Protective effect of modified Huangqi Chifeng decoction(加味黄芪赤风汤)on immunoglobulin A nephropathy through toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-kappa B signaling pathway

OBJECTIVE:To examine the nephroprotective mechanism of modified Huangqi Chifeng decoction(加味黄芪赤风汤,MHCD)in immunoglobulin A nephropathy(IgAN)rats.METHODS:To establish the IgAN rat model,the bovine serum albumin,lipopolysaccharide,and carbon tetrachloride 4 method was employed.The rats were then randomly assigned to the control,model,telmisartan,and high-,medium-,and low-dose MHCD groups,and were administered the respective treatments via intragastric administration for 8 weeks.The levels of 24-h urinary protein,serum creatinine(CRE),and blood urea nitrogen(BUN)were measured in each group.Pathological alterations were detected.IgA deposition was visualized through the use of immunofluorescence staining.The ultrastructure of the kidney was observed using a transmission electron microscope.The expression levels of interleukin-6(IL-6),monocyte chemoattractant protein-1(MCP-1),and transforming growth factor-β1(TGF-β1)were examined by immunohistochemistry and quantitative polymerase chain reaction.Levels of toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),and nuclear factor-kappa B(NF-κB)P65,were examined by immunohistochemistry,Western blotting,and quantitative polymerase chain reaction.RESULTS:The 24-h urine protein level in each group increased significantly at week 6,and worsen from then on.But this process can be reversed by treatments of telmisartan,and high-,medium-,and low-dose of MHCD,and these treatments did not affect renal function.Telmisartan,and high-,and medium-dose of MHCD reduced IgA deposition.Renal histopathology demonstrated the protective effect of high-,medium-,and low-dose of MHCD against kidney injury.The expression levels of MCP-1,IL-6,and TGF-β1 in kidney tissues were downregulated by low,medium and high doses of MHCD treatment.Additionally,treatment of low,medium and high doses of MHCD decreased the protein and mRNA levels of TLR4,MyD88,and NF-κB.CONCLUSIONS:MHCD exerted nephroprotective effects on IgAN rats,and MHCD regulated the expressions of key targets in TLR4/MyD88/NF-κB signaling pathway,thereby alleviating renal inflammation by inhibiting MCP-1,IL-6 expressions,and ameliorating renal fibrosis by inhibiting TGF-β1 expression.

Taohong Siwu decoction(桃红四物汤)ameliorates atherosclerosis in rats possibly through toll-like receptor 4/myeloid differentiation primary response protein 88/nuclear factor-κB signal pathway

OBJECTIVE:To investigate the effect of Taohong Siwu decoction(桃红四物汤,TSD)on atherosclerosis in rats as well as investigate the underlying mechanism based on molecular docking.METHODS:Sixty healthy male Sprague-Dawley rats were randomly divided into 6 groups with 10 rats in each group:control group,model group,atorvastatin group(AT,2.0 mg/kg),and TSD groups(20,10,5 g/kg)after 7 d of acclimation.The model of atherosclerosis was successfully established except the control group by high fat diet(HFD)and vitamin D2.Biochemical analyzers were used to detect the levels of triglyceride(TG),total cholestero(TC),low density lipoprotein-cholesterol(LDLC)and high density lipid-cholesterol(HDL-C)in blood lipid.The levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)were determined by enzyme-linked immunosorbent assay.Sudan IV staining and Hematoxylin and eosin staining(HE staining)were performed to observe the pathological changes in aortic tissue.Molecular docking technology was used to predict the best matching between the main components of TSD and the target proteins.The expression of target proteins was further detected by quantitative real time polymerase chain reaction(q RTPCR)and Western blot analysis.RESULTS:The results showed that TSD restricted atherosclerosis development and decreased the inflammatory cytokines in plasma.Molecular docking results predicted that the main components of TSD showed a strong binding ability with toll-like receptor(TLR4),myeloid differentiation primary response protein 88(My D88),and nuclear factor kappa-B(NF-κB).The results of q RT-PCR and Western blot analysis showed that the m RNA and protein expressions of TLR4,My D88 and NF-κB p65 in the aorta were reduced in atorvastatin group and TSD group.CONCLUSIONS:TSD can ameliorate atherosclerosis in rats,and the underlying mechanism is supposed be related to the suppression of inflammatory response by regulating TLR4/My D88/NF-κB signal pathway.

Puerarin protects brain tissue against cerebral ischemia/reperfusion injury by inhibiting the inflammatory response

Puerarin, a traditional Chinese medicine, exerts a powerful neuroprotective effect in cerebral ischemia/reperfusion injury, but its mechanism is unknown. Here, we established rat models of middle cerebral artery ischemia/reperfusion injury using the suture method. Puerarin （100 mg/kg） was administered intraperitoneally 30 minutes before middle cerebral artery occlusion and 8 hours after reperfusion. Twenty-four hours after reperfusion, we found that puerarin significantly improved neurological deficit, reduced infarct size and brain water content, and notably diminished the expression of Toll-like receptor-4, myeloid differentiation factor 88, nuclear factor kappa B and tumor necrosis factor-α in the ischemic region. These data indicate that puerarin exerts an anti-inflammatory protective effect on brain tissue with ischemia/reperfusion damage by downregulating the expression of multiple inflammatory factors.

Compound Gaoziban tablet(复方高滋斑片) alleviates depression via toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-kappa B pathway

OBJECTIVE: To evaluate the effect of compound Gaoziban tablet(复方高滋斑片, CGZBT) on depression, and to investigate the underlying mechanism. METHODS: The components of CGZBT were analysed by high-performance liquid chromatography. Then, we assessed the effects of varying doses of CGZBT on an established chronic unpredictable mild stress(CUMS) model in rats. Whether animals were depressed was evaluated by sucrose preference test, open field test and forced swimming test. Neurotransmitters of hippocampus were detected by liquid chromatography-mass spectrometry. Serum levels of tumor necrosis factor-alpha(TNF-α), interleukin(IL)-1β, IL-6, IL-4, and IL-10 were measured by enzyme-linked immunosorbent assay. Expressions of toll-like receptor 4(TLR4), myeloid differentiation factor 88(My D88), phospho-nuclear factorkappa B(p-NF-κB), cyclooxygenase-2(COX-2), ionized calcium binding adapter molecule-1(IBA-1) were assessed by immunohistochemical staining and western blotting. RESULTS: Eight compounds were identified from CGZBT, moreover, our results showed that CGZBT effectively reversed the CUMS-induced decrease in sucrose preference, shortened the movement distance and prolonged immobility time. CGZBT significantly increased levels of 5-hydroxytryptamine, dopamine, norepinephrine, 5-hydroxyindoleacetic acid levels, and reduced the expression of TNF-α, IL-1β, IL-6, yet increased IL-4 and IL-10. Furthermore, the expressions of TLR4, My D88, COX-2, p-NF-κB and IBA-1 in hippocampus were effectively reversed after treatment with CGZBT. CONCLUSIONS: These results indicated that CGZBT could, at least in part, alleviate depression induced by CUMS via the TLR4/My D88/NF-κB pathway, suggesting its potential as an antidepressant drug.

Efficacy of Liangxue Guyuan decoction(凉血固元汤)on radiation-induced intestinal injury in rats via the toll-like receptor 4/myeloid differentiation primary response 88/nuclear factor-kappa B pathway

OBJECTIVE:To evaluate the efficacy of Liangxue Guyuan decoction(凉血固元汤,LGD)on radiation-induced intestinal injury in rats,and the possible underlying mechanism of action.METHODS:A total of 255 male Sprague-Dawley rats were used.15 rats were assigned to the control group and the remaining 240 rats were exposed to a^(60)Co source at a dose of 11 Gy.Irradiated rats were randomly divided into model,dexamethasone(DXM),low-dose LGD(LGDl),and high-dose LGD(LGDh)groups and treated for 11 d.The survival rate,weight of body,intestinal pathology and the expression of toll-like receptor 4(TLR4),myeloid differentiation primary response 88(MyD88),and nuclear`factor-kappa B(NF-κB)were recorded.RESULTS:Radiation reduced the survival rate and weight of rats,destroyed the intestinal structure,induced an inflammatory reaction,and increased both protein and mRNA expression of TLR4,MyD88,and NF-κB in ileum.However,LGDh increased the survival rate,inhibited weight loss,alleviated inflammation and improve the expression of TLR4 pathway.CONCLUSIONS:LGD increased the survival rate and inhibit weight loss of irradiated rats,and reduced inflammation and intestinal injury.The underlying mechanism may involve regulation of the TLR4/MyD88/NF-κB pathway.

Expression and significance of myeloid differentiation factor 88 in marrow dendritic cells in asthmatic rats with cigarette smoke exposure

Background Smoking causes frequent asthma attacks, leading to a rapid decline in lung function in patients with asthma, and it can also reduce the therapeutic effect of glucocorticoids in patients with asthma. Therefore, the present study aimed to investigate the effect of cigarette smoke on the expression of myeloid differentiation factor 88 （MyD88） in marrow dendritic cells （DCs） in asthmatic rats, and to explore the molecular mechanism of cigarette smoke exposure on asthma by DCs. Methods Forty Wistar rats were randomly divided into the following groups： control, smoke exposure, asthma, and asthma combined with smoke exposure. The animal model was established, and then rat bone marrow-derived DCs were collected. Additionally, rat spleen lymphocytes and bone marrow-derived DCs were cultured together for mixed lymphocyte responses. Interferon （IFN）-gamma and interleukin （IL）-4, IL-10, and IL-12 expressions were determined by enzyme-linked immunosorbent assay （ELISA）. MyD88 expression was determined by Western blotting. The proliferation of lymphocytes was examined with methyl thiazolyl tetrazolium （MTT） colorimetric assay. Results MyD88 expression was decreased in the asthma combined with smoke exposure group compared to the asthma group （P 〈0.01）, and IL-10 and IL-12 expressions were decreased in the asthma combined with smoke exposure group compared to control group （P 〈0.01）. In addition, DCs stimulating activity on allogeneic lymphocytes were significantly decreased in the smoke exposure combined with asthma group compared to the control and asthma groups （P 〈0.01）. After allogeneic mixed lymphocyte responses, IL-4 expression was increased and IFN-gamma was decreased in the asthma group and the asthma combined with smoke exposure group compared to control group （P 〈0.01）. IL-4 expression was increased and IFN-gamma was decreased in the asthma combined with smoke exposure group compared to the asthma group （P 〈0.01）. The study also showed that MyD88 expression was positively correlated with IL-12 and IFN-gamma expressions and the activity of lymphocytes （P 〈0.01）, and negatively correlated with IL-4 expression （P 〈0.01）. Conclusions Smoking aggravates asthma by weankening immunological mechanism. MyD88-dependent pathways may play a role in the immunological balance and activation of lymphocytes.

Potassium dehydroandrographolide succinate regulates the MyD88/CDH13 signaling pathway to enhance vascular injuryinduced pathological vascular remodeling

Pathological vascular remodeling is a hallmark of various vascular diseases.Previous research has established the significance of andrographolide in maintaining gastric vascular homeostasis and its pivotal role in modulating endothelial barrier dysfunction,which leads to pathological vascular remodeling.Potassium dehydroandrographolide succinate(PDA),a derivative of andrographolide,has been clinically utilized in the treatment of inflammatory diseases precipitated by viral infections.This study investigates the potential of PDA in regulating pathological vascular remodeling.The effect of PDA on vascular remodeling was assessed through the complete ligation of the carotid artery in C57BL/6 mice.Experimental approaches,including rat aortic primary smooth muscle cell culture,flow cytometry,bromodeoxyuridine(BrdU)incorporation assay,Boyden chamber cell migration assay,spheroid sprouting assay,and Matrigel-based tube formation assay,were employed to evaluate the influence of PDA on the proliferation and motility of smooth muscle cells(SMCs).Molecular docking simulations and co-immunoprecipitation assays were conducted to examine protein interactions.The results revealed that PDA exacerbates vascular injury-induced pathological remodeling,as evidenced by enhanced neointima formation.PDA treatment significantly increased the proliferation and migration of SMCs.Further mechanistic studies disclosed that PDA upregulated myeloid differentiation factor 88(MyD88)expression in SMCs and interacted with T-cadherin(CDH13).This interaction augmented proliferation,migration,and extracellular matrix deposition,culminating in pathological vascular remodeling.Our findings underscore the critical role of PDA in the regulation of pathological vascular remodeling,mediated through the MyD88/CDH13 signaling pathway.

Suppression of MyD88 disturbs gut microbiota and activates the NLR pathway and hence fails to ameliorate DsS-inducedcolitis

Background:Myeloid differentiation factor 88(MyD88)is the core adaptor for Toll-like receptors defending against microbial invasion and initiating a downstream immune response during microbiota-host interaction.However,the role of MyD88 in the pathogenesis of inflammatory bowel disease is controversial.This study aims to investigate the impact of MyD88 on intestinal inflammation and theunderlyingmechanism.Methods:MyD88 knockout(MyD88^(-/-))mice and the MyD88 inhibitor(TJ-M2010-5)were used to investigate the impact of MyD88 on acute dextran sodium sulfate(Dss)-induced colitis.Disease activity index,colon length,histological score,and inflammatory cytokines were examined to evaluate the severity of colitis.RNA transcriptome analysis and 16S rDNA sequencing were used to detect the potential mechanism.Results:In an acute DSS-colitis model,the severity of colitis was not alleviated in MyD88^(-/-)mice and TJ-M2010-5-treated mice,despite significantly lower levels of NF-kB activation being exhibited compared to control mice.Meanwhile,16S rDNA sequencing and RNA transcriptome analysis revealed a higher abundance of intestinal Proteobacteria and an up-regulation of the nucleotide oligomerization domain-like receptors(NLRs)signaling pathway in colitis mice following MyD88 suppression.Further blockade of the NLRs signaling pathway or elimination of gut microbiota with broad-spectrum antibiotics in DsS-induced colitis mice treated with TJ-M2010-5 ameiorated the disease severity,which was not improved solely by MyD88 inhibition.After treatment with broad-spectrum antibiotics,downregulation of the NLR signaling pathway was observed.Conclusion:Our study suggests that the suppression of MyD88 might be associated with unfavorable changes in the composition of gut microbiota,leading to NLR-mediated immune activation and intestinal inflammation.

Electroacupuncture-induced activation of GABAergic system alleviates airway inflammation in asthma model by suppressing TLR4/MyD88/NF-κB signaling pathway

Background: Electroacupuncture (EA) has been shown to attenuate airway inflammation in asthmatic mice;however, the underlying mechanism is not fully understood. Studies have shown that EA can significantly increase the inhibitory neurotransmitter γ-aminobutyric acid (GABA) content in mice, and can also increase the expression level of GABA type A receptor (GABAAR). Furthermore, activating GABAAR may relieve inflammation in asthma by suppressing toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-kappa B (NF-κB) signaling pathway. Therefore, this study aimed to investigate the role of GABAergic system and TLR4/MyD88/NF-κB signaling pathway in asthmatic mice treated with EA. Methods: A mouse model of asthma was established, and a series of methods including Western blot and histological staining assessment were employed to detect the level of GABA, and expressions of GABAAR and TLR4/MyD88/NF-κB in lung tissue. In addition, GABAAR antagonist was used to further validate the role and mechanism of GABAergic system in mediating the therapeutic effect of EA in asthma. Results: The mouse model of asthma was established successfully, and EA was verified to alleviate airway inflammation in asthmatic mice. The release of GABA and the expression of GABAAR were significantly increased in asthmatic mice treated with EA compared with untreated asthmatic mice ( P < 0.01), and the TLR4/MyD88/NF-κB signaling pathway was down-regulated. Moreover, inhibition of GABAAR attenuated the beneficial effects of EA in asthma, including the regulation of airway resistance and inflammation, as well as the inhibitory effects on TLR4/MyD88/NF-κB signaling pathway. Conclusion: Our findings suggest that GABAergic system may be involved in mediating the therapeutic effect of EA in asthma, possibly by suppressing the TLR4/MyD88/NF-κB signaling pathway.

Therapeutic Effect of Crocin on Diabetic Retinopathy in Rats Based on TLR4/My D88/NF-κB Pathway

Objective:To study the therapeutic effect of crocin on diabetic retinopathy(DR)in rats based on toll-like receptor 4(TLR4)/myeloid differentiation factor 88(My D88)/nuclear factor-κB(NF-κB)pathway.Methods:Thirty SPF SD rats were used in the experiment,which were randomly divided into DR group,control group and crocin group,with 10 rats in each group.The DR rat model was established by feeding the rats in both the DR group and crocin group with a high glucose and high fat diet,along with intraperitoneal injection(IP)of streptozotocin.Crocin IP was administered to the rats in the crocin group,whereas the rats in the DR group and control group received an equivalent dosage of saline IP for 12 weeks.A comparison was made among the three groups regarding retinal thickness,vascular permeability,expression of TLR4/My D88/NF-κB pathway protein,levels of inflammatory factors,and levels of Bcl-2,Bax,and Bcl-2/Bax.Results:The DR group and crocins group exhibited a lower retinal thickness compared to the control group,while the crocins group displayed a higher thickness than the DR group.The DR group and crocins group had higher retinal vascular permeability than the control group,and the crocins group had lower retinal vascular permeability than the DR group(P<0.05).TLR4,My D88,and P-NF-κB relative expressions were higher in the DR and crocin groups than in the control group,whereas TLR4,My D88,and P-NF-κB relative expressions were lower in the crocin group than in the DR group(P<0.05).The DR group and crocin group exhibited elevated levels of inflammatory cytokines compared to the control group,while the crocin group displayed decreased levels in comparison to the DR group(P<0.05).The DR group and crocin group exhibited lower levels of Bcl-2 and Bcl-2/Bax compared to the control group,whereas the control group displayed higher levels of Bax.The crocin group exhibited elevated levels of Bcl-2 and Bcl-2/Bax compared to the DR group,whereas the DR group displayed diminished levels of Bax(P<0.05).Conclusion:Crocin has the potential to enhance the retinal thickness and vascular permeability of DR rats,and the inhibition of the TLR4/My D88/NF-κB pathway by crocin could play a crucial role in impeding the advancement of DR.

Electroacupuncture Attenuates Immune-Inflammatory Response in Hippocampus of Rats with Vascular Dementia by Inhibiting TLR4/MyD88 Signaling Pathway

Objective:To investigate whether electroacupuncture(EA)alleviates cognitive impairment by suppressing the toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)signaling pathway,which triggers immune-inflammatory responses in the hippocampus of rats with vascular dementia(VaD).Methods:The experiments were conducted in 3 parts and in total the Sprague-Dawley rats were randomly divided into 8 groups by a random number table,including sham,four-vessel occlusion(4-VO),4-VO+EA,4-VO+non-EA,sham+EA,4-VO+lipopolysaccharide(LPS),4-VO+LPS+EA,and 4-VO+TAK-242 groups.The VaD model was established by the 4-VO method.Seven days later,rats were treated with EA at 5 acupoints of Baihui(DV 20),Danzhong(RN 17),Geshu(BL 17),Qihai(RN 6)and Sanyinjiao(SP 6),once per day for 3 consecutive weeks.Lymphocyte subsets,lymphocyte transformation rates,and inflammatory cytokines interleukin-6(IL-6)and tumor necrosis factorα(TNF-α)were measured to assess immune function and inflammation in VaD rats.Transmission electron microscopy was used to observe the ultrastructure of nerve cells in the hippocampus.The levels of TLR4,MyD88,IL-6,and TNF-αwere detected after EA treatment.TLR4/MyD88 signaling and cognitive function were also assessed after intracerebroventricular injection of TLR4 antagonist TAK-242 or TLR4 agonist LPS with or without EA.Results:Compared with the 4-VO group,EA notably improved immune function of rats in the 4-VO+EA group,inhibited the protein and mRNA expressions of TLR4 and MyD88 in the hippocampus of rats,reduced the expressions of serum IL-6 and TNF-α(all P<0.05 or P<0.01),and led to neuronal repair in the hippocampus.There were no significant differences between the 4-VO+LPS+EA and 4-VO+EA groups,nor between the 4-VO+TAK-242 and 4-VO+EA groups(P>0.05).Conclusions:EA attenuated cognitive impairment associated with immune inflammation by inhibition of the TLR4/MyD88 signaling pathway.Thus,EA may be a promising alternative therapy for the treatment of VaD.

Zebrafish facilitates drug screening: potential of 3-deoxy-andrographoside from Chuanxinlian(Herba Andrographitis Paniculatae) as an anti-inflammatory agent

OBJECTIVE: To systematically evaluate the in vivo antiinflammatory potential of diterpene lactones from Chuanxinlian(Herba Andrographitis Paniculatae)(AP). METHODS: We firstly adopted zebrafish, a novel and ideal animal model for high-throughput drug screening, to investigate the in vivo anti-inflammatory activities of 17 diterpene lactones isolated from AP.RESULTS: The results showed that most of diterpene lactones displayed significant anti-inflammatory effects in lipopolysaccharide microinjection-, copper sulfate exposure-or tail transection-induced zebrafish inflammation models. Moreover, diterpene lactone 3-deoxy-andrographoside(AP-5) was firstly found to attenuate inflammatory responses, which was closely associated with the myeloid differentiation primary response 88/nuclear factor-kappa B and signal transducer and activator of transcription 3 pathways. CONCLUSION: Our research sheds light on the inestimable roles of zebrafish in high-throughput drug screening, elucidates the potent inhibitory effects of diterpene lactones against inflammation and indicates that AP-5 may serve as a potential alternative agent for the treatment of inflammatory diseases.