OBJECTIVE: To investigate the effect of aquaporin 5 (AQP5) on Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor κB (NF-κB) signaling pathway in Sjögren syndrome (SS) rats. METHODS: The SS gene expression data sets GSE406611 and GSE84844 were extracted from the Gene Expression Omnibus (GEO), and the AQP5 mRNA expression was analyzed by R software. The rat SS model was constructed. The successfully modeled rats were divided into SS group, SS+NC group, and SS+pc group, 10 rats in each group; and 10 rats were set as Normal group. The rats in the SS+NC group were injected with 10 μg of rno-pcDNA3.1-AQP5-NC at the submandibular gland, subcutaneously every day for 28 days. The rats in the SS+pc group were injected with 10 μg of rno-pcDNA3.1-AQP5 at the submandibular gland, subcutaneously every day for 28 days. The enzyme-linked immunosorbent assay (ELISA) kit was used to detect the content of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the serum. High-throughput sequencing was used to identify the target genes. Quantitative real-time PCR (qPCR) and Western blot were used to detect the mRNA and protein expressions of AQP5, TLR4, MyD88, and NF-κB in the rat submandibular gland tissue. RESULTS: In the SS dataset GSE406611 and GSE84844, the mRNA expression of AQP5 in SS was significantly reduced. Compared with the Normal group, the content of TNF-α and IL-1β in the serum, the mRNA and protein expressions of TLR4, MyD88, and NF-κB in the SS group were significantly increased, the mRNA and protein expressions of AQP5 were significantly decreased. After overexpression of AQP5, the content of TNF-α and IL-1β in the serum, the mRNA and protein expressions of TLR4, MyD88, and NF-κB in the SS+pc group were significantly decreased, the mRNA and protein expressions of AQP5 were significantly increased. The differences were statistically significant (all P < 0.05). CONCLUSION The expression of AQP5 is involved in the progression of SS. Increasing the expression of AQP5 can significantly inhibit inflammatory stress and reduce the pathological damage of submandibular gland tissue. This may be related to the inhibition of TLR4/MyD88/NF-κB conduction.
Animals ; Toll-Like Receptor 4/genetics* ; Myeloid Differentiation Factor 88/genetics* ; Aquaporin 5/metabolism* ; Sjogren's Syndrome/genetics* ; Signal Transduction ; NF-kappa B/metabolism* ; Rats ; Rats, Sprague-Dawley ; Interleukin-1beta/metabolism* ; Female

The study aims to observe the effects and explore the mechanisms of Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination in the treatment of the inflammatory response of mice with atherosclerosis(AS) via the Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/nuclear factor-κB(NF-κB) signaling pathway. Male ApoE~(-/-) mice were randomly assigned into a model group, a Buyang Huanwu Decoction group, an Astragali Radix-Angelicae Sinensis Radix combination group, and an atorvastatin group, and male C57BL/6J mice of the same weeks old were used as the control group. Other groups except the control group were given high-fat diets for 12 weeks to establish the AS model, and drugs were administrated by gavage. Aortic intimal hyperplasia thickness, blood lipid level, plasma inflammatory cytokine levels, M1/M2 macrophage markers, and expression levels of proteins in TLR4/MyD88/NF-κB pathway in the vessel wall were measured to evaluate the effects of drugs on AS lesions and inflammatory responses. The results showed that the AS model was successfully established with the ApoE~(-/-) mice fed with high-fat diets. Compared with the control group, the model group showed elevated plasma total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-c) levels(P<0.05), thickened intima(P<0.01), and increased plasma tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) levels(P<0.01). Moreover, the model group showed increased expression of vascular cell adhesion molecule-1(VCAM-1) and inducible nitric oxide synthase(iNOS)(P<0.01), inhibited expression of endothelial nitric oxide synthase(eNOS) and cluster of differentiation 206(CD206)(P<0.01), and up-regulated mRNA and protein levels of TLR4, MyD88, NF-κB inhibitor alpha(IκBα), and NF-κB in the vessel wall(P<0.05). Compared with the model group, Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination lowered the plasma TC and LDL-c levels(P<0.01), alleviated the intimal hyperplasia(P<0.01), and reduced the plasma TNF-α and IL-6 levels(P<0.05). Moreover, the two interventions promoted the expression of eNOS and CD206(P<0.05), inhibited the expression of VCAM-1 and iNOS(P<0.01), and down-regulated the mRNA and protein levels of TLR4, MyD88, IκBα, and NF-κB(P<0.05) in the vessel wall. This study indicated that Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination could delay the progression of AS, inhibit the polarization of vascular wall macrophages toward M1 type, and attenuate vascular inflammatory response by inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway in the vascular wall. Astragali Radix and Angelicae Sinensis Radix were the main pharmacological substances in Buyang Huanwu Decoction for alleviating the AS vascular inflammatory response.
Mice ; Male ; Animals ; NF-kappa B/metabolism* ; Toll-Like Receptor 4/metabolism* ; NF-KappaB Inhibitor alpha/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/metabolism* ; Myeloid Differentiation Factor 88/metabolism* ; Vascular Cell Adhesion Molecule-1/metabolism* ; Cholesterol, LDL ; Hyperplasia ; Mice, Inbred C57BL ; Atherosclerosis/genetics* ; Apolipoproteins E/therapeutic use* ; RNA, Messenger

This study investigated the therapeutic effect of Shegan Mahuang Decoction(SGMHD) on cold-induced asthma in rats and explored its underlying mechanism. Seventy-two healthy male SD rats of specific pathogen free(SPF) grade were randomly divided into a blank group, a model group, a positive control group(dexamethasone, 0.4 mg·kg~(-1)), and low-, medium-, and high-dose SGMHD groups(3.2, 6.4, and 12.8 g·kg~(-1)). The blank group received saline, while the other groups were sensitized by intraperitoneal injection of ovalbumin(OVA) solution. Subsequently, the rats were placed in a cold chamber adjustable to 0-2 ℃, and OVA solution was ultrasonically nebulized to induce cold-induced asthma in rats. After three weeks of treatment, the general behaviors of rats were observed. Hematoxylin-eosin(HE) staining was used to evaluate pathological changes in lung tissues, periodic acid-Schiff(PAS) staining assessed mucin changes, and Masson staining was performed to examine collagen deposition. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of the inflammatory factors interleukin-4(IL-4) and vascular endothelial growth factor(VEGF) in serum and bronchoalveolar lavage fluid(BALF). Real-time quantitative polymerase chain reaction(RT-PCR) was employed to assess the mRNA expression levels of transient receptor potential vanilloid subfamily member 1(TRPV1), nuclear respiratory factor 1(NRF-1), and mitochondrial transcription factor A(mtTFA) in lung tissues. Western blot was used to measure the protein expression levels of TRPV1, NRF-1, and mtTFA in lung tissues. Compared with the blank group, the model group exhibited signs of rapid respiration, increased frequency of defecation with looser stools, and disheveled and dull fur. Pathological results showed significant infiltration of inflammatory cells in lung tissues, narrowing of bronchial lumens, increased mucin secretion, and enhanced collagen deposition in the model group. Additionally, the levels of IL-4 and VEGF in serum and BALF were significantly elevated, and the mRNA and protein expression levels of TRPV1, NRF-1, and mtTFA in lung tissues were significantly increased. Compared with the model group, SGMHD improved the behaviors of rats, alleviated pathological changes in lung tissues, mucin production, and collagen deposition, significantly decreased the levels of IL-4 and VEGF in serum and BALF, and reduced the mRNA expression levels of TRPV1, NRF-1, and mtTFA in lung tissues, with the medium-dose SGMHD group showing the most significant effect. Moreover, the protein expression levels of TRPV1, NRF-1, and mtTFA in lung tissues were also reduced, with the medium-dose SGMHD group exhibiting the most significant effect. In conclusion, this study demonstrates that SGMHD can alleviate airway inflammation and inhibit airway remodeling in cold-induced asthma rats. These effects may be associated with the modulation of the TRPV1/NRF-1/mtTFA signaling pathway.
Rats ; Male ; Animals ; Mice ; Interleukin-4/metabolism* ; Vascular Endothelial Growth Factor A/metabolism* ; Rats, Sprague-Dawley ; Asthma/genetics* ; Lung ; Bronchoalveolar Lavage Fluid ; RNA, Messenger/metabolism* ; Collagen/metabolism* ; Mucins/therapeutic use* ; Ovalbumin ; Disease Models, Animal ; Mice, Inbred BALB C ; TRPV Cation Channels/metabolism* ; Drugs, Chinese Herbal

Objective To investigate the role of microRNA-18a (miR-18a) in the pathogenesis of allergic rhinitis in mice. Methods Twenty-two BALB/c mice were randomly divided into a blank group, a model group and a miR-18a group. Mice in the model group and the miR-18a group were injected intraperitoneally with obumin (OVA) suspension to prepare allergic rhinitis models, and mice in the miR-18a group were simultaneously given lentiviral vector plasmid for overexpression of miR-18a. Allergy symptoms were evaluated by the behavioral score and HE staining. The plasma levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor α (TNF-α) were measured by ELISA. The distribution of CD45⁺ cells in nasal mucosa was measured by immunofluorescence histochemistry, and CD45⁺ cells in nasal lavage fluid were measured by flow cytometry. The mRNA expression levels of IL-1β, IL-6 and TNF-α in nasal mucosa tissues were measured by fluorescence quantitative PCR, and the protein expressions of Toll like receptor 4 (TLR4), nuclear factor κB p65 (NF-κB p65), inhibitor of NF-κB α (IκBα) and phosphorylated IκBα (p-IκBα) in nasal mucosa were measured by Western blot analysis. Results Compared with the blank group, the plasma levels of IL-1β, IL-6, and TNF-α in the model group increased significantly. The number of CD45⁺ cells in both nasal mucosa tissue and nasal irrigation fluid increased, and the mRNA levels of IL-1β, IL-6 and TNF-α and the protein expression levels of TLR4, NF-κB p65 and p-IκBα in nasal mucosa increased. Compared with the model group, the plasma levels of IL-1β, IL-6 and TNF-α in the miR-18a group decreased significantly. The number of CD45⁺ cells in both nasal mucosa tissue and nasal lavage fluid decreased, and the mRNA levels of IL-1β, IL-6 and TNF-α and the exprotein expression levels of TLR4, NF-κB p65 and p-IκBα in nasal mucosa decreased. Conclusion miR-18a can inhibit the occurrence and development of allergic rhinitis, and its molecular mechanism is related to the inhibition of TLR4/NF-κB pathway activation.
Animals ; Mice ; Disease Models, Animal ; Inflammation ; Interleukin-6/genetics* ; MicroRNAs/genetics* ; NF-kappa B/metabolism* ; NF-KappaB Inhibitor alpha ; Rhinitis, Allergic ; RNA, Messenger ; Toll-Like Receptor 4/metabolism* ; Tumor Necrosis Factor-alpha/genetics*

This study aims to investigate the effect of modified Danggui Shaoyao Powder on the suppressor of cytokine signaling 3(SOCS3)/Toll-like receptor 4(TLR4) signaling pathway in gastric tissue of rats with chronic atrophic gastritis(CAG).Sixty SPF-grade SD rats were randomly assigned into the normal group, model group, Moluo Pills group, and high-, medium-, and low-dose groups of modified Danggui Shaoyao Powder.The rats in other groups except the normal group were treated with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) to establish the CAG model.After 12 weeks of modeling, the rats in each group were administrated with corresponding drugs by gavage for 8 weeks.After the last administration, the histopathological changes of rat gastric mucosa were observed via hematoxylin-eosin(HE) staining.The serum levels of IL-6, TNF-α, and CRP were determined by enzyme-linked immunosorbent assay(ELISA).The mRNA levels of SOCS3 and TLR4 were determined by real-time PCR.The protein levels of SOCS3, TLR4, JAK2, p-JAK2, STAT3, and p-STAT3 in rat gastric tissue were measured by Western blot.Immunohistochemical method was employed to determine the protein levels of NF-κB, MyD88, NLRP3, Bcl-2, Bax, and Bad in rat gastric tissue.The results showed that modified Danggui Shaoyao Powder alleviated gastric mucosal atrophy of rats, significantly lowered the levels of IL-6, TNF-α, and CRP in rat serum, up-regulated the mRNA level of SOCS3, and down-regulated the mRNA level of TLR4 in rat gastric tissue.Furthermore, modified Danggui Shaoyao Powder up-regulated the protein level of SOCS3, down-regulated the protein levels of TLR4, p-JAK2, p-STAT3, NF-κB, MyD88, NLRP3, Bax, and Bad, and promoted the expression of Bcl-2 protein.Therefore, modified Danggui Shaoyao Powder may mitigate the gastric mucosal atrophy of rats by regulating the SOCS3/TLR4 signaling pathway.
Animals ; Atrophy ; Gastritis, Atrophic/genetics* ; Interleukin-6/metabolism* ; Myeloid Differentiation Factor 88/metabolism* ; NF-kappa B/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Powders ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Suppressor of Cytokine Signaling 3 Protein/metabolism* ; Toll-Like Receptor 4/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; bcl-2-Associated X Protein/metabolism*

Based on network pharmacology and in vivo experiment, this study explored the therapeutic effect of Tetrastigma hemsle-yanum(SYQ) on sepsis and the underlying mechanism. The common targets of SYQ and sepsis were screened out by network pharmacology, and the "SYQ-component-target-sepsis" network was constructed. The protein-protein interaction(PPI) network was established by STRING. Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment were performed based on DAVID to predict the anti-sepsis mechanism of SYQ. The prediction results of network pharmacology were verified by animal experiment. The network pharmacology results showed that the key anti-sepsis targets of SYQ were tumor necrosis factor(TNF), interleukin(IL)-6, IL-1β, IL-10, and cysteinyl asparate specific proteinase 3(caspase-3), which were mainly involved in Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor kappaB(NF-κB) signaling pathway. The results of animal experiment showed that SYQ can decrease the content of C-reactive protein(CRP), procalcitonin(PCT), lactate dehydrogenase(LDH), IL-6, TNF-α, and IL-1β, increase the content of IL-10, and down-regulate the protein levels of Bcl-2-associa-ted X(Bax)/B-cell lymphoma 2(Bcl2), cleaved caspase-3, TLR4, MyD88, and p-NF-κB p65/NF-κB p65. In summary, SYQ plays an anti-inflammatory role in the treatment of sepsis by acting on the key genes related to inflammation and apoptosis, such as TNF-α, IL-6, IL-lβ, IL-10, Bax, Bcl2, and cleaved caspase-3. The mechanism is the likelihood that it suppresses the TLR4/MyD88/NF-κB signaling pathway, which verifies relative prediction results of network pharmacology.
Animals ; Anti-Inflammatory Agents/therapeutic use* ; C-Reactive Protein ; Caspase 3/metabolism* ; Interleukin-10 ; Interleukin-6/metabolism* ; Lactate Dehydrogenases/metabolism* ; Myeloblastin/metabolism* ; Myeloid Differentiation Factor 88/metabolism* ; NF-kappa B/metabolism* ; Network Pharmacology ; Procalcitonin/therapeutic use* ; Sepsis/genetics* ; Toll-Like Receptor 4/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; bcl-2-Associated X Protein/metabolism*

High-throughput transcriptome sequencing was used to study the mechanism of Shenling Baizhu Powder(SLBZP) in the alleviation of the dextran sulfate sodium(DSS)-induced ulcerative colitis(UC) in mice. The mouse model of DDS-induced UC was treated with SLBZP by gavage. The changes in general state, disease activity index(DAI), and colon length were observed. The hematoxylin-eosin(HE) staining was used to observe the pathological changes in the colon tissues of mice. Enzyme-linked immunosorbent assay(ELISA) was used to determine the expression levels of tumor necrosis factor-α(TNF-α), interleukin(IL)-1β, IL-6, IL-4, and IL-10 in the serum and tissues of mice. The differentially expressed genes in the control group, the model group, and the SLBZP group were analyzed by high-throughput transcriptome sequencing, and the Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were conducted on the differentially expressed genes. The results showed that after intragastric administration of SLBZP, the symptoms of diarrhea and bloody stool were improved, and the disease active index(DAI) score was reduced. SLBZP effectively reduced the inflammatory cell infiltration and goblet cell loss in the colonic mucosal tissue, reduced the levels of TNF-α, IL-1β, IL-6 in the serum and colon tissue, and increased the levels of IL-4 and IL-10 in the serum and colon tissue. There were 25 differential genes in SLBZP vs the model group, which were significantly enriched in immune response, immune system process, immunoglobulin production, and other biological processes. KEGG pathway analysis showed that the differential genes were enriched in signaling pathways such as neomycin, kanamycin, and gentamicin biosynthesis, cytokine-cytokine receptor interaction, primary immunodeficiency, and IgA synthesis of the intestinal immune network. This study shows that SLBZP may alleviate UC through immune regulation.
Animals ; Mice ; Colitis, Ulcerative/genetics* ; Colon ; Dextran Sulfate/adverse effects* ; Disease Models, Animal ; Interleukin-10/genetics* ; Interleukin-4/genetics* ; Interleukin-6/genetics* ; Mice, Inbred C57BL ; Powders ; Transcriptome ; Tumor Necrosis Factor-alpha/metabolism* ; Drugs, Chinese Herbal/therapeutic use*

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*

This study aims to explore the effect of Sini Decoction on Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) signaling pathway in the mice with allergic asthma(AA). Forty-eight SPF-grade BALB/c mice were randomly assigned into a blank control group, a model group, a dexamethasone group, and high-, medium-, and low-dose Sini Decoction groups, with 8 mice in each group. The sensitization solution made of ovalbumin and aluminum hydroxide powder was injected intraperitoneally in other groups except the blank control group which was injected with an equal volume of normal saline. The solution(or normal saline) was injected three times in total with an interval of 7 days. At the same time of sensitization, external cold stimulation and ice water were administered in a 4 ℃ climate box for 20 min every day. After modeling, the mice in each group were administrated with corresponding drugs by gavage for 3 weeks. At the end of administration, pentobarbital sodium(30 mg·kg~(-1)) was used for anesthesia, and then the samples were collected for the determination of various indexes. The phenol red test was conducted to evaluate tracheal excretion function. The histopathological changes of lung tissue were observed via hematoxylin-eosin(HE) staining. Masson staining was employed to reveal the deposition of blue collagen fibers around bronchi in lung tissue and the area occupied by blue collagen fibers was calculated. Immunofluorescence method was used to measure the expression of bronchial type Ⅰ collagen(Col-Ⅰ) and α-smooth muscle actin(α-SMA). The protein and mRNA levels of TLR4, NF-κB, cysteinyl aspartate specific proteinase-1(caspase-1), and interleukin-13(IL-13) were determined by Western blot and real-time fluorescence quantitative polymerase chain reaction(real-time PCR), respectively. Compared with the model group, Sini Decoction significantly increased the phenol red excretion from trachea, lowered the lung inflammation score, reduced subepithelial collagen deposition, and decreased Col-Ⅰ and α-SMA levels. Furthermore, the decoction down-regulated the protein and mRNA levels of TLR4, NF-κB, caspase-1, and IL-13 in mouse lung tissue. In conclusion, Sini Decoction can improve air remodeling by inhibiting the TLR4/NF-κB signaling pathway.
Mice ; Animals ; NF-kappa B/metabolism* ; Airway Remodeling ; Interleukin-13/pharmacology* ; Toll-Like Receptor 4/genetics* ; Saline Solution/pharmacology* ; Phenolsulfonphthalein/pharmacology* ; Asthma/genetics* ; Signal Transduction ; Mice, Inbred BALB C ; RNA, Messenger ; Caspases

To investigated the mechanisms underlying the effects of modified Kaixin San(MKXS) on improving memory and synaptic damage of Alzheimer's disease(AD) mouse model with conditional presenilin 1/2 conditional double knockout(PS cDKO). Specifically, 60 PS cDKO mice(3-3.5 months old) and their age-matched wild-type(WT) littermates were randomized into three groups: WT group(n=20), PS cDKO group(n=20), and PS cDKO+MKXS group(n=20). Mice in WT and PS cDKO groups were fed with standard chow and those in PS cDKO+MKXS group were given chow containing MKXS(at 2.55 g·kg~(-1)) for 60 days. Novel object reco-gnition task was employed to detect the recognition memory of mice, and Western blot to detect the protein levels of synapse-associated proteins in the hippocampus(HPC) of mice, such as NR1, NR2 A, NR2 B, p-αCaMKⅡ, tau, and p-tau. Microglial morphology in the HPC CA1 of mice was observed based on immunohistochemistry. Quantitative real time-PCR(qRT-PCR) was employed to detect the mRNA levels of the pro-inflammatory factors and synapse-associated proteins in the HPC of mice, including COX-2, iNOS, IL-1β, IL-6, TNF-α, PSD95, NR1, NR2 A, NR2 B, and MAP2. The protein levels of IL-1β, TNF-α, and IL-6 were tested by enzyme-linked immunosorbent assay(ELISA). The interaction between PSD95 and αCaMKⅡ and between PSD95 and p-αCaMKⅡ was tested by co-immunoprecipitation(Co-IP). The results showed that PS cDKO+MKXS demonstrated significantly higher preference index and recognition index of the new objects, lower protein level of p-tau(ser 396/404) and mRNA levels of COX-2, iNOS, TNF-α, IL-1β, and IL-6 in HPC, higher protein levels of NR1, NR2 A, NR2 B, and p-αCaMKⅡ and mRNA levels of NR1, NR2 A, NR2 B, PSD95, and MAP2, and stronger interaction of αCaMKⅡ with PSD95 and interaction of p-αCaMKⅡ with PSD95 than the PS cDKO group. Immunohistoche-mical staining showed that MKXS inhibited the activation of microglia. In conclusion, MKXS improves memory and synaptic damage in mice with AD by modulating αCaMKⅡ-PSD95 protein binding through inhibition of neuroinflammation.
Animals ; Mice ; Alzheimer Disease/genetics* ; Disks Large Homolog 4 Protein/metabolism* ; Neuroinflammatory Diseases ; Tumor Necrosis Factor-alpha/metabolism* ; Cyclooxygenase 2/metabolism* ; Interleukin-6/metabolism* ; Protein Binding ; Mice, Knockout ; Hippocampus/metabolism* ; Disease Models, Animal ; RNA, Messenger/metabolism*