Objective To investigate the key targets and pathways of Juhongtai formula granules in improving acute lung injury(ALI).Methods Juhongtai formula granules and their drug-containing serum components were analyzed by UPLC-QTOF-MS/MS,and the active ingredients were screened based on the"Lipinski Rule of Five".The action targets of the above active ingredients were obtained through the TCMSP,Swiss Target Prediction and SuperPred databases,and the ALI-related targets were obtained from the GeneCards,OMIM,PharmGKB,CTD databases and GEO chip.The target protein-protein interaction network was constructed using the String database and Cytoscape to analyze the key targets.The DAVID database was used for GO functional annotation and KEGG pathway enrichment analysis,and the CB-dock2 platform was used for molecular docking verification.After one week of adaptive feeding,the experimental SD rats were randomly divided into the blank group,the model group,the positive control group,the negative control group and the Juhongtai formula granules group,with 6 rats in each group.The rat model of ALI was replicated by tracheal infusion of lipopolysaccharide.The pathological morphology of lung tissues in each group of rats was observed by HE staining,and the mRNA expressions of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-1 β(IL-1β)and nitric oxide were detected by reverse transcription quantitative polymerase chain reaction(RT-qPCR).Results In total,44 components of the Juhongtai formula granules were identified,including 26 drug-containing serum components,and 21 key compounds were screened.A total of 22 intersection targets were obtained.The pathway enrichment results indicated the action of these targets on the advanced glycation end product receptor(AGE-RAGE)and TNF signaling pathways.The docking results showed that limonin,kaempferol and naringenin could be matched with prostaglandin peroxidase 2,nitric oxide synthase 2 and TNF.Animal experiments confirmed that the Juhongtai formula granules can alleviate inflammatory infiltration in lung tissue and reduce the expressions of TNF-α,IL6,IL-1β mRNA and nitric oxide.Conclusion Juhongtai formula granules can inhibit ALI-related inflammatory targets through the synergistic effects of multiple components such as limonin and kaempferol,regulate signaling pathways such as AGE-RAGE and TNF,reduce the production of inflammatory factors and nitric oxide,and improve ALI.

Objective To investigate the key targets and pathways of Juhongtai formula granules in improving acute lung injury(ALI).Methods Juhongtai formula granules and their drug-containing serum components were analyzed by UPLC-QTOF-MS/MS,and the active ingredients were screened based on the"Lipinski Rule of Five".The action targets of the above active ingredients were obtained through the TCMSP,Swiss Target Prediction and SuperPred databases,and the ALI-related targets were obtained from the GeneCards,OMIM,PharmGKB,CTD databases and GEO chip.The target protein-protein interaction network was constructed using the String database and Cytoscape to analyze the key targets.The DAVID database was used for GO functional annotation and KEGG pathway enrichment analysis,and the CB-dock2 platform was used for molecular docking verification.After one week of adaptive feeding,the experimental SD rats were randomly divided into the blank group,the model group,the positive control group,the negative control group and the Juhongtai formula granules group,with 6 rats in each group.The rat model of ALI was replicated by tracheal infusion of lipopolysaccharide.The pathological morphology of lung tissues in each group of rats was observed by HE staining,and the mRNA expressions of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-1 β(IL-1β)and nitric oxide were detected by reverse transcription quantitative polymerase chain reaction(RT-qPCR).Results In total,44 components of the Juhongtai formula granules were identified,including 26 drug-containing serum components,and 21 key compounds were screened.A total of 22 intersection targets were obtained.The pathway enrichment results indicated the action of these targets on the advanced glycation end product receptor(AGE-RAGE)and TNF signaling pathways.The docking results showed that limonin,kaempferol and naringenin could be matched with prostaglandin peroxidase 2,nitric oxide synthase 2 and TNF.Animal experiments confirmed that the Juhongtai formula granules can alleviate inflammatory infiltration in lung tissue and reduce the expressions of TNF-α,IL6,IL-1β mRNA and nitric oxide.Conclusion Juhongtai formula granules can inhibit ALI-related inflammatory targets through the synergistic effects of multiple components such as limonin and kaempferol,regulate signaling pathways such as AGE-RAGE and TNF,reduce the production of inflammatory factors and nitric oxide,and improve ALI.

Objective:To investigate the effect of mild moxibustion on transient receptor potential vanilloid type 1(TRPV1)channel expression in primary dysmenorrhea(PD)rats and explore its mechanism in alleviating central pain sensitization.Methods:Thirty-two female non-pregnant Wistar rats were randomized into a blank group,a model group,a mild moxibustion group,and a capsazepine group,with 8 rats in each group.Except for the blank group,the other three groups used estradiol benzoate,ice-water bath,and oxytocin to establish the rat PD model of cold-dampness stagnation pattern.The interventions began on day 1 of modeling,once a day,and lasted 10 d.The mild moxibustion group received mild moxibustion at Shenque(CV8)and Guanyuan(CV4),20 min/time;in the capsazepine group,capsazepine was injected at a dose of 2 mg/(kg·bw).The abdominal pain threshold was measured 10-30 min after oxytocin injection on day 11;enzyme-linked immunosorbent assay was used to detect serum prostaglandin F2α(PGF2α)level;the expression of TRPV1,cluster of differentiation 11B(CD11B),and proto-oncogene c-Fos in the spinal dorsal horn and hypothalamus was detected by immunofluorescence and Western blotting.Results:Compared to the blank group,the model group showed a decreased pain threshold(P<0.05)and an increased serum PGF2α level with elevated TRPV1,CD11B,and c-Fos protein expression in the spinal dorsal horn and hypothalamus(P<0.05).Compared to the model group,both the mild moxibustion group and capsazepine group showed significantly increased pain thresholds(P<0.05),along with decreased serum PGF2α levels and reduced protein expression levels of TRPV1,CD11B,and c-Fos in the spinal dorsal horn and hypothalamus(P<0.05).Rat pain threshold in the capsazepine group was higher than that in the mild moxibustion group(P<0.05).Serum PGF2α level,the expression levels of CD11B and c-Fos proteins in the spinal dorsal horn,as well as TRPV1,CD11B,and c-Fos proteins in the hypothalamus of the capsazepine group were lower than those in the mild moxibustion group(P<0.05).Conclusion:Mild moxibustion at Shenque(CV8)and Guanyuan(CV4)may alleviate the central pain sensitization in PD rats by down-regulating TRPV1 channel expression in the spinal dorsal horn and hypothalamus,thus playing an analgesic effect.

Objective:To investigate the effect of mild moxibustion on transient receptor potential vanilloid type 1(TRPV1)channel expression in primary dysmenorrhea(PD)rats and explore its mechanism in alleviating central pain sensitization.Methods:Thirty-two female non-pregnant Wistar rats were randomized into a blank group,a model group,a mild moxibustion group,and a capsazepine group,with 8 rats in each group.Except for the blank group,the other three groups used estradiol benzoate,ice-water bath,and oxytocin to establish the rat PD model of cold-dampness stagnation pattern.The interventions began on day 1 of modeling,once a day,and lasted 10 d.The mild moxibustion group received mild moxibustion at Shenque(CV8)and Guanyuan(CV4),20 min/time;in the capsazepine group,capsazepine was injected at a dose of 2 mg/(kg·bw).The abdominal pain threshold was measured 10-30 min after oxytocin injection on day 11;enzyme-linked immunosorbent assay was used to detect serum prostaglandin F2α(PGF2α)level;the expression of TRPV1,cluster of differentiation 11B(CD11B),and proto-oncogene c-Fos in the spinal dorsal horn and hypothalamus was detected by immunofluorescence and Western blotting.Results:Compared to the blank group,the model group showed a decreased pain threshold(P<0.05)and an increased serum PGF2α level with elevated TRPV1,CD11B,and c-Fos protein expression in the spinal dorsal horn and hypothalamus(P<0.05).Compared to the model group,both the mild moxibustion group and capsazepine group showed significantly increased pain thresholds(P<0.05),along with decreased serum PGF2α levels and reduced protein expression levels of TRPV1,CD11B,and c-Fos in the spinal dorsal horn and hypothalamus(P<0.05).Rat pain threshold in the capsazepine group was higher than that in the mild moxibustion group(P<0.05).Serum PGF2α level,the expression levels of CD11B and c-Fos proteins in the spinal dorsal horn,as well as TRPV1,CD11B,and c-Fos proteins in the hypothalamus of the capsazepine group were lower than those in the mild moxibustion group(P<0.05).Conclusion:Mild moxibustion at Shenque(CV8)and Guanyuan(CV4)may alleviate the central pain sensitization in PD rats by down-regulating TRPV1 channel expression in the spinal dorsal horn and hypothalamus,thus playing an analgesic effect.

Objective To explore the regulatory effect of miR-6838-5p on DDR1 gene expression and proliferation of breast cancer cells.Methods The expression levels of miR-6838-5p in normal breast epithelial cells and breast cancer cells were detected using qRT-PCR,and the potential target genes of miR-6838-5p was predicted using TargetscanV 8.0.Double luciferase reporter gene experiment was performed to verify the binding between miR-6838-5p and DDR1.Breast cancer MCF-7 cells were transfected via liposome,miR-6838-5p mimic,miR-6838-5p inhibitor,DDR1 siRNA,DDR1-overexpresisng vector,or both miR-6838-5p mimic and DDR1-overexpressing vector,and the changes in cell proliferation were examined with CCK-8 and EdU assays;Western blotting was used to detect the expression of DDR1.The mediating role of DDR1 in miR-6838-5p overexpression-induced inhibition of MCF-7 cell proliferation was verified in a nude mouse model bearing MCF-7 cell xenografts.Results The expression of miR-6838-5p was significantly lower in breast cancer cells than in normal breast epithelial cells.In MCF-7 cells,miR-6838-5p overexpression induced significant inhibition of cell proliferation.Dual luciferase reporter gene experiment demonstrated a binding relationship between miR-6838-5p and DDR1(P<0.01).Western blotting showed that miR-6838-5p overexpression significantly lowered DDR1 expression in MCF-7 cells,and DDR1 overexpression promoted proliferation of the cells;co-transfection of the cells with DDR1-overexpressing vector significantly attenuated the inhibitory effect of miR-6838-5p mimic on cell proliferation.In the tumor-bearing nude mice,the xenografts overexpressing miR-6838-5p showed a significantly smaller volum with obviously the expression of DDR1.Conclusion Overexpression of miR-6838-5p inhibits breast cancer cell proliferation by regulating DDR1 expression.

ObjectiveTo observe the effect of Shengxiantang （SXT） on cell senescence mediated by wingless/integrated （Wnt）3a/β-catenin pathway in rats with idiopathic pulmonary fibrosis （IPF） and reveal the possible mechanism in improving lung function of IPF rats. MethodA total of 32 SPF level SD rats were randomly divided into sham group， model group， pirfenidone group， and SXT group. The IPF rat model was established by intratracheal instillation of bleomycin （0.005 g·kg^-1）. The following day after surgery， rats in the SXT group were given the aqueous solution of SXT granules （0.78 g·kg^-1）， and the pirfenidone group was given pirfenidone suspension （0.05 g·kg^-1）. The other groups were given deionized water （10 mL·kg^-1） for 28 consecutive days. Lung tissue was collected after the lung function was measured. The pathological changes of the lung tissue were observed by hematoxylin-eosin （HE） and Masson staining， and then the Szapiel score and Ashcroft score were performed. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect telomere length. Western blot was applied to detect the expressions of epithelial-mesenchymal transformation （EMT） markers ［α-smooth muscle actin （α-SMA） and E-cadherin］， telomere reverse transcriptase （TRET）， aging-related proteins （p53 and p21）， senescence-associated secretory phenotype ［interleukin-6 （IL-6） and matrix metalloproteinase-1 （MMP-1）］， and key proteins of Wnt signaling pathway ［Wnt3a， glycogen synthase kinase-3β （GSK-3β）， β-catenin， Cyclin D₁， and c-Myc］. ResultCompared with those in the Sham group， peak expiratory flow （PEF） and minute ventilation volume （MV） in the model group were significantly decreased （P<0.01）， and the frequency of respiratory （f） was significantly increased （P<0.01）. The Szapiel score， Ashcroft score， and protein expression of α-SMA， p53， p21， IL-6， MMP-1， Wnt3a， GSK3β， β-catenin， Cyclin D₁， and c-Myc were increased （P<0.01）. The expressions of E-cadherin and TERT， as well as telomere length were significantly decreased （P<0.01）. Compared with those in the model group， PEF and MV in the SXT group were significantly increased （P<0.01）， while f was significantly decreased （P<0.01）. The Szapiel score， Ashcroft score， and protein expression of α-SMA， p53， p21， IL-6， MMP-1， Wnt3a， GSK3β， β-catenin， Cyclin D₁， and c-Myc were significantly decreased （P<0.05， P<0.01）. Nevertheless， the expression of E-cadherin and TERT， as well as telomere length were significantly increased （P<0.01）. ConclusionSXT presents a significant protective effect on lung function in IPF rats， and the prescription may act on the Wnt3a/β-catenin signaling pathway to regulate cell senescence induced by TERT to inhibit EMT.

Objective To observe the analgesic effects of mild moxibustion on primary dysmenorrhea(PD)rats with cold and dampness stagnation syndrome and its effect on BDNF/TrkB signaling pathway in hypothalamus;To explore its mechanism for the treatment of PD.Methods A total of 32 Wistar non-pregnant female rats were randomly divided into blank group,model group,Western medicine group and mild moxibustion group,with 8 rats in each group.Except for the blank group,the other groups received estradiol benzoate intraperitoneal injection combined with ice bath treatment + oxytocin intraperitoneal injection to establish PD with cold and dampness stagnation syndrome model.The mild moxibustion group received treatment at"Shenque"and"Guanyuan"from the eighth day of modeling for 10 min,and the Western medicine group was given ibuprofen solution intragastically for 4 days.The latency period of rats twisting was observed and the twisting score was calculated,Western blot and PCR were used to detect the expressions of c-fos,BDNF,TrkB protein and mRNA in hypothalamic tissue.Results Compared with the blank group,the model group showed a shortened latency period and an increased twisting score(P<0.01),the expressions of c-fos,BDNF,TrkB protein and mRNA in hypothalamic tissue increased(P<0.01,P<0.05).Compared with the model group,the mild moxibustion group had a longer latency period and lower twisting score(P<0.01),while the expressions of c-fos,BDNF,TrkB protein and mRNA in hypothalamic tissue increased(P<0.01,P<0.05).Conclusion Mild moxibustion may effectively improve the pain state of PD rats with cold and dampness stagnation syndrome.This mechanism may be related to downregulating c-fos expression,inhibiting BDNF/TrkB signaling pathway activation,thereby inhibiting pain signal transmission,regulating pain occurrence and maintenance.

Objective To investigate the mechanism of astragaloside Ⅰ,the active constituent of milkvetch root,in inhibiting podocyte injury and improving diabetic kidney disease.Methods According to the body weight,60 male db/db mice were randomly divided into the model group,astragaloside Ⅰ low-dose group(10 mg/kg),astragaloside Ⅰ medium-dose group(20 mg/kg),astragaloside Ⅰ high-dose group(40 mg/kg),and valsartan group(10mg/kg),with 12 mice per group.Twelve db/db littermate control db/m mice were used as the control group.The drug was administered by gavage for 8 weeks.Transmission electron microscope was used to observe the ultrastructure of the kidney;immunohistochemistry and Western blotting were used to detect the expression of nephrotic protein(nephrin),a marker of renal podocytes;enzyme-linked immunosorbent assay was used to detect the contents of interleukin-1β(IL-1β)and interleukin-18(IL-18)in the serum of mice;Western blotting was used to detect the protein expressions of NOD-like receptor thermoprotein domain-related protein 3(NLRP3),cysteinyl aspartate specific proteinase 1(Caspase-1),and Gasdermin D(GSDMD)in kidney tissue.Results Compared with the control group,the glomeruli of the model group showed obvious podocyte loss and foot process fusion;the protein expression of nephrin was decreased(P＜0.05);the contents of IL-1 β and IL-18 in serum were increased(P＜0.05);the protein expressions of NLRP3,Cleaved-Caspase-1,and GSDMD-N were increased(P＜0.05).Compared with the model group,the renal pathological damage in the astragaloside Ⅰ administration groups were alleviated;the protein expression of nephrin was increased(P＜0.05);the contents of IL-1β and IL-18 in serum were decreased(P＜0.05);the protein expressions of NLRP3,Cleaved-Caspase-1,and GSDMD-N were decreased(P＜0.05).Conclusion Astragaloside Ⅰ may play a role in intervening diabetic kidney disease by inhibiting pyroptosis and improving podocyte injury.

Objective To explore the regulatory effect of miR-6838-5p on DDR1 gene expression and proliferation of breast cancer cells.Methods The expression levels of miR-6838-5p in normal breast epithelial cells and breast cancer cells were detected using qRT-PCR,and the potential target genes of miR-6838-5p was predicted using TargetscanV 8.0.Double luciferase reporter gene experiment was performed to verify the binding between miR-6838-5p and DDR1.Breast cancer MCF-7 cells were transfected via liposome,miR-6838-5p mimic,miR-6838-5p inhibitor,DDR1 siRNA,DDR1-overexpresisng vector,or both miR-6838-5p mimic and DDR1-overexpressing vector,and the changes in cell proliferation were examined with CCK-8 and EdU assays;Western blotting was used to detect the expression of DDR1.The mediating role of DDR1 in miR-6838-5p overexpression-induced inhibition of MCF-7 cell proliferation was verified in a nude mouse model bearing MCF-7 cell xenografts.Results The expression of miR-6838-5p was significantly lower in breast cancer cells than in normal breast epithelial cells.In MCF-7 cells,miR-6838-5p overexpression induced significant inhibition of cell proliferation.Dual luciferase reporter gene experiment demonstrated a binding relationship between miR-6838-5p and DDR1(P<0.01).Western blotting showed that miR-6838-5p overexpression significantly lowered DDR1 expression in MCF-7 cells,and DDR1 overexpression promoted proliferation of the cells;co-transfection of the cells with DDR1-overexpressing vector significantly attenuated the inhibitory effect of miR-6838-5p mimic on cell proliferation.In the tumor-bearing nude mice,the xenografts overexpressing miR-6838-5p showed a significantly smaller volum with obviously the expression of DDR1.Conclusion Overexpression of miR-6838-5p inhibits breast cancer cell proliferation by regulating DDR1 expression.

Objective To explore the mechanism of the treatment of primary dysmenorrhea(PD)by mild moxibustion on"Shenque"and"Guanyuan"acupoints based on the regulation of cAMP-PKA signaling pathway on TRPV1.Methods Totally 32 female non-pregnant Wistar rats were randomly divided into blank group,model group,mild moxibustion group and capsazepine group,with 8 rats in each group.Except for the blank group,the other groups all used estradiol benzoate intraperitoneal injection combined with ice water bath to establish a PD cold-dampness stagnation syndrome rat model.Intervention began on the first day of modeling,the mild moxibustion group selects"Shenque"and"Guanyuan"for mild moxibustion,20 min per time,the capsazepine group was injected capsazepine 2 mg/kg,once a day for 10 consecutive days.ELISA was used to detect uterine PGF2α and cAMP content,immunofluorescence staining was used to detect TRPV1 expression in uterine tissue,Western blot was used to detect PKA,p-PKA and TRPV1 protein expression.Results Compared with the blank group,the latency period of body twisting in the model group rats decreased,and the body twisting score increased(P<0.01);the contents of PGF2α and cAMP in uterine tissue increased(P<0.01),and the expressions of TRPV1 and p-PKA proteins increased(P<0.01).Compared with the model group,the mild moxibustion group and capsazepine group showed an increase in the latency period of body twisting and a decrease in the body twisting score(P<0.01);the content of PGF2α and cAMP in uterine tissue decreased(P<0.01),and the expressions of TRPV1 and p-PKA proteins decreased(P<0.05,P<0.01).Compared with the mild moxibustion group,the capsazepine group showed an increase in the latency period of body twisting and a decrease in the body twisting score(P<0.01);the contents of PGF2α and cAMP in uterine tissue decreased(P<0.05,P<0.01),and the expressions of TRPV1 protein decreased(P<0.05).Conclusion Mild moxibustion at"Shenque"and"Guanyuan"acupoints has obvious analgesic effect on PD rats,and its mechanism may be related to the regulation of uterine cAMP-PKA signaling pathway mediated TRPV1 protein expression.