）， and up-regulated the mRNA and protein levels of IκBα （P<0.05， P<0.01） in the hypothalamus. ConclusionChaihu Longgu Mulitang can mitigate anxiety-like behaviors and relieve anxiety in GAD rats by inhibiting the p38 MAPK/NF-κB signaling pathway and reducing the activation of microglia and the levels of pro-inflammatory cytokines in the hypothalamus.

Effect of different parts of Pinelliae Rhizoma Decoction against airway inflammation and analysis of effective components / 中国中药杂志

This study investigated the material basis and mechanism of Pinelliae Rhizoma Decoction in the treatment of airway inflammation. The cigarette smoke combined with lipopolysaccharide(LPS) was used to induce an airway inflammation model in mice. The expression levels of IL-6 and IL-8 in the bronchoalveolar lavage fluid(BALF) and the phosphorylation levels of p38 and IκB in the lungs of mice were taken as indexes to screen the effective extracts by system solvent extraction from Pinelliae Rhizoma Decoction(dichloromethane extract, ethyl acetate extract, n-butanol extract, etc.). Meanwhile, the human bronchial epithelial(16-HBE) cell model of cigarette smoke extract(CSE)-induced injury was established, and the mRNA expression levels of IL-6 and IL-8 and the phosphorylation levels of p38 and IκB proteins were also taken as indexes to evaluate the anti-inflammatory effect of different extracts of Pinelliae Rhizoma Decoction. The results showed that Pinelliae Rhizoma Decoction significantly antagonized airway inflammation in mice by down-regulating the expression levels of IL-6 and IL-8 in mice with airway inflammation and 16-HBE cells with CSE-induced injury and inhibiting the phosphorylation levels of p38 and IκB. The dichloromethane and ethyl acetate extracts of Pinelliae Rhizoma Decoction showed significant anti-inflammatory effects, while such effects of other extracts were not prominent. Furthermore, the database of Pinelliae Rhizoma composition was constructed, and the components in effective extracts were analyzed by HPLC-TOF-MS and Nano-LC-MS/MS. As revealed by the results, the compositions of the two effective extracts were similar with 36 common components. They were combined and then divided into Pinelliae Rhizoma alkaloids(PTAs) and Pinelliae Rhizoma non-alkaloids(PTNAs) by 732 cation-exchange resin. Further in vitro investigation confirmed the significant anti-inflammatory effect of PTNAs, while such effect of PTAs was not manifest. The MS analysis showed 172 peptides and 7 organic acids in PTNAs. The peptide content in PTNAs was 63.5% measured by quantitative analysis of BCA assay, and the organic acid content was 9.92% by potentiometric titration method. The findings of this study suggested that Pinelliae Rhizoma Decoction could antagonize airway inflammation in mice by inhibiting phosphorylation of p38 and IκB and blocking the activation of MAPK and NF-κB signaling pathways, and the effective components were related to the peptides and organic acids in PTNAs. The above results lay a foundation for the research on the mechanism and material basis of Pinelliae Rhizoma in antagonizing airway inflammation.

Effects of p38MAPK inhibitor on fetal lung injury in a rat model of acute pancreatitis in late pregnancy / 中华急诊医学杂志

Objective To investigate the effect and underlying mechanisms of p38 mitogenactivated protein kinase inhibitor SB203580 on fetal lung injury in a rat model of acute pancreatitis in late pregnancy.Methods Twenty-four pregnant Sprague-Dawley rats in last gestation were randomly(random number) divided into the SO group,APILP group,and SB203580 treatment (SB) group.APILP model was induced by retrograde injection of 5％ sodium taurocholate into the biliary-pancreatic duct.SB203580 administration (10 mg/kg body weight,intraperitoneal injection) was performed 0.5 h before surgery.All the rats in the SO and APILP groups received intraperitoneal injection of equivoluminal solvent at the same time point.Animals were sacrificed at 12 h after the induction of APILP,then the blood and tissue samples were harvested.Serum levels of AMY and TNF-α were analyzed.Histopathological changes of maternal pancreas and fetal lung were observed and evaluated.The expression and location of NF-κB in fetal lungs were detected by immunohistochemistry and MPO expression in fetal lungs was examined by immunofluorescence.The expression ofp-p38MAPK,p38MAPK,TNF-α and ICAM-1 was determined by Western blot.One-way ANOVA and Tukey's multiple comparison tests were used for statistical analysis.Results The levels of AMY and TNF-α in maternal serum were markedly increased after APILP [(7871.3±623.5) vs (1 915.3±452.3),(193.8±25.4) vs (107.0±±13.3),(P＜0.05)].Obvious pathological changes presented in matemal pancreas and fetal lung after the attack of APILP,and their pathological scores were significantly higher than those of the SO group [(12.44±1.08) vs (1.56±0.56),(2.50±0.53) vs (0.88±0.64),(P＜0.05)].The number of NF-κB and MPO positive cells in fetal lungs were significantly higher than those in the SO group [(150.63±34.58) vs(29.50±8.80),(53.38±8.30) vs (11.75±3.33);P＜0.05)].In addition,the expression and nuclear translocation were pervasive in fetal lungs in the APILP group.Furthermore,the levels of p-p38MAPK [(0.6367±0.0386) vs (0.2282±0.0220)],TNF-α [(0.6313±0.0395) vs (0.0725±0.0076)],ICAM-1 [(0.8958±0.0776) vs (0.1372±0.0388)] and HMGB1 [(0.6478±0.0209) vs (0.2825±0.0533)] expression in fetal lungs were significantly increased after the establishment of APILP model (P＜0.05).However,with the pre-administration of SB203580,the pathological scores of matemal pancreases (9.38±1.58) and fetal lungs (1.63±0.52) were decreased significantly (P＜0.05),as well as the levels of AMY (4162.1±642.1) and TNF-α (139.6±21.1) in maternal serum (P＜0.05).The number of NF-κB (93.00±18.88) and MPO (27.38±4.75) positive cells in fetal lungs were dramatically reduced (P＜0.05) and fewer nuclear translocation was observed in the SB group.Interestingly,the expression levels of p-p38MAPK (0.2578±0.0170),TNF-α (0.3240±0.0326),ICAM-1 (0.4177±0.0823) and HMGB1 (0.4923±0.0457) in fetal lungs were markedly decreased with the treatment of SB203580 (P＜0.05).Conclusions P38MAPK and its downstream inflammatory signaling pathway were involved in the process of APILP-related fetal lung injury;SB203580 administration could significantly attenuate fetal lung injury induced by APILP,which may be closely related to the inhibition of p38MAPK phosphorylation and inflammatory cascade caused by the activation of downstream signal pathways.

The effects of IGF-1 on the proliferation and apoptosis of cultured condylar chondrocytes / 实用口腔医学杂志

Objective:To investigate the effects of IGF-1 and IL-1β on the proliferation and apoptosis of cultured human condylar chondrocytes( CCs) of temporomandibular joint( TMJ) . Methods:Cultured CCs were derived from human TMJ condylar cartilage tis-sue, and identified by immunocytochemistry staining. The cultured cells were divided into 6 groups:control group, IL-1β(10 μg/L) group and IL-1 group (10 μg/L) + IGF-1 group (0, 1, 10, 50 and 100 μg/L, respectively). The cell proliferation ability was de-tected by MTT assay. The cell cycle and apoptosis were detected by flow cytometry. The expression of apoptosis-associated factors Bcl-2, Bax and p38 MAPK/NF-κB proteins were detected by Western blot. Results:Type II collagen was positively expressed in cultured CCs. IL-1β treatment decreased cell proliferation, increased cell apoptosis with concomitant increase of the percentage of early apopto-sis and late apoptotic cells, increased Caspase-3 expression, decreased Bcl-2/Bax ratio, and increased the expression of p38 MAPK/NF-κB proteins. Whereas, with 1-100 μg/L IGF-1 pretreatment, the proliferation ability and Bcl-2/Bax ratio of the cells were in-creased(P<0. 05), the apoptotic cells were decreased, the expression of Caspase-3 and p38 MAPK/NF-κB proteins was decreased ( P<0. 05) in a dose-dependent manner. Conclusion:IGF-1 may inhibit IL-1β-induced cell apoptosis and attenuate the activation of p38 MAPK/NF-κB of human condylar chondrocytes.

Berberine regulates MMPs/TIMP via inhibition of P38 MAPK/NF-κB phosphorylation and anti-inflammation in rat experimental periodontitis / 西安交通大学学报(医学版)

ABSTRACT:Objective To investigate the effects of berberine on matrix metalloproteinases/tissue inhibitors of matrix metalloproteinases (MMPs/TIMPs) in rat periodontitis .Methods Adult male Sprague‐Dawley rats were subjected to thread ligation and porphyromonas gingivalis ( P . gingivalis ) inoculation resulting in periodontitis . Berberine [150 mg/(kg · d)] or vehicle was administered by oral gavage for 4 weeks .Alveolar bone loss and soft‐tissue destruction were determined by micro‐computed tomography (μ‐CT) and HE staining .The contents of tumor necrosis factor‐α(TNF‐α) and interleukin‐1β(IL‐1β) of gingival tissue were examined by ELISA .The expressions of MMP‐2 ,MMP‐9 and TIMP‐2 as well as the phosphorylation of P38 MAPK and NF‐κB were determined by Western blot .Results Significant increases were observed in alveolar bone loss and periodontal soft‐tissue destruction with higher levels of TNF‐α, IL‐1β and MMP‐2/9 expressions and the activities of P38 MAPK and NF‐κB in the experiment periodontitis group .Berberine of [150 mg/(kg · d)] not only improved the periodontal tissue and decreased alveolar bone loss ,but also reduced the contents of TNF‐α,IL‐1βand MMP‐2/9 and increased TIMP‐2 .In addition ,berberine inhibited the phosphorylation of P38 MAPK/NF‐κB .Conclusion Berberine protects against periodontal tissue damage via decreasing MMP‐2 and MMP‐9 expressions but increasing TIMP‐2 expression , which may be induced by inhibition of P38 MAPK/NF‐κB and the anti‐inflammatory effect on rat periodontitis .