Neuropathic pain is one of the common complications of diabetes. Tetrahydropalmatine(THP) is a main active component of Corydalis Rhizoma with excellent anti-inflammatory and pain-alleviating properties. This study aims to investigate the therapeutic effect of THP on diabetic neuropathic pain(DNP) and the underlying mechanism. High-fat and high-sugar diet(4 weeks) and streptozotocin(STZ, 35 mg·kg~(-1), single intraperitoneal injection) were employed to induce type-2 DNP in rats. Moreover, lipopolysaccharide(LPS) was used to induce the activation of BV2 microglia in vitro to establish an inflammatory cellular model. Fasting blood glucose(FBG) was measured by a blood glucose meter. Mechanical withdrawal threshold(MWT) was assessed with von Frey filaments, and thermal withdrawal latency(TWL) with hot plate apparatus. The protein expression levels of OX42, inducible nitric oxide synthase(iNOS), CD206, p38, and p-p38 were determined by Western blot, the fluorescence expression levels of OX42 and p-p38 in the dorsal horn of the rat spinal cord by immunofluorescence, the mRNA content of p38 and OX42 in rat spinal cord tissue by qRT-PCR, and levels of nitric oxide(NO), interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), interleukin-10(IL-10), and serum fasting insulin(FINS) by enzyme-linked immunosorbent assay(ELISA). RESULTS:: showed that the mo-del group demonstrated significant decrease in MWT and TWL, with pain symptoms. THP significantly improved the MWT and TWL of DNP rats, inhibited the activation of microglia and p38 MAPK signaling pathway in rat spinal cord, and ameliorated its inflammatory response. Meanwhile, THP promoted the change of LPS-induced BV2 microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, suppressed the activation of the p38 MAPK signaling pathway, decreased the expression levels of inflammatory factors NO, IL-1β, IL-6, and TNF-α, and increased the expression level of anti-inflammatory factor IL-10. The findings suggested that THP can significantly ameliorate the pain symptoms of DNP rats possibly by inhibiting the inflammatory response caused by M1 polarization of microglia via the p38 MAPK pathway.
Animals ; Berberine Alkaloids ; Blood Glucose/metabolism* ; Diabetes Mellitus ; Diabetic Neuropathies/genetics* ; Interleukin-10 ; Interleukin-6/metabolism* ; Lipopolysaccharides/pharmacology* ; Microglia ; Neuralgia/metabolism* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Spinal Cord/metabolism* ; Streptozocin/therapeutic use* ; Tumor Necrosis Factor-alpha/metabolism* ; p38 Mitogen-Activated Protein Kinases/metabolism*

This paper aims to explore the activity of Codonopsis canescens extract against rheumatoid arthritis(RA) based on the Toll-like receptors(TLRs)/mitogen-activated protein kinases(MAPKs)/nuclear factor kappa B(NF-κB) signaling pathways and its mechanism. The ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry(UPLC-Q-TOF-MS) was used to identify the components of C. canescens extract. Forty-eight male SD rats were randomly divided into six groups, namely the normal group, the model group, the methotrexate(MTX) tablet group, and the low, medium, and high-dose C. canescens extract(ZDS-L, ZDS-M, and ZDS-H) groups, with 8 rats in each group. The model of collagen-induced arthritis in rats was induced by injection of bovine type Ⅱ collagen emulsion. MTX(2.5 mg·kg~(-1)), ZDS-L, ZDS-M, and ZDS-H(0.3 g·kg~(-1), 0.6 g·kg~(-1), and 1.2 g·kg~(-1)) were administrated by gavage. Rats in the normal group and the model group received distilled water. MTX was given once every three days for 28 days, and the rest medicines were given once daily for 28 days. Body weight, degree of foot swelling, arthritis index, immune organ index, synovial histopathological changes, and serum levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6(IL-6) were observed. Protein expressions of TLR2, TLR4, NF-κB p65, p38 MAPK, and p-p38 MAPK in rats were determined by Western blot. Thirty-four main components were identified by UPLC-Q-TOF-MS, including 15 flavonoids, 7 phenylpropanoids, 4 terpenoids, 4 organic acids, 2 esters, and 2 polyalkynes. As compared with the normal group, the body weight of the model group was significantly decreased(P<0.01), and foot swelling(P<0.05, P<0.01), arthritis index(P<0.01), and the immune organ index(P<0.01) were significantly increased. The synovial histopathological injury was obviously observed in the model group. The serum levels of inflammatory factors TNF-α, IL-1β, and IL-6 were significantly increased(P<0.01), and the protein expression levels of TLR2, TLR4, NF-κB p65, p-p38 MAPK/p38 MAPK in the synovial tissue were significantly increased(P<0.01) in the model group. As compared with the model group, the body weights of the ZDS dose groups were increased(P<0.01), and the degree of foot swelling(P<0.01) and the arthritis index were decreased(P<0.05, P<0.01). The immune organ index was decreased(P<0.01) in the ZDS dose groups, and the synovial tissue hyperplasia and inflammatory cell infiltration were alleviated. The serum levels of TNF-α, IL-1β, and IL-6 were significantly decreased(P<0.05, P<0.01), and the protein expression levels of TLR2, TLR4, NF-κB p65, p-p38 MAPK/p38 MAPK were decreased(P<0.05, P<0.01) in the ZDS dose groups. C. canescens extract containing apigenin, tricin, chlorogenic acid, aesculin, ferulic acid, caffeic acid, and oleanolic acid has a good anti-RA effect, and the mechanism may be related to the inhibition of TLRs/MAPKs/NF-κB signaling pathways.
Animals ; Cattle ; Male ; Rats ; Arthritis, Experimental/drug therapy* ; Arthritis, Rheumatoid/drug therapy* ; Body Weight ; Codonopsis/chemistry* ; Interleukin-6/blood* ; NF-kappa B/genetics* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; Plant Extracts/therapeutic use* ; Rats, Sprague-Dawley ; Signal Transduction ; Toll-Like Receptor 2/metabolism* ; Toll-Like Receptor 4/metabolism* ; Tumor Necrosis Factor-alpha/pharmacology*

OBJECTIVE: : To investigate the effect of polysaccharides (LBPs) on TLR/NF-κB independent pathway and serum tumor necrosis factor (TNF-α) level in diabetic MyD88-knockout mice. METHODS: : Diabetes was induced by feeding high-fat/high-sugar diet and injection of low-dose streptozotocin in MyD88-knockout mice. The diabetic mice were randomly divided into model group, positive control group and LBPs group. The expressions of TRAM, TRIF, TRAF6, RIP1 and TNF-α mRNA and proteins in mouse peritoneal macrophages were detected by real-time RT-PCR and Western blotting after LBPs treatment for 3 month. Serum TNF-α was determined with ELISA kit. RESULTS: : Real time RT-PCR showed that compared with model group, the relative expressions of and mRNA in macrophages of LBPs group were significantly decreased and expression of was significantly increased (all <0.05). Expression of TRAM, TRIF, TRAF6, RIP1 and TNF-α proteins as well as serum TNF-α level had no significant difference between LBPs group and model group (all >0.05). CONCLUSIONS : LBPs may not inhibit serum TNF-α level through TLR/NF-κB independent pathway.
Animals ; Diabetes Mellitus, Experimental ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation ; drug effects ; Macrophages, Peritoneal ; drug effects ; Mice ; Mice, Knockout ; Myeloid Differentiation Factor 88 ; genetics ; NF-kappa B ; genetics ; Random Allocation ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; blood ; metabolism

OBJECTIVE: To investigate the role of cathepsin B in hepatic Kupffer cells (KCs) in activating Toll-like receptor 4(TLR- 4)-independent inflammatory pathways in mice with lipopolysaccharide (LPS)-induced sepsis. METHODS: Eighteen wild-type (WT) mice and 18 TLR4-knockout (TLR4) mice were both divided into 3 groups for intraperitoneal injections of a lethal dose (54 mg/kg) of LPS, LPS and CA-074(a cathepsin B inhibitor), or normal saline, and the survival of the mice were observed. Another 36 WT mice and 36 TLR4mice were also divided into 3 groups and subjected to intraperitoneal injections of normal saline, 20 mg/kg LPS, or LPS with CA-074 pretreatment.After the treatments, KCs were collected from the mice for assessing the protein level and activity of cathepsin B.The histopathological changes of the liver were observed with HE staining, and the serum levels of IL-1α, IL-1β, TNF-α and IL-18 were detected. RESULTS: Compared with the WT mice,TLR4mice receiving the lethal dose of LPS had significantly longer survival time (up to 84 h) after the injection,but were still unable to fully resist LPS challenge.CA-074 pretreatment prolonged the survival time of WT mice and TLR4mice to 60 h and 132 h,respectively.In the mouse models of sepsis,20 mg/kg LPS induced significantly enhanced activity of cathepsin B without affecting its expression level in the KCs (<0.05) and increased the serum levels of the inflammatory cytokines.CA-074 pretreatment of the mice obviously lessened the detrimental effects of LPS in TLR4mice by significantly lowering cathepsin B activity in the KCs,alleviating hepatocyte apoptosis and reducing the serum levels of inflammatory cytokines. CONCLUSIONS Cathepsin B plays an important role in activating TLR4-independent inflammatory pathways in mice with LPS-induced sepsis.
Animals ; Cathepsin B ; antagonists & inhibitors ; physiology ; Dipeptides ; pharmacology ; Gene Knockout Techniques ; Hepatocytes ; Inflammation ; metabolism ; Interleukin-18 ; blood ; Interleukin-1alpha ; blood ; Interleukin-1beta ; blood ; Kupffer Cells ; metabolism ; Lipopolysaccharides ; Liver ; pathology ; Mice ; Sepsis ; etiology ; metabolism ; Toll-Like Receptor 4 ; genetics ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo explore the possible mechanism and protective effect of BMSCs (bone mesenchymal stem cells) carrying superoxide dismutase (SOD) gene on mice with paraquat-induced acute lung injury.

METHODSTo establish the cell line of BMSCs bringing SOD gene, lentiviral vector bringing SOD gene was built and co-cultured with BMSCs. A total of 100 BALB/c mice were randomly divided into five groups, namely Control group, poisoning group (PQ group) , BMSCs therapy group (BMSC group) , BMSCs-Cherry therapy group (BMSC-Cherry group) , BMSCs-SOD therapy group (BMSC-SOD group) . PQ poisoning model was produced by stomach lavaged once with 1 ml of 25 mg/kg PQ solution, and the equal volume of normal saline (NS) was given to Control group mice instead of PQ. The corresponding BMSCs therapy cell lines were delivered to mice through the tail vein of mice 4h after PQ treatment.Five mice of each group were sacrificed 3 d, 7 d, 14 d and 21 days after corresponding BMSCs therapy cell lines administration, and lung tissues of mice were taken to make sections for histological analysis. The serum levels of glutathione (GSH) , malondialdehyde (MDA) , SOD, and the levels of transforming growth factor-β (TGF-β) and tumor necrosis factor-&alpha; (TNF-&alpha;) in lung tissue were determined. The level of SOD was assayed by Westen-blot.

RESULTSCompared with Control group, the early (3 days) levels of SOD protein in lung tissue of PQ group obviously decreased, and the late (21 days) levels of SOD obviously increased, while in therapy groups, that was higher than that in PQ group, and the BMSCs-SOD group showed most obvious (all P<0.05) . Compared with Control group, the levels of plasma GSH and SOD of PQ group and each therapy group wae significantly lower than those in Control group, while in therapy groups, those were higher than those of PQ group, and the BMSCs-SOD group showed most obvious (all P<0.05) .Compared with Control group, the level of plasma MDA, TNF-&alpha; and TGF-β in PQ group and therapy groups were significantly higher, while in therapy groups, that was lower than that in PQ group, and the BMSCs-SOD group showed most obvious (all P<0.05) . Lung biopsy showed that, the degree of lung tissue damage in each therapy group obviously reduced.

CONCLUSIONSOD is the key factor of the removal of reactive oxygen species (ROS) in cells, that can obviously inhibit the oxidative stress damage and the apoptosis induced by PQ, thus significantly increasing alveolar epithelial cell ability to fight outside harmful environment.

Acute Lung Injury ; chemically induced ; therapy ; Animals ; Antioxidants ; metabolism ; Cell Line ; Glutathione ; blood ; Lung ; pathology ; Malondialdehyde ; blood ; Mesenchymal Stem Cell Transplantation ; Mice ; Mice, Inbred BALB C ; Oxidative Stress ; Paraquat ; poisoning ; Superoxide Dismutase ; blood ; genetics ; Transforming Growth Factor beta ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

PURPOSE: High mobility group box 1 (HMGB1) plays a central role in the pathogenesis of sepsis and multiple organ dysfunction syndromes. We investigated the associations of a single nucleotide polymorphism (SNP; rs1045411) in HMGB1 with various clinical parameters, severity, and prognosis in patients with sepsis, severe sepsis, or septic shock. MATERIALS AND METHODS: We enrolled 212 adult patients followed for 28 days. All patients were genotyped for rs1045411, and the serum levels of HMGB1 and several cytokines were measured. RESULTS: The proportions of patients according to genotype were GG (71.2%), GA (26.4%), and AA (2.4%). Among patients with chronic lung disease comorbidity, patients with a variant A allele had higher positive blood culture rates and higher levels of various cytokines [interleukin (IL)-1beta, IL-6, IL-10, IL-17, and tumor necrosis factor-alpha] than those with the GG genotype. In the analysis of those with diabetes as a comorbidity, patients with a variant A allele had higher blood culture and Gram-negative culture rates than those with GG genotypes; these patients also had a higher levels of IL-17. In the analysis of those with sepsis caused by a respiratory tract infection, patients with a variant A allele had higher levels of IL-10 and IL-17 (all p<0.05). This polymorphism had no significant impact on patient survival. CONCLUSION: The variant A allele of rs1045411 appears to be associated with a more severe inflammatory response than the GG genotype under specific conditions.
Adult ; Aged ; Alleles ; Asian Continental Ancestry Group/genetics ; China/epidemiology ; Cytokines/*blood/*genetics ; Female ; Genotype ; HMGB1 Protein/blood/*genetics ; Humans ; Interleukin-10/genetics ; Interleukin-17/genetics ; Interleukin-6/blood ; Male ; Middle Aged ; Polymorphism, Genetic/*genetics ; Polymorphism, Single Nucleotide/*genetics ; Prognosis ; Republic of Korea ; Sepsis/immunology/*metabolism/mortality ; Shock, Septic/immunology/*metabolism/mortality ; Survival ; Tumor Necrosis Factor-alpha/genetics

Alkaloids from Ba lotus seeds (ABLS) are a kind of important functional compounds in lotus seeds. The present study was designed to determine its hypertension prophylactic effects in the L-NNA-induced mouse hypertension model. The mice were treated with ABLS, the serum and tissues levels of NO, MDA, ET-1, VEGF, and CGRP were determined using the experimental kits, the mRNA levels of various genes in the heart muscle and blood vessel tissues were further determined by RT-PCR assay. ABLS could reduce the systolic blood pressure (SBP), mean blood pressure (MBP), and diastolic blood pressure (DBP), compared to that of the model control group. After ABLS treatment, the NO (nitric oxide) contents in serum, heart, liver, kidney and stomach of the mice were higher than that of the control mice, but the MDA (malonaldehyde) contents were lower than that of the control mice. The serum levels of ET-1 (endothelin-1), VEGF (vascular endothelial growth factor) were decreased after ABLS treatment, but CGRP (calcium gene related peptide) level was increased. The ABLS treated mice had higher mRNA expressions of HO-1, nNOS, and eNOS and lower expressions of ADM, RAMP2, IL-1β, TNF-α, and iNOS than the control mice. Higher concentration of ABLS had greater prophylactic effects, which were close to that of the hypertension drug captopril. These results indicated the hypertension prophylactic effects of ABLS could be further explored as novel medicine or functional food in the future.
Alkaloids ; administration & dosage ; Animals ; Blood Pressure ; drug effects ; Disease Models, Animal ; Humans ; Hypertension ; chemically induced ; drug therapy ; metabolism ; physiopathology ; Interleukin-1beta ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred ICR ; Nitroarginine ; Nymphaeaceae ; chemistry ; Seeds ; chemistry ; Tumor Necrosis Factor-alpha ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

A main symptom of equine metabolic syndrome (EMS) in ponies is pathological obesity characterized by abnormal accumulation of fat deposits and inflammation. In this study, we analyzed the expression of two pro-inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), in subcutaneous adipose tissue and the correlation with serum concentrations in peripheral blood of Welsh ponies. Based on clinical examination findings, the animals were divided into two groups: ponies affected with EMS (n = 8) and obese ponies (n = 8). The adipose tissue was examined using immunohistochemical analysis while concentrations IL-6 and TNF-alpha were measured using enzyme-linked immunosorbent assays (ELISAs). Additionally, histological characterization of the adipose tissue was performed. The results obtained showed that IL-6 expression in adipose tissue biopsies derived from animals with EMS was enhanced while TNF-alpha levels of both groups were comparable. Compared to the obese ponies, EMS animals also had significantly elevated levels of serum IL-6 and TNF-alpha. Histological analysis revealed macrophage infiltration and fibrosis in adipose tissue preparations from the EMS group. These data suggest that IL-6 may play a key role in the course of EMS in Welsh ponies. Our findings also demonstrated that analysis of pro-inflammatory cytokines levels in serum may serve as an additional tool for diagnosing EMS.
Adipose Tissue/*metabolism ; Animals ; Female ; Horse Diseases/blood/*metabolism ; Horses ; Interleukin-6/blood/genetics/*metabolism ; Male ; Metabolic Syndrome X/metabolism/*veterinary ; Tumor Necrosis Factor-alpha/blood/genetics/*metabolism

[To explore the effect of Humifuse Euphorbia Herb ( HEH) on alleviating insulin resistance in type 2 diabetic KK-Ay mice. Totally 40 KK-Ay mice fed with high-fat diet were divided into four groups: the metformin group, the model group, the HEH low-dose group and the HEH high-dose group, and orally administrated with metformin hydrochloride (250 mg x kg(-1)), distilled water, humifuse euphorbia herb 1 g x kg(-1) and 2 g x kg(-1). Besides, C57BL/6J mice with ordinary feed were taken as the normal control group and orally administrated with equal distilled water. The oral administration for the five groups lasted for eight weeks. Before and after the experiment, weight, fasting glucose and insulin tolerance were determined. The morphological changes in pancreas were observed through hematoxylin-eosin (HE) staining on pancreatic tissue sections. The serum insulin, TNF-&alpha;, IL-6, adiponectin (ADPN) and leptin (LEP) were detected by ELISA. The results showed that HEH could reduce weight and fasting glucose in KK-Ay mice, alleviate hyperinsulinemia, reduce blood glucose-time AUC, increase 30-min blood glucose decline rate, relieve insulin resistance, significantly ameliorate the pathomorphological changes in pancreas in each group, decrease serum TNF-&alpha;, IL-6 and leptin levels in KK-Ay mice and rise serum ADPN level. This study proved that humifuse euphorbia herb can ameliorate the insulin resistance in KK-Ay mice, and its mechanism may be related to the effect on inflammatory factors and adipocytokines.
Animals ; Blood Glucose ; metabolism ; Diabetes Mellitus, Type 2 ; drug therapy ; genetics ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Euphorbia ; chemistry ; Humans ; Insulin ; metabolism ; Insulin Resistance ; Interleukin-6 ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVETo observe the effects of docosahexaenoic acid (DHA) on the expressions of TNF-&alpha;, IL-6, and leukotriene B4 (LTB4) in serum and expression of NF-κB in pulmonary tissue of rats with severe scald injury.

METHODSOne hundred and sixty SD rats were divided into sham injury (A), sham injury+DHA (B), scald (C), and scald+DHA (D) groups according to the random number table, with 40 rats in each group. Rats in groups A and B were sham injured, while rats in groups C and D were inflicted with 30% TBSA full-thickness scald on the back. Rats in groups B and D were injected with 0.5 mg/mL DHA solution with the dosage of 1 mL/kg via tail vein 5 minutes post injury, while rats in groups A and C with normal saline solution 1 mL/kg. At post injury hour (PIH) 3, 6, 12, 24, and 48, pulmonary tissue and abdominal aorta blood were collected from 8 rats in each group. The serum levels of TNF-&alpha;, IL-6, and LTB4 were determined with ELISA, and the protein expression of NF-κB p65 in pulmonary tissue was determined with Western blotting. Data were processed with analysis of variance of factorial design and LSD-t test.

RESULTS(1) The serum levels of TNF-&alpha; and IL-6 of rats in group A were similar to those of group B at each time point (with tTNF-&alpha; values from 0.223 to 0.947, tIL-6 values from 0.767 to 2.084, P values above 0.05). Compared with those of group A, the serum levels of TNF-&alpha; and IL-6 of rats in groups C and D were significantly higher at each time point (with tTNF-&alpha; values from 11.800 to 40.357, tIL-6 values from 10.334 to 39.321, P values below 0.01). The serum levels of TNF-&alpha; and IL-6 of rats in group D were significantly lower than those of group C at each time point (with tTNF-&alpha; values from -17.643 to -8.331, tIL-6 values from -21.596 to -6.332, P values below 0.01). The serum levels of TNF-&alpha; and IL-6 in groups C and D both showed a trend of increase earlier and decrease later, and they peaked at PIH 12, respectively (360.4 ± 13.2), (306.8 ± 7.2) pg/mL and (265.4 ± 12.3), (230.5 ± 2.2) pg/mL. (2) The serum level of LTB4 in group A was similar to that of group B at each time point (with t values from 0.787 to 1.096, P values above 0.05). The serum level of LTB4 was significantly higher in groups C and D than in group A at each time point (with t values from 7.501 to 38.962, P values below 0.01). The serum level of LTB4 in group D was obviously lower than that of group C at each time point (with t values from -19.244 to -2.532, P values below 0.01). The serum level of LTB4 in groups C and D both showed a trend of increase earlier and decrease later, and it peaked at PIH 12, (4.59 ± 0.29) and (2.85 ± 0.32) ng/mL respectively. (3) The protein expression of NF-κB p65 in pulmonary tissue in group A was similar to that of group B at each time point (with t values from 0.847 to 1.256, P values above 0.05). The protein expression of NF-κB p65 was significantly higher in groups C and D than in group A at each time point (with t values from 15.167 to 98.074, P values below 0.01). The protein expression of NF-κB p65 in group D was obviously lower than that of group C at each time point (with t values from -37.190 to -14.415, P values below 0.01). The protein expression of NF-κB p65 in groups C and D both showed a trend of increase earlier and decrease later, and it peaked at PIH 12, respectively 4.46 ± 0.12 and 2.94 ± 0.21.

CONCLUSIONSParenteral supply of DHA to rats with severe scald injury can reduce the levels of TNF-&alpha;, IL-6, and LTB4 in serum and decrease the expression of NF-κB in pulmonary tissue, thus alleviating the inflammation response.

Animals ; Blotting, Western ; Burns ; Cytokines ; Docosahexaenoic Acids ; Enzyme-Linked Immunosorbent Assay ; Inflammation ; Interleukin-6 ; blood ; Leukotriene B4 ; blood ; Lung ; metabolism ; pathology ; NF-kappa B ; metabolism ; Rats ; Rats, Sprague-Dawley ; Serum ; Soft Tissue Injuries ; Tumor Necrosis Factor-alpha ; blood ; genetics ; Up-Regulation ; physiology