PURPOSE: Rheumatoid arthritis (RA) is an inflammatory joint disorder, the progression of which leads to the destruction of cartilage and bone. Chemokines are involved in RA pathogenesis. In this study, we investigated the chemokine signaling pathway associated with CCL2 in peripheral blood (PB) and synovial tissues (ST) of RA patients based on our previous work about chemokine signaling pathway involved in the activation of CCL2 production in collagen-induced arthritis rat ST. MATERIALS AND METHODS: Total RNA was isolated from PB leukocytes and synovium of the knee joint in both RA patients and control populations. Real-time polymerase chain reaction was used to determine CCL4, CCR5, c-Jun, c-Fos, and CCL2 expressions. Serum level of CCL2 was assessed by enzyme-linked immunosorbent assay, and the production of CCL2 in ST was analyzed immunohistochemically. RESULTS: The expressions of CCL4, CCR5, c-Jun, c-Fos, and CCL2 messenger RNA in RA patients were significantly higher than those in healthy controls, both in ST and on PB leukocyte. Serum CCL2 levels were elevated in RA patients. Histological examination of rheumatoid joints revealed extensive CCL2 expression in RA ST. CONCLUSION: CCL2, CCL4, c-Jun, c-Fos, and CCR5 may play an important role in the recruitment of PB leukocytes into the RA joints. These data provide evidence that the chemokine signaling pathway is involved in CCL2 expression in RA patient tissues, which may contribute to chronic inflammation associated with RA. Targeting this signaling pathway may provide a novel therapeutic avenue in RA.
Adult ; Animals ; Arthritis, Rheumatoid/*blood/metabolism ; Case-Control Studies ; Chemokine CCL2/*blood/metabolism ; Chemokines/metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Gene Expression ; Humans ; Male ; Middle Aged ; RNA, Messenger/genetics/metabolism ; Rats ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Synovial Membrane/*metabolism

Objective:To evaluate the relationship between intestinal microbiota heterogeneity and acquired myasthenia in septic mice.Methods:Eighty SPF healthy male C57BL/6 mice, weighing 18-20 g, aged 6-8 weeks, were included. Forty mice were selected to prepare a sepsis model by intraperitoneal injection of lipopolysaccharide (LPS) 10 mg/kg. Sepsis-sensitive mice (state of dying or even death within 24 h after developing the model) and sepsis-resistant mice (survival for 7 days and recovery) were screened. The feces from donor mice were collected to make fecal bacteria fluid. Forty mice were selected and divided into 4 groups ( n=10 each) by the random number table method: control group (C group), antibiotic group (ABX group), resistant group (Res group), and sensitive group (Sen group). Group C received no treatment. In ABX group, compound antibiotics were given by intragastric gavage once a day for 5 consecutive days. In Res group, the fecal solution from sepsis-resistant mice 150 μl was given by gavage once a day for 3 consecutive days starting from 5 days after gavage administration of compound antibiotics, and then LPS 15 mg/kg was intraperitoneally injected. In Sen group, the fecal solution from sepsis-sensitive mice was given by gavage for 3 days (using the same method as previously described in Res group) starting from 5 days after gavage administration of compound antibiotics, and then LPS 15 mg/kg was intraperitoneally injected. The severity of sepsis was assessed and scored at 24 h after developing the model. The four limb grip strength was measured after fecal bacteria transplantation and at 24 h after developing the sepsis model. The Compound Muscle Action Potential (CMAP) of the gastrocnemius was measured at 24 h after developing the sepsis model. Then blood samples were collected for determination of the levels of interleukin-1 (IL-1), IL-6 and tumor necrosis factor α (TNF-α) in serum by enzyme-linked immunosorbent assay. The tissues of anterior tibial muscle and gastrocnemius muscle were obtained for determination of the expression of muscle-specific ring finger protein 1 (MuRF-1) and muscle atrophy box F protein (MAFbx) by Western blot. The diameter and cross-sectional area of gastrocnemius muscle fibers were measured after HE staining. The feces of mice were collected at 3 days after fecal bacteria transplantation, and DNA was extracted from mouse fecal samples for 16S rDNA sequencing and untargeted metabolomics analysis. Results:Compared with C group, the score for severity of sepsis was significantly increased, the four limb grip strength was decreased after developing the sepsis model, the serum IL-6 concentration was increased, and the diameter and cross-sectional area of gastnemius muscle fibers were decreased ( P<0.05), and no statistically significant changes were found in the other parameters in Res group, and no statistically significant changes were found in the indexes mentioned above in ABX group ( P>0.05). Compared with C group and Res group, the score for severity of sepsis was significantly increased, the four limb grip strength was decreased, the latency of CMAP was prolonged, and the amplitude of CMAP was decreased, the serum concentrations of IL-1, IL-6 and TNF-α were increased, the diameter and cross-sectional area of gastrocnemius muscle fibers were decreased, and the expression of MuRF-1 and MAFbx in anterior tibial muscle was up-regulated after developing the sepsis model in Sen group ( P<0.05). 16S rDNA sequencing of intestinal flora: Compared with Sen group, no significant change was found in Chao1 index, Good-coverage index and Simpson index in Res group ( P>0.05), Shannon index was increased ( P<0.05), and no significant change was found in β diversity in Res group ( P>0.05). LDA analysis showed that f_Akkermarisiaceae, o_Verrucomicrobiales, g_Akmansia, c_Verrucomicrobiae and p_Verrucomicrobiota were significantly enriched in Sen group, and g_Enterococcus and f_Enterococcaceae were significantly enriched in Res group ( P<0.05). Nontargeted metabolic analysis: The metabolite profiles in Res group and Sen group suggested that the model was well developed (R2Y>Q2Y). Compared with Sen group, 90 metabolites was significantly up-regulated and 88 down-regulated, significantly up-regulated metabolic substances including vitamin K1, gamma-tocopherol, and taurine in Res group ( P<0.05). The differential metabolite KEGG enrichment pathway included 2-oxocarboxylic acid metabolism and ubiquinone and other terpenoid-quinone biosynthesis ( P<0.05). Conclusions:Intestinal flora heterogeneity may be involved in the pathogenesis of acquired myasthenia in septic mice.

ObjectiveTo explore the effective components， targets， and mechanism of Houttuynia cordata against lung cancer by means of systems pharmacology and further to provide a reference for the further development and clinical application of this medicinal. MethodChemical components of H. cordata were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） and the active components were screened based on oral bioavailability （OB） and drug-likeness （DL）. Then the potential targets were predicted， followed by enrichment analysis. Finally， sodium houttuyfonate （SH） was selected for verifying the anti-tumor mechanism. Cell counting kit-8 （CCK-8） assay was used to evaluate the effect of SH on the in vitro proliferation of two lung cancer cell lines： A549 and LLC， and the regulation of tumor-related proteins by SH was verified by Western blot. ResultA total of 7 active compounds and 352 targets of the active components were screened out. According to the enrichment analysis of targets， H. cordata had potential therapeutic effects on cancer. SH had inhibitory effect on both A549 and LLC. Western blot results showed that G₁/S-specific Cyclin D₁， E₁ and cyclin-dependent kinase （CDK）2， CDK4 all tended to be down-regulated， and Janus kinase 2 （JAK2）/signal transducer and activator of transcription 3 （STAT3） also changed significantly. ConclusionH. cordata has the potential anti-tumor effects by arresting the tumor cells in the G₁ phase through the JAK2/STAT3 pathway.

Objective:To investigate the effect of proprotein convertase subtilisin/kexin type 9 (PCSK9) on platelet activation in sepsis.Methods:① Clinical trial: a prospective study was conducted. Patients with sepsis and septic shock aged ≥ 18 years old who met the diagnostic criteria of Sepsis-3 admitted to the department of intensive care medicine of the Affiliated Hospital of Binzhou Medical College from January to October in 2021 were selected as subjects. Healthy subjects in the same period were taken as healthy control group. Platelet count (PLT) in the first routine blood test after admission was recorded. Venous blood was taken 1 day after diagnosis, and serum PCSK9 level was determined by enzyme-linked immunosorbent assay (ELISA). The differences of PCSK9 level and PLT between the two groups were compared, and subgroup analysis was conducted based on PLT for patients with sepsis. The correlation between PCSK9 level and PLT in septic patients was analyzed by Pearson correlation method. ② Animal experiment: 80 male C57BL/6 mice were randomly divided into control group, sepsis model group [lipopolysaccharide (LPS) group], PCSK9 inhibitor pretreatment group (PCSK9 inhibitor+LPS group) and PCSK9 inhibitor control group (PCSK9 inhibitor group), with 20 mice in each group. The mouse model of sepsis was reproduced by intraperitoneal injection of LPS 12 mg/kg, and the control group and PCSK9 inhibitor group were intraperitoneally injected with the same amount of sterile normal saline. PCSK9 inhibitor+LPS group and PCSK9 inhibitor group were pretreated with PCSK9 inhibitor 5 mg/kg intraperitoneal injection for 7 days before injection of LPS or normal saline, respectively, and the control group and LPS group were injected with an equal amount of sterile normal saline. The lung tissues were taken for pathological and immunohistochemical observation 24 hours after modeling. Blood was taken from the heart for determining PLT. Platelet activation was detected by flow cytometry. The expression level of platelet-activation marker CD40L was detected by Western blotting.Results:① Clinical trial: there were 57 cases in the sepsis group and 27 cases in the healthy control group. Serum PCSK9 level in the sepsis group was significantly higher than that in the healthy control group (μg/L: 232.25±72.21 vs. 191.72±54.92, P < 0.05), and PLT was significantly lower than that in the healthy control group [×10 9/L: 146.00 (75.50, 204.50) vs. 224.00 (194.00, 247.00), P < 0.01]. Subgroup analysis showed that the serum PCSK9 level in the thrombocytopenia patients ( n = 20) was significantly higher than that in the non-thrombocytopenia patients ( n = 37; μg/L: 264.04±60.40 vs. 215.06±72.95, P < 0.01). Correlation analysis showed a significant negative correlation between serum PCSK9 levels and PLT in septic patients ( r = -0.340, P = 0.010). ② Animal experiment: there were no significant pathological changes in lung tissue in the control group and PCSK9 inhibitor group under light microscope, and no significant differences in PLT, platelet activation and plasma CD40L protein expression was found between the two groups. In the LPS group, a large number of inflammatory cells were infiltrated in the pulmonary interstitium, the alveolar structure was damaged obviously, the alveolar septum was widened, the alveolar cavity was extensively bleeding, the capillary dilatation with bleeding and platelet aggregation were found, the PLT was significantly decreased, the platelet activation and the expression level of CD40L protein in plasma were significantly increased. The infiltration of inflammatory cells in lung tissue of mice in the PCSK9 inhibitor+LPS group was reduced to a certain extent, the thickening of alveolar septa was reduced, the platelet aggregation in lung tissue was decreased as compared with the LPS group, the PLT was significantly increased (×10 9/L: 515.83±46.60 vs. 324.83±46.31, P < 0.05), the platelet activation and the expression level of CD40L protein in plasma were significantly decreased [positive expression rate of platelet activation dependent granule surface facial mask protein CD62P: (12.15±1.39)% vs. (18.33±2.74)%, CD40L protein (CD40L/β-actin): 0.77±0.08 vs. 1.18±0.10, both P < 0.05]. Conclusion:PCSK9 level has a certain effect on promoting platelet activation in sepsis, and inhibition of PCSK9 level may have potential research value in improving adverse outcomes caused by sepsis thrombocytopenia.