ABSTRACT
This study explored whether Sagittaria sagittifolia polysaccharides(SSP) activates the nuclear factor erythroid-2-related factor2(Nrf2)/heme oxygenase-1(HO-1) signaling pathway to protect against liver damage jointly induced by multiple heavy metals. First, based on the proportion of dietary intake of six heavy metals in rice available in Beijing market, a heavy metal mixture was prepared for inducing mouse liver injury and HepG2 cell injury. Forty male Kunming mice were divided into five groups: control group, model group, glutathione positive control group, and low-and high-dose SSP groups, with eight mice in each group. After 30 days of intragastric administration, the liver injury in mice was observed by HE staining. In the in vitro experiment, MTT assay was conducted to detect the effects of SSP at 0.25, 0.5, 1, and 2 mg·mL~(-1) on HepG2 cell survival at different time points. The content of alanine transaminase(ALT) and aspartate aminotransferase(AST) in the 48-h cell culture fluid was measured using micro-plate cultivation method, followed by the detection of the change in reactive oxygen species(ROS) content by flow cytometry. The mRNA expression levels of Nrf2 and HO-1 in cells were determined by RT-PCR, and their protein expression by Western blot. HE staining results showed that compared with the model group, the SSP administration groups exhibited significantly alleviated inflammatory cell infiltration and fatty infiltration in the liver, with better outcomes observed in the high-dose SSP group. In the in vitro MTT assay, compared with the model group, SSP at four concentrations all significantly increased the cell survival rate, decreased the ALT, AST, and ROS content(P<0.05), and down-regulated Nrf2 and HO-1 mRNA and protein expression(P<0.05). SSP significantly improves inflammatory infiltration in the liver tissue of mice exposed to a variety of heavy metals and corrects the liver fat degeneration, which may be related to its regulation of the Nrf2/HO-1 signaling pathway, reduction of ROS, and alleviation of oxidative damage.
Subject(s)
Animals , Male , Mice , Heme Oxygenase-1/metabolism , Liver , Metals, Heavy/metabolism , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Polysaccharides/pharmacology , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Sagittaria/metabolismABSTRACT
Objective:To investigate the therapeutic effect of polydatin on ulcerative colitis （UC） in mice and its regulation of protein kinase C<italic>θ</italic>（PKC<italic>θ</italic>）/signal transducer and activator of transcription 3（STAT3） signaling on T helper cell 17（Th17） and its mechanism in the treatment of UC. Method:The 32 male C57BL/6 mice were randomly divided into normal group， model group， polydatin group （0.045 g·kg<sup>-1</sup>） and sulfasalazine group （0.5 g·kg<sup>-1</sup>）. The UC model was established by giving 3% dextran sodium sulfate （DSS） solution to free drinking water in mice. Polydatin and sulfasalazine groups were given by gavage 0.5 h before modeling for 7 days. The normal group and model group were given the same amount of normal saline. After the last administration， the colonic tissue was taken and hematoxylin-eosin （HE） was used to observe the pathological changes of colonic tissue. Flow cytometry was used to detect the proportion of Th17 in the lamina propria of colonic mucosa. The expression of interleukin-17A （IL-17A） in serum was detected by enzyme-linked immunosorbent assay （ELISA）. Polydatin was added to CD4<sup>+ </sup>T cells purified from spleen of C57BL/6 mice by magnetic-activated cell sorting （MACS） under the stimulation of cell stimulation cocktail <italic>in vitro </italic>in order to detect its impact on PKC<italic>θ</italic> and STAT3 phosphorylation. Result:Compared with normal group， the body weight was significantly decreased， and disease activity index （DAI） scores of the model group was significantly increased （<italic>P</italic><0.01）， the colonic mucosal epithelium was damaged and inflammatory cells infiltration in the mucosa and submucosa was obvious， the proportion of Th17 in the lamina propria of colonic mucosa was significantly increased （<italic>P</italic><0.01）， and the content of serum IL-17A was significantly increased （<italic>P</italic><0.01）. Compared with the model group， the weight and DAI score of polydatin and sulfasalazine groups were significantly improved （<italic>P</italic><0.01）， the degree of colon tissue damage was significantly improved， the proportion of Th17 in colon mucosa lamina propria was significantly decreased （<italic>P</italic><0.01）， and the content of IL-17A in serum was significantly decreased （<italic>P</italic><0.01）. <italic>In vitro</italic> experiments showed that polydatin could significantly inhibit the phosphorylation of PKC<italic>θ</italic> and STAT3 in Th17 （<italic>P</italic><0.01） as well as IL-17A secretion. Conclusion:Polydatin can improve the ulcerative colitis in mice via inhibiting the phosphorylation of PKC<italic>θ</italic> and STAT3 to preclude IL-17A secreting in Th17.
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Objective:To explore the protective effect of Gegen Qinliantang on the intestinal mucosal epithelial barrier function of ulcerative colitis （UC） mice， and to explore its mechanism of action in the treatment of ulcerative colitis via matrix metallopeptidase-9 （MMP-9）/p38 mitogen-activated protein kinase （p38 MAPK） signaling pathway. Method:The 48 female C57BL/6 mice were randomly divided into normal group， model group， sulfasalazine group （0.3 g·kg<sup>-1</sup>） and Gegen Qinliantang high， medium and low dose groups （2.84，1.42，0.71 g·kg<sup>-1</sup>）. The UC murine model was established by 3% dextran sulfate sodium （DSS）. Gegen Qinliantang and sulfasalazine were intragastrically administered on the 8<sup>th</sup> day after the model was established for 7 days， and the normal group was treated with the same amount of normal saline. Colon tissues were collected after the last administration， and the pathological changes of colon tissues were detected by hematoxylin-eosin （HE） staining. The expression of tight junction （TJ） proteins such as Occludin and zonula occludens-1（ZO-1） in colon tissues was detected by immunohistochemistry （IHC）， and the expression levels of tumor necrosis factor-alpha （TNF-<italic>α</italic>）， interleukin-1<italic>β</italic> （IL-1<italic>β</italic>）， and MMP-9 mRNA in colon tissues were detected by Real-time polymerase chain reaction （Real-time PCR）. The expression of phosphorylated p38 MAPK （p-p38 MAPK）， p38 MAPK and MMP-9 protein in colon tissues was detected by Western blot. Result:Compared with normal group， the body weight of mice decreased （<italic>P</italic><0.01） and disease activity index （DAI） score increased significantly （<italic>P</italic><0.01） in model group， the colon tissues of the model group were damaged more obviously， the expression of occludin and ZO-1 proteins in model group was significantly reduced （<italic>P</italic><0.01）， and the relative expression levels of TNF-<italic>α</italic>， IL-1<italic>β</italic>， and MMP-9 mRNA in model group were significantly increased （<italic>P</italic><0.01）， the expression of p-p38 MAPK and MMP-9 in model group was significantly increased （<italic>P</italic><0.01）. Compared with model group， the body mass and DAI score of the sulfasalazine group and Gegen Qinliantang group were significantly improved （<italic>P</italic><0.05，<italic>P</italic><0.01）， the colonic tissues damage were significantly improved， and the expression of Occludin and ZO-1 protein was significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）， the relative expression levels of TNF-<italic>α</italic>， IL-1<italic>β</italic>， and MMP-9 mRNA were significantly decreased （<italic>P</italic><0.01）， and the expression of p-p38 MAPK and MMP-9 was significantly decreased （<italic>P</italic><0.01）. The changes in the middle dose group were the most obvious among the various dose groups of Gegen Qinliantang. Conclusion:Gegen Qinliantang repairs the intestinal mucosal barrier function by inhibiting the expressions of MMP-9 and inflammatory cytokines such as TNF-<italic>α</italic> and IL-1<italic>β</italic>， blocking the activation of the p38 MAPK signaling pathway， and increasing the expressions of tight junction protein.
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OBJECTIVE@#This study tests whether long-term intake of Allium tuberosum (AT) can alleviate pulmonary inflammation in ovalbumin (OVA)-induced asthmatic mice and evaluates its effect on the intestinal microbiota and innate lymphoid cells (ILCs).@*METHODS@#BALB/c mice were divided into three groups: phosphate buffer saline, OVA and OVA + AT. The asthmatic murine model was established by sensitization and challenge of OVA in the OVA and OVA + AT groups. AT was given to the OVA + AT group by oral gavage from day 0 to day 27. On day 28, mice were sacrificed. Histopathological evaluation of lung tissue was performed using hematoxylin and eosin, and periodic acid-Schiff staining. The levels of IgE in serum, interleukin-5 (IL-5) and IL-13 from bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay. The ILCs from the lung and gut were detected by flow cytometry. 16S ribosomal DNA sequencing was used to analyze the differences in colon microbiota among treatment groups.@*RESULTS@#We found that long-term intake of AT decreased the number of inflammatory cells from BALF, reduced the levels of IL-5 and IL-13 in BALF, and IgE level in serum, and rescued pulmonary histopathology with less mucus secretion in asthmatic mice. 16S ribosomal DNA sequencing results showed that AT strongly affected the colonic bacteria community structure in asthmatic mice, although it had no significant effect on the abundance and diversity of the microbiota. Ruminococcaceae and Desulfovibrionaceae were identified as two biomarkers of the treatment effect of AT. Moreover, AT decreased the numbers of ILCs in both the lung and gut of asthmatic mice.@*CONCLUSION@#The results indicate that AT inhibits pulmonary inflammation, possibly by impeding the activation of ILCs and adjusting the homeostasis of gut microbiota in asthmatic mice.
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ObjectiveThere are many methods for the detection of hydroxyproline (HYP), but few of them are suitable for the detection of lung tissue in mice. We intend to establish an accurate and reliable method for measuring HYP levels based on mouse lung tissues to assess the degree of fibrosis development more effectively.MethodsBased on the alkali hydrolysis method, the effects of the concentration of alkali hydrolysate and hydrolysis time on the determination results of HYP level in mice lung tissue were compared; the effects of the changes of experimental conditions on the determination results of HYP standard were compared; and the results of the determination of HYP level in mice lung tissue under dry and wet conditions were compared on the basis of the above experimental results.ResultsThe optimum concentration of alkali hydrolysate is 2 mol/L and the optimum hydrolysis time is 20 min. The optimum pH value of citric acid buffer is 6.0-6.5. The optimum solvent for chloramine T is methanol, the optimum reaction time for chloramine T solution is 15 min, the optimum reaction time for perchloric acid solution is 5 min, and the optimum reaction time for 4-(dimethylamino) benzoyl toluene is 5 min. The optimum condition of aldehyde solution color development is that it is bathed in water at 85 for 3 minutes. Some related reagents are stored in suitable environment after preparation, and the experimental data will not be affected within 7 days. Dry lung tissue of mice can improve the detection level of HYP. The improved experimental protocol was applied to the bleomycin-induced mouse pulmonary fibrosis model, and the HYP measurement results were significantly higher than that of the original protocol.ConclusionAn accurate and reliable method for the determination of hydroxyproline in lung tissue of mice was established.
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Objective:To investigate the renal protective effect of Tangshenping capsule (Tangshenping) on diabetic nephropathy (DN) KKAy mice and its effect on Wnt/β-catenin signaling pathway. Method:Sixty female Sprague-Dawley KKAy mice aged 10 weeks old were induced with KKAy rat feed for 10 weeks. The DN animal model was successfully determined with blood glucose (>16.7 mmol·L-1) and 24 hour urine protein (>0.4 mg). The model mice were randomly divided into a model group, an irbesartan group, and low, medium and high-dose Tangshenping group, with 10 female C57BL/6J mice as a control group. The treatment groups were given the corresponding drugs by gavage. The normal group and the model group were given an equal volume of deionized water by gavage. The intragastric dose was 0.01 mL·g-1 body weight coefficient once a day. The general conditions of the mice were observed, the body mass was weighed every 4 weeks, and 24 h urine protein was quantified. At the 26th week, the blood was collected from eyeballs, and the mice were put to death. The quality of the kidneys, serum blood urea nitrogen (BUN), serum creatinine (SCr), triglyceride (TG), malondialdehyde (MDA), nitric oxide (NO) and superoxide dismutase (SOD) content were measured. In situ hybridization and immunohistochemistry were used to detect the expressions of Wnt4, glycogen synthase kinase 3β(GSK3β) and β-catenin in kidney tissues. Result:Compared with model group, body mass, kidney mass/body mass, and 24 h urine protein were significantly lower in high-dose Tangshenping group (PPPβ and β-catenin were decreased (PConclusion:Tangshenping may inhibit the activation of Wnt/β-catenin signaling pathway, reverse the transdifferentiation of renal tubular epithelial cells in DN KKAy mice, delay the progression of renal interstitial fibrosis, and then exert renal protection.
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Objective: To explore the effect of Da Chengqitang on the lung index,lung index inhibition rate,lung histological morphology,classification changes of inflammatory cells and mitogen-activated protein kinase (MAPK) signal pathway in mice with allergic asthma.Method: Forty female C57BL/6 mice were randomly divided into normal control group,model group,dexamethasone group (0.005 g·kg-1) and Da Chengqitang group (19 g·kg-1).Murine allergic asthma model was established by sensitization and nebulization of ovalbumin (OVA).In brief,asthmatic mice were first sensitized by OVA and Al (OH)3 mixture ip on day 0 and day 14,and then nebulized by OVA from day 21 to 27.At the same time,each mouse in the dexamethasone and Da Chengqitang groups were intragastrically administered with 0.2 mL corresponding medicine one hour before the nebulization challenge,while the normal control group was given with the same amount of normal saline.On day 28,pulmonary morphology was detected by htoxylin eosin (HE) staining and inflammatory cells from the brachial alveolar lavage fluid were counted by Diff staining.The expression levels of key proteins in MAPK signaling pathway were detected by Western blot.Result: As compared with the normal control group,the lung indexes were significantly increased in model group (PP0.01),with a predominant percentage of eosinophils,moreover,the expression levels of phosphorylated p38 MAPK and extracellular signal-regulated kinase 1/2(ERK1/2) were increased obviously in asthmatic mice.After treatment by Da Chengqitang,lung indexes and pulmonary inflammation were significantly decreased,with an inhibitory rate of 68.4% for lung indexes,and inflammatory pathology of lung tissues was obviously improved and inflammatory cell exudation was alleviated,with the obviously lower levels of phosphorylated p38 MAPK and ERK1/2 protein.Conclusion: Da Chengqitang based on "Pulmonary Intestinal Treatment" can effectively improve lung inflammation in mice with allergic asthma,which may be related to the expression of phosphorylated p38 MAPK and ERK1/2 protein.
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<p><b>OBJECTIVE</b>To investigate the effect of electroacupuncture (EA) treatment on the expression of cyclooxygenase (COX) 2 and microglia in spinal cord by using rat model of neuropathic pain, and to probe into the relationship between COX 2 and microglia.</p><p><b>METHODS</b>The rats were randomly divided into 6 groups, including normal control group, model group, sham group, EA 1 group (distant acupoints + local acupoints), EA 2 group (local acupoints), and EA 3 group (distant acupoints). Thermal withdrawal latencies were evaluated at 1 day preoperatively and 3, 5 and 7 days postoperatively. At 7 days postoperatively, the spinal COX 2 mRNA was detected by reverse-transcription polymerase chain reaction. Double immunofluorescent staining technology was applied to screen and verify the relationship between altered COX 2 and microglia.</p><p><b>RESULTS</b>Compared with the model group, thermal withdrawal latencies increased after EA treatment (P<0.01). The expressions of COX 2 mRNA were up-regulated in spinal cord of rat on day 7 after surgery (P<0.05). Compared with the model group, EA stimulation (EA 1 and EA 2 groups) reversed the up-regulation of COX 2 mRNA expression (P<0.05). EA 1 and EA 2 groups might have better treatment effect compared with the EA 3 group. Fluorescent images displayed COX 2 and microglia expressed at common areas.</p><p><b>CONCLUSIONS</b>EA was effective in analgesic and anti-inflammatory. EA has decreased the expression of spinal COX 2 mRNA in the trend of the therapeutic effect of "distant acupoints + local acupoints", and "local acupoints" intervention may be superior to that of "distant acupoints" intervention. Microglia may be related to the formation of COX 2.</p>