The dynamic shifts of IL-10-producing Th17 and IL-17-producing Treg in health and disease: a crosstalk between ancient "Yin-Yang" theory and modern immunology.

The changes in T regulatory cell (Treg) and T helper cell (Th) 17 ratios holds paramount importance in ensuring internal homeostasis and disease progression. Recently, novel subsets of Treg and Th17, namely IL-17-producing Treg and IL-10-producing Th17 have been identified. IL-17-producing Treg and IL-10-producing Th17 are widely considered as the intermediates during Treg/Th17 transformation. These "bi-functional" cells exhibit plasticity and have been demonstrated with important roles in multiple physiological functions and disease processes. Yin and Yang represent opposing aspects of phenomena according to the ancient Chinese philosophy "Yin-Yang" theory. Furthermore, Yin can transform into Yang, and vice versa, under specific conditions. This theory has been widely used to describe the contrasting functions of immune cells and molecules. Therefore, immune-activating populations (Th17, M1 macrophage, etc.) and immune overreaction (inflammation, autoimmunity) can be considered Yang, while immunosuppressive populations (Treg, M2 macrophage, etc.) and immunosuppression (tumor, immunodeficiency) can be considered Yin. However, another important connotation of "Yin-Yang" theory, the conversion between Yin and Yang, has been rarely documented in immune studies. The discovery of IL-17-producing Treg and IL-10-producing Th17 enriches the meaning of "Yin-Yang" theory and further promotes the relationship between ancient "Yin-Yang" theory and modern immunology. Besides, illustrating the functions of IL-17-producing Treg and IL-10-producing Th17 and mechanisms governing their differentiation provides valuable insights into the mechanisms underlying the dynamically changing statement of immune statement in health and diseases.

[Mechanism and compatibility efficacy of Astragali Radix-Chuanxiong Rhizoma drug pair in treating ischemic stroke based on network regulation].

Based on the association network of "drug pair-disease", the effect characteristics of Astragali Radix-Chuanxiong Rhizoma drug pair in the treatment of ischemic stroke(IS) with Qi deficiency and blood stasis and the matching mechanism of the two were explored. Through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and SwissTargetPrediction Database, the effective chemical components of the drug pair were screened, and the candidate targets were predicted. Databa-ses such as GeneCards, DrugBank, Online Mendelian Inheritance in Man(OMIM), and Therapeutic Target Database(TTD) were searched to obtain gene targets related to IS. Through STRING and Cytoscape 3.9.1 software, the protein-protein interaction(PPI) network was constructed by using the interaction information of disease syndrome-related genes and candidate targets of drug pairs, and the core targets were screened according to the network topological feature values. Based on the Metascape platform and DAVID database, the biomolecular interaction information was integrated to analyze the Kyoto Encyclopedia of Genes and Genomes(KEGG) and mine biological functions, so as to further explore the mechanism of action and compatibility characteristics of Astragali Radix-Chuan-xiong Rhizoma. The results showed that the candidate biological process was mainly involved in the regulation of functional modules such as immune, blood circulation, neurotransmitter, and oxidative stress, and it was enriched in lipid and atherosclerosis, calcium signaling pathway, and platelet activation. Astragali Radix and Chuanxiong Rhizoma have their own characteristics. Astragali Radix has a regulatory response to growth factors while maintaining the body's immune balance, while Chuanxiong Rhizoma mainly improves the circulatory system and participates in hormone metabolism, so as to indicate the compatibility mechanism of Astragali Radix-Chuanxiong Rhizoma drug pair for multi-target and multi-pathway synergistic treatment of IS. Through further experimental verification, it was found that the Astragali Radix-Chuanxiong Rhizoma drug pair could significantly down-regulate the expression of key targets including TLR4, NF-κB, IL-1ß, F2R, PLCß1, and MYLK. This study preliminarily reveals that the Astragali Radix-Chuanxiong Rhizoma drug pair may play the three replenishing effects of promoting blood circulation, benefiting Qi, and clearing collaterals by correcting immune di-sorders, blood circulation disorders, and inflammation, which provide support for the clinical research on the subsequent improvement of Qi deficiency and blood stasis in the treatment of IS and provide a new idea for the analysis of modern biological connotation of the compatibility of seven emotions of traditional Chinese medicine.

[Effect of triptolide on reproductive toxicity in female rats with â ¡ type collagen induced arthritis].

In order to study the toxic effect and mechanism of triptolide(TP) on the reproductive system of female rats with Ⅱ type collagen induced arthritis(CIA), 50 SD rats were randomly divided into normal control group, CIA model group, and three groups receiving TP tablets at clinically equivalent doses of 0. 5, 1, and 2 times, respectively(with TP dosages of 3. 75, 7. 5, and 15 µg·kg~(-1)·d~(-1)), each comprising 10 rats. Intragastric administration was started on the day after the first immunization, once a day, for 42 days.The results were taken on the 21st and 42nd days to calculate the uterine and ovarian organ indexes; pathological and morphological changes in uterus and ovaries were observed under a light microscope; and the levels of estradiol(E_2) and cytochrome P450A1(aromatase,CYP19A1) in ovarian homogenate were detected by ELISA. Furthermore, immunohistochemistry was employed to detect the expression levels of transforming growth factor ß3( TGFß3) pathway-related proteins, mothers against decapentaplegic homolog 3(Smad3) and steroidogenic factor-1(SF-1) in ovarian tissues. In vitro, the mouse Chinese hamster ovary(CHO) cell line was established, and after 24 hours of TP administration(30, 60, 120 nmol·L~(-1)), cell proliferation was detected by the thiazolyl blue tetrazolium bromide(MTT) method, apoptosis by the flow cytometry, and TGFß3, Smad3 and SF-1 protein expression in cells by the Western blot method, and the nuclear entry of SF-1 was detected by immunofluorescence. The results showed that compared with the CIA model group, all TP administration groups showed decreased number of uterine glands, total follicles, mature follicles, and corpus luteum on days 21 and 42 of administration, but there was no statistical difference, and only the administration of 2 times the clinically equivalent dose of TP could significantly increase the number of atretic follicles at 42 days of administration. TP at 3. 75 µg·kg-1·d-1significantly reduced the level of E_2 at 21 days of administration and the expression of TGFß3 and Smad3 factors in ovarian tissues,but had no significant effect on the rate-limiting enzyme in estrogen synthesis CYP19A1. TP at 7. 5 and 15 µg·kg~(-1)·d~(-1) significantly reduced the expression of SF-1 regardless of administration for 21 days or 42 days. TP can significantly promote ovarian cell apoptosis in vitro, with apoptosis mainly concentrated in the late stage of apoptosis after 24 hours of administration. In addition, 60 nmol·L~(-1) TP significantly reduced the protein expression of TGFß3, Smad3 and SF-1 in a dose-dependent manner. In summary, intragastric administration of TP at less than 2 times the clinically equivalent dose for 21 days and 42 days did not cause obvious reproductive damage to the uterus and ovarian tissues of CIA rats, and the number of atretic follicles changed significantly only when the 2 times the clinically equivalent dose was administered for 42 days. TP exerted reproductive toxicity in vivo on reproductive target organs and in vitro on ovarian cells by inhibiting the expression of TGFß3/Smad3/SF-1 pathway.

[Mechanism of tetramethylpyrazine intervention with ischemia-reperfusion rats based on Nrf2/HO-1/CXCR4 pathway through regulating neural stem cell migration].

This study aims to decipher the mechanism of tetramethylpyrazine(TMP) in regulating the migration of neural stem cells(NSCs) in the rat model of middle cerebral artery occlusion(MCAO) via the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase 1(HO-1)/C-X-C motif chemokine receptor 4(CXCR4) pathway. SD rats were randomized into sham, MCAO(model), and tetramethylpyrazine(TMP, 20 mg·kg~(-1) and 40 mg·kg~(-1)) groups. The neurological impairment was assessed by the modified neurological severity score(mNSS). The immunofluorescence assay was employed to detect the cells stained with both 5-bromodeoxyuridine(BrdU) and doublecortin(DCX) in the brain tissue. The effect of TMP on the migration of C17.2 cells was observed. Western blot was employed to determine the protein levels of Nrf2, HO-1, p62, NAD(P)H quinone oxidoreductase 1(NQO1), stromal cell-derived factor 1(SDF-1), and CXCR4 in the brain tissue and C17.2 cells. The results showed that after 7 days and 21 days of mode-ling, the mNSS and BrdU~+/DCX~+ cells were increased, and the expression of Nrf2 and CXCR4 in the brain tissue was up-regulated. Compared with the model group, TMP(40 mg·kg~(-1)) reduced the mNSS, increased the number of BrdU~+/DCX~+ cells, and up-regulated the expression of Nrf2, CXCR4, and SDF-1. In addition, TMP promoted the migration of C17.2 cells and up-regulated the expression of p62, Nrf2, HO-1, and NQO1 in a time-and dose-dependent manner. The expression was the highest at the time point of 12 h in the TMP(50 µg·mL~(-1)) group(P<0.01). In conclusion, TMP activates the Nrf2/HO-1/CXCR4 pathway to promote the migration of NSCs to the ischemic area, thus exerting the therapeutic effect on the ischemia-reperfusion injury. This study provides experimental support for the application of TMP in ischemic stroke.

[Mechanism of tetramethylpyrazine combined with neural stem cells transplantation on cerebral ischemia-reperfusion rats].

This study aimed to investigate the intervention effect of tetramethylpyrazine(TMP) combined with transplantation of neural stem cells(NSCs) on middle cerebral artery occlusion(MCAO) rat model and to explore the mechanism of TMP combined with NSCs transplantation on ischemic stroke based on the regulation of stem cell biological behavior. MCAO rats were randomly divided into a model group, a TMP group, an NSCs transplantation group, and a TMP combined with NSCs transplantation group according to neurological function scores. A sham group was set up at the same time. The neurological function score was used to evaluate the improvement of neurological function in MCAO rats after TMP combined with NSCs transplantation. The proliferation, migration, and differentiation of NSCs were evaluated by BrdU, BrdU/DCX, BrdU/NeuN, and BrdU/GFAP immunofluorescence labeling. The protein expression of stromal cell-derived factor 1(SDF-1), C-X-C motif chemokine receptor 4(CXCR4), as well as oxidative stress pathway proteins nuclear factor erythroid 2-related factor 2(Nrf2), Kelch-like ECH-associated protein 1(KEAP1), heme oxygenase 1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1) was detected by Western blot to study the migration mechanism of TMP combined with NSCs. The results showed that TMP combined with NSCs transplantation significantly improved the neurological function score in MCAO rats. Immunofluorescence staining showed a significant increase in the number of BrdU~+, BrdU~+/DCX~+, BrdU~+/NeuN~+, and BrdU~+/GFAP~+ cells in the TMP, NSCs transplantation, and combined treatment groups, with the combined treatment group showing the most significant increase. Further Western blot analysis revealed significantly elevated expression of CXCR4 protein in the TMP, NSCs transplantation, and combined treatment groups, along with up-regulated protein expression of Nrf2, HO-1, and NQO1, and decreased KEAP1 protein expression. This study showed that both TMP and NSCs transplantation can promote the recovery of neurological function by promoting the proliferation, migration, and differentiation of NSCs, and the effect of TMP combined with NSCs transplantation is superior. The mechanism of action may be related to the activation of the Nrf2/HO-1/CXCR4 pathway.

[Optimization and verification of conditions for induction of neutrophil extracellular traps in vitro].

This study aims to optimize the conditions for the formation of neutrophil extracellular traps(NETs) in vitro, so as to establish a relatively stable experimental research platform. Different conditions were compared, including commonly used laboratory animals(rats and mice) and a variety of cell sources(bone marrow neutrophils and peripheral blood neutrophils separated by percoll density gradient centrifugation). Different inducers like lipopolysaccharide(LPS) and phorbol 12-myristate 13-acetate(PMA) were used for induction in vitro. Myeloperoxidase(MPO)/citrullinated histone H3(CitH3)/DAPI immunofluorescence and cell free DNA(cf-DNA) content determination were used for comprehensive evaluation to screen the optimal conditions for the formation of NETs induced in vitro. Furthermore, the stability of the selected conditions for inducing the formation of NETs in vitro was evaluated by tetramethylpyrazine(TMP), an active component in Chinese herbal medicines. The results showed that coated poly-D-lysine(PDL) induced the formation of NETs in bone marrow neutrophils of mice to a certain extent. Both LPS and PMA significantly up-regulated the protein levels of MPO and CitH3 in mouse bone marrow neutrophils and elevated the cfDNA level in the supernatant of rat peripheral blood neutrophils. The cfDNA level in the PMA-induced group increased more significantly than that in the LPS-induced group(P<0.05). The results of immunofluorescence staining showed that the expression of MPO and CitH3 in mouse bone marrow neutrophils, rat bone marrow neutrophils, and rat peripheral blood neutrophils were significantly increased after PMA induction, especially in rat peripheral blood neutrophils. TMP significantly down-regulated the protein levels of MPO, CitH3, and neutrophil elastase(NE) in rat peripheral blood neutrophils induced by PMA. In conclusion, treating the peripheral blood neutrophils of rats with PMA is the optimal condition for inducing the formation of NETs in vitro. This study provides an optimal platform for in vitro studies based on NETs and a basis for studying the effects of traditional Chinese medicines targeting NETs.

[Effect and mechanism of aqueous extract of Strychni Semen on bone destruction in rats with type â ¡ collagen-induced rheumatoid arthritis].

To investigate the mechanism of action of aqueous extract of Strychni Semen(SA) on bone destruction in rats with type Ⅱ collagen-induced arthritis(CIA), the SD rats were randomly divided into normal group, model group, low, medium, and high dose(2.85, 5.70, and 11.40 mg·kg~(-1)) groups of SA, and methotrexate group. Except for the normal group, the CIA model was prepared for the other groups. After the second immunization, different doses of SA were given to the low, medium, and high dose groups of SA once a day, and the methotrexate group was given once every three days. 0.3% sodium hydroxymethylcellulose(CMC-Na) was given once a day to the normal and model groups for 28 d. The clinical score of arthritis was evaluated every three days. Micro computed tomography(Micro-CT) method was used to evaluate the degree of bone destruction. Histopathological changes in the joint tissue and the number of osteoclasts in CIA rats were evaluated by hematoxylin-eosin(HE) staining and tartrate-resistant acid phosphatase(TRAP) staining. The expression of interleukin-1ß(IL-1ß) in the joint tissue of rats was detected by immunohistochemistry. Western blot was used to detect key protein expression in mitogen-activated protein kinase(MAPK) and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt) signaling pathways in the joint tissue of rats. The results showed that different doses of SA were able to improve the red and swollen inflammatory joint and joint deformity in CIA rats to varying degrees, reduce the clinical score, inhibit synovial inflammation, vascular opacification, cartilage erosion, and bone destruction, and reduce the number of TRAP-positive cells in bone tissue. Micro-CT results showed that the SA was able to increase bone mineral density, bone volume fraction, trabecular reduce, and trabecular number and reduce bone surface/bone volume and trabecular separation/spacing. Different doses of SA could down-regulate the protein expression of IL-1ß, p-JNK, p-ERK, p-p38, PI3K, and p-Akt to varying degrees. In conclusion, SA can improve disease severity, attenuate histopathological and imaging changes in joints, and have osteoprotective effects in CIA rats, and its mechanism of action may be related to the inhibition of the overactivation of MAPK and PI3K/Akt signaling pathways.

[Mechanism of Yuxuebi Tablets in treating synovial inflammation in rheumatoid arthritis based on transcriptomics].

This study investigated the mechanism of Yuxuebi Tablets(YXB) in the treatment of synovial inflammation in rheumatoid arthritis(RA) based on transcriptomic analysis. Transcriptome sequencing technology was employed to analyze the gene expression profiles of joint tissues from normal rats, collagen-induced arthritis(CIA) rats(an RA model), and YXB-treated rats. Common diffe-rentially expressed genes(DEGs) were subjected to Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses. RA synovial inflammation-related target genes were retrieved from the OMIM and GeneCards databases. Venny 2.1 software was used to identify the intersection of YXB target genes and RA synovial inflammation-related target genes, and GO and KEGG enrichment analyses were performed on the intersecting target genes. Immunohistochemistry was used to assess the protein expression levels of the inflammatory factors interleukin-1ß(IL-1ß) and tumor necrosis factor-α(TNF-α) in rat joint tissues. Western blot analysis was employed to measure the expression levels of key proteins in the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt) signaling pathway. A total of 2 058 DEGs were identified by intersecting the genes from the normal group vs model group and the model group vs YXB treatment group. A search in OMIM and GeneCards databases yielded 1 102 RA synovial inflammation-related target genes. After intersecting with the DEGs in the YXB treatment group, 204 intersecting target genes were identified, primarily involving biological processes such as immune response, signal transduction, and inflammatory response; cellular components including plasma membrane, extracellular space, and extracellular region; molecular functions like protein binding, identical protein binding, and receptor binding. These target genes were mainly enriched in signaling pathways such as PI3K/Akt, cytokine-cytokine receptor interaction, and Janus kinase/signal transducer and activator of transcription(JAK/STAT). Western blot results showed that YXB at low, medium, and high doses could significantly inhibit the expression levels of key proteins in the PI3K/Akt signaling pathway in rat joint tissues in a dose-dependent manner. Immunohistochemistry further confirmed these findings, showing that YXB not only suppressed the protein expression levels of the inflammatory factors IL-1ß and TNF-α in the joint synovial tissues of CIA rats, but also inhibited p-Akt protein expression. In conclusion, this study used transcriptomic analysis to uncover the key mechanisms of YXB in inhibiting synovial inflammation and alleviating the progression of RA, with a focus on its role in suppressing the PI3K/Akt signaling pathway.

[Mechanism of aqueous extract of Strychni Semen in improving pain in rats with rheumatoid arthritis based on TLR4/TNF-α/MMP-9 signaling pathway].

This study aims to explore the mechanism of aqueous extract of Strychni Semen(SA) in relieving pain in the rat model of rheumatoid arthritis(RA) via Toll-like receptor 4(TLR4)/tumor necrosis factor-α(TNF-α)/matrix metalloproteinase-9(MMP-9) signaling pathway. Firstly, the main chemical components of Strychni Semen were searched against TCMSP, TCMID, ETCM, and related literature, and the main targets of the chemical components were retrieved from TargetNet and SwissTargetPrediction. The main targets of RA and pain were searched against GeneCards, Online Mendelian Inheritance in Man(OMIM), and Therapeutic Target Database(TTD). Venny 2.1.0 was used to obtain the common targets shared by Strychni Semen, RA, and pain, and STRING and Cytoscape 3.6.1 were used to build the protein-protein interaction network. Then, molecular docking was carried out in AutoDock Vina. Finally, the rat model of type Ⅱ collagen-induced arthritis(CIA) was established. The up-down method and acetone method were employed to examine the mechanical pain threshold and cold pain threshold of rats, and the pain-relieving effect of SA on CIA rats was evaluated comprehensively. Hematoxylin-eosin(HE) staining was employed to evaluate the histopathological changes of joints in CIA rats. The expression levels of key target proteins was determined by immunohistochemistry and Western blot, and the mRNA levels of key targets were determined by real-time fluorescence quantitative polymerase chain reaction(real-time PCR). The results of network prediction showed that Strychni Semen may act on the TLR4/TNF-α/MMP-9 signaling pathway to exert the pain-relieving effect. The results of molecular docking showed that brucine, the main active component of SA, had strong binding ability to TLR4, TNF-α, and MMP-9. The results of animal experiments showed that SA improved the mechanical and cold pain sensitivity(P<0.05, P<0.01) and reduced the joint histopathological score of CIA rats(P<0.01). In addition, medium and high doses of SA down-regulated the protein and mRNA levels of TNF-α, TLR4, and MMP-9(P<0.05,P<0.01). In conclusion, SA alleviated the mechanical pain sensitivity, cold pain sensitivity, and joint histopathological changes in CIA rats by inhibiting the over activation of TLR4/TNF-α/MMP-9 signaling pathway.

[Mechanism of bulleyaconitine A in inhibiting bone destruction of rheumatoid arthritis via Src/PI3K/Akt signaling pathway].

Based on the sarcoma receptor coactivator(Src)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt) signaling pathway, the mechanism of action of bulleyaconitine A in the treatment of bone destruction of experimental rheumatoid arthritis(RA) was explored. Firstly, key targets of RA bone destruction were collected through GeneCards, PharmGKB, and OMIM databa-ses. Potential targets of bulleyaconitine A were collected using SwissTargetPrediction and PharmMapper databases. Next, intersection targets were obtained by the Venny 2.1.0 platform. Protein-protein interaction(PPI) network and topology analysis were managed by utilizing the STRING database and Cytoscape 3.8.0. Then, Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were conducted in the DAVID database. AutoDock Vina was applied to predict the molecular docking and binding ability of bulleyaconitine A with key targets. Finally, a receptor activator of nuclear factor-κB(RANKL)-induced osteoclast differentiation model was established in vitro. Quantitative real-time polymerase chain reaction(qRT-PCR) was used to detect the mRNA expression levels of related targets, and immunofluorescence and Western blot were adopted to detect the protein expression level of key targets. It displayed that there was a total of 29 drug-disease targets, and Src was the core target of bulleyaconitine A in anti-RA bone destruction. Furthermore, KEGG enrichment analysis revealed that bulleyaconitine A may exert an anti-RA bone destruction effect by regulating the Src/PI3K/Akt signaling pathway. The molecular docking results showed that bulleyaconitine A had better bin-ding ability with Src, phosphatidylinositol-4,5-diphosphate 3-kinase(PIK3CA), and Akt1. The result of the experiment indicated that bulleyaconitine A not only dose-dependently inhibited the mRNA expression levels of osteoclast differentiation-related genes cathepsin K(CTSK) and matrix metalloproteinase-9(MMP-9)(P<0.01), but also significantly reduced the expression of p-c-Src, PI3K, as well as p-Akt in vitro osteoclasts(P<0.01). In summary, bulleyaconitine A may inhibit RA bone destruction by regulating the Src/PI3K/Akt signaling pathway. This study provides experimental support for the treatment of RA bone destruction with bulleyaconitine A and lays a foundation for the clinical application of bulleyaconitine A.

Reactive oxygen species-mediated oxidative stress accelerates glycolysis via activation of the CaMKKß/AMPK pathway in the yak longissimus dorsi postmortem.

BACKGROUND: Adenosine monophosphate-activated protein kinase (AMPK) is instrumental in the initiation of early postmortem glycolysis and the advent of pale, soft, and exudative (PSE) meat when cellular energy is altered. However, conflicting studies show that AMPK activation without corresponding energy level changes in PSE meat challenges this long-held notion. Here, we examined the effects of reactive oxygen species (ROS)-mediated oxidative stress on AMPK activation in the context of glycolysis, protein solubility, and water-holding capacity (WHC) in the postmortem yak longissimus dorsi (LD) muscle. Further, we explored the mechanisms underlying these effects. RESULTS: Hydrogen peroxide (H2 O2 ) significantly augmented the degree of oxidative stress, increasing the production of ROS and malondialdehyde excessive production and reducing the activity of the anti-oxidants superoxide dismutase and glutathione peroxidase. In turn, oxidative stress dramatically promoted AMPK activation and glycolysis by increasing glycogen depletion and promoting hexokinase and phosphofructokinase activity. Subsequently, lactic acid accumulation increased, leading to a rapid decline in pH, which aggravated protein solubility degree and centrifugal loss in the early postmortem yak LD muscle. Importantly, these changes caused by oxidative stress were eliminated by the AMPK inhibitor. Mechanistically, oxidative stress elevated calcium ion (Ca2+ ) levels, which mobilized calcium/calmodulin-dependent protein kinase ß (CaMKKß) and AMPK. Rescue experiments confirmed that the increases were attenuated using Ca2+ and CaMKKß chelators, respectively. CONCLUSION: These results indicated that oxidative stress caused by ROS hastened early-stage postmortem glycolysis and reduced the WHC of yak meat. These effects were likely mediated by the alternative and energy-independent CaMKKß/AMPK signaling pathway. © 2022 Society of Chemical Industry.

[Mechanism of artesunate on bone destruction in experimental rheumatoid arthritis based on transcriptomics and network pharmacology].

The present study investigated the mechanism of artesunate in the treatment of bone destruction in experimental rheumatoid arthritis(RA) based on transcriptomics and network pharmacology. The transcriptome sequencing data of artesunate in the inhibition of osteoclast differentiation were analyzed to obtain differentially expressed genes(DEGs). GraphPad Prism 8 software was used to plot volcano maps and heat maps were plotted through the website of bioinformatics. GeneCards and OMIM were used to collect information on key targets of bone destruction in RA. The DEGs of artesunate in inhibiting osteoclast differentiation and key target genes of bone destruction in RA were intersected by the Venny 2.1.0 platform, and the intersection target genes were analyzed by Gene Ontology(GO)/Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment. Finally, the receptor activator of nuclear factor-κB(RANKL)-induced osteoclast differentiation model and collagen-induced arthritis(CIA) model were established. Quantitative real time polymerase chain reaction(q-PCR), immunofluorescence, and immunohistochemistry were used to verify the pharmacological effect and molecular mechanism of artesunate in the treatment of bone destruction in RA. In this study, the RANKL-induced osteoclast differentiation model in vitro was established and intervened with artesunate, and transcriptome sequencing data were analyzed to obtain 744 DEGs of artesunate in inhibiting osteoclast differentiation. A total of 1 291 major target genes of bone destruction in RA were obtained from GeneCards and OMIM. The target genes of artesunate in inhibiting osteoclast differentiation and the target genes of bone destruction in RA were intersected to obtain 61 target genes of artesunate against bone destruction in RA. The intersected target genes were analyzed by GO/KEGG enrichment. According to the results previously reported, the cytokine-cytokine receptor interaction signaling pathway was selected for experimental verification. Artesunate intervention in the RANKL-induced osteoclast differentiation model showed that artesunate inhibited CC chemokine receptor 3(CCR3), CC chemokine receptor 1(CCR1) and leukemia inhibitory factor(LIF) mRNA expression in osteoclasts in a dose-dependent manner compared with the RANKL-induced group. Meanwhile, the results of immunofluorescence and immunohistochemistry showed that artesunate could dose-dependently reduce the expression of CCR3 in osteoclasts and joint tissues of the CIA rat model in vitro. This study indicated that artesunate regulated the CCR3 in the cytokine-cytokine receptor interaction signaling pathway in the treatment of bone destruction in RA and provided a new target gene for the treatment of bone destruction in RA.

[Anti-rheumatoid arthritis mechanism of Sophorae Tonkinesis Radix et Rhizoma based on network pharmacology and experimental verification].

Based on the network pharmacology, molecular docking, and animal experiment, this study explored the anti-rheumatoid arthritis(RA) mechanism of Sophorae Tonkinesis Radix et Rhizoma(STRR). The active components of STRR were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), Traditional Chinese Medicine Integrative Database(TCMID), and previous research, main targets of STRR from TCMSP and SwissTargetPrediction, and targets of RA from GeneCards, DrugBank, Online Mendelian Inheritance in Man(OMIM), and Therapeutic Target Database(TTD). The common targets of the two were screened by Venny 2.1.0. Cytoscape 3.6.0 was used to generate the "component-target" network, and STRING and Cytoscape were used to construct the protein-protein interaction(PPI) network. DAVID 6.8 was employed for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and AutoDock Vina for molecular docking. Finally, collagen-induced rheumatoid arthritis(CIA) mouse model was constructed, and the expression of core target proteins was detected by Western blot. A total of 27 active components, including quercetin, genistein, kaempferol, subprogenin C, and daidzein, and 154 anti-RA targets, such as signal transducer and activator of transcription 3(STAT3), tumor necrosis factor(TNF), mitogen-activated protein kinase 1(MAPK1), AP-1 transcription factor subunit(JUN), and interleukin 6(IL6), of STRR were screened out. It was preliminarily indicated that STRR may regulate phosphatidylinositol-3-kinase-protein kinase B(PI3 K-AKT) signaling pathway and TNF signaling pathway to modulate the positive regulation of RNA polymerase Ⅱ promoter transcription, inflammatory response, and other biological processes, thus exerting the anti-RA effect. The results of molecular docking showed that the main active components in STRR had high binding affinity to the core targets. Animal experiment suggested that the water extract of STRR can significantly reduce the levels of p-STAT3, p-MAPK1, and TNF. This study demonstrated the multi-component, multi-target and multi-pathway synergistic effect of STRR in the treatment of RA, laying an experimental basis for clinical application of this medicine.

[Inhibitory effect of artesunate on bone destruction in rheumatoid arthritis: an exploration based on AhR/ARNT/NQO1 signaling pathway].

This study aimed to explore the effect of artesunate(ARS) on bone destruction in rheumatoid arthritis(RA) based on the aryl hydrocarbon receptor(AhR)/AhR nucleart ranslocator(ARNT)/NAD(P)H quinone dehydrogenase 1(NQO1) signaling pathway. Macrophage-colony stimulating factor(M-CSF) and receptor activator of nuclear factor-κB(RANKL) were used to induce the differentiation of primary bone marrow-derived mouse macrophages into osteoclasts. After intervention with ARS(0.2, 0.4, and 0.8 µmol·L~(-1)), the formation and differentiation of osteoclasts were observed by tartrate-resistant acid phosphatase(TRAP) and F-actin staining. The protein expression levels of AhR and NQO1 were detected by Western blot, and their distribution in osteoclasts was observed by immunofluorescence localization. Simultaneously, the collagen induced arthritis(CIA) rat model was established using type Ⅱ bovine collagen emulsion and then treated with ARS(7.5, 15, and 30 mg·kg~(-1)) by gavage for 30 days. Following the observation of spinal cord and bone destruction in CIA rats by Masson staining, the expression of AhR and ARNT in rat knee joint tissue was measured by immunohistochemistry and the NQO1 protein expression in the knee joint tissue by Western blot. The results showed that a large number of TRAP-positive cells were present in RANKL-induced rats. Compared with the RANKL-induced group, ARS(0.2, 0.4, and 0.8 µmol·L~(-1)) inhibited the number of TRAP-positive cells in a dose-dependent manner. F-actin staining results showed that the inhibition of F-actin formation was enhanced with the increase in ARS dose. As revealed by Western blot and immunofluorescence assay, ARS significantly promoted the expression of AhR and its transfer to the nucleus, thereby activating the protein expression of downstream ARNT and antioxidant enzyme NQO1. At the same time, the CIA rat model was successfully established. Masson staining revealed serious joint destruction in the model group, manifested by the failed staining of surface cartilage, disordered arrangement of collagen fibers, and unclear boundaries of cartilage and bone. The positive drug and ARS at different doses all improved cartilage and bone destruction to varying degrees, with the best efficacy detected in the high-dose ARS group. According to immunohistochemistry, ARS promoted AhR and ARNT protein expression in knee cartilage and bone of CIA rats and also NQO1 protein expression in rat knee and ankle joint tissues. In conclusion, ARS inhibited osteoclast differentiation by activating the AhR/ARNT/NQO1 signaling pathway, thus alleviating RA.

Characterization of an Acidic Polysaccharides from Carrot and Its Hepatoprotective Effect on Alcoholic Liver Injury in Mice.

The characteristics of acidic polysaccharides extracted from Daucus carota L. var. sativa Hoffm were investigated and its hepatoprotective effects on alcoholic liver injury were determined in the mice model. A carrot polysaccharide (CPS-I: Carrot polysaccharide-I) with the molecular weight of 3.40×104  kDa was isolated from Daucus carota L. and purified by diethylaminoethyl-52 and Sephadex G-150 column chromatography. The components were analyzed by HPLC, which revealed that CPS-I consisted of galacturonic acid, rhamnose, xylose, arabinose, fructose, and galactose at a relative ratio of 1 : 3.16 : 1.13 : 5.53 : 3.45 : 7.76. Structural characterization analysis suggested that CPS-I was mainly composed of →6)-ß-D-Galp-(1→ and →5)-α-L-Araf-(1→. The hepatoprotective effect of CPS-I was evaluated by alcoholic liver injury mice model. The results showed that the administration of CPS-I (300 mg/kg/day) alleviated the alcoholic liver injury in mice by increasing the levels of ADH and ALDH and reducing oxidative stress. CPS-I ameliorated the pathological changes of liver characterized by lipid accumulation, and reduced the number of lipid droplets.

[Effect of neutrophil extracellular traps on ischemic stroke and intervention of traditional Chinese medicine].

Ischemic stroke is the leading cause of death and disability in adults in China. Recent studies have shown that neutrophil extracellular traps play a crucial role in occurrence and development of ischemic stroke. This paper reviewed the literatures on NETs since the discovery of NETs more than a decade ago, and summarized the composition of NETs, the effects of NETs on stroke, the intervention targets of NETs, and the effects of traditional Chinese medicine on NETs. NETs are an important cause of brain injury after stroke. Platelets, peptidylarginine deiminase 4, reactive oxygen species and histones are the targets to regulate NET formation in stroke. There are few researches on traditional Chinese medicine targeting NETs for stroke. Studies on the intervention of traditional Chinese medicine mainly target on neutrophils, which are the main components of NETs, and platelets, which induce the formation of NETs. The paper provided a comprehensive overview of current studies of NETs in ischemic stroke, so as to provide new ideas for the treatment and drug development of ischemic stroke.

[Visual analysis of research progress of Sophorae Tonkinensis Radix et Rhizoma based on CiteSpace].

To analyze the study advance of Sophorae Tonkinensis Radix et Rhizoma, this study utilized CiteSpace 5.6.R5 software to conduct bibliometrics analysis on the Chinese literatures of Sophorae Tonkinensis Radix et Rhizoma from 1990 to 2020 included in the CNKI database retrieval platform. The analysis contents involved the number of published papers, co-authors, cooperative institutions, emergence, co-occurrence and clustering of keywords. A total of 808 Chinese literatures were included in the study, of which 17 were published by SUN Rong, the author with the most published papers, and formed a research team centered on SUN Rong; the analysis of the cooperation of publishing institutions showed that the Drug Safety Evaluation Research Center, Shanghai University of Traditional Chinese Medicine was the organization with the largest number of publications, with a total of 29 articles. It also formed a scientific research coorperation institution with Shandong Academy of Traditional Chinese Medicine as the core, and formed a relatively close cooperative network relationship. The analysis of literature keywords showed that the research direction was concentrated on the traditional Chinese medicine of Sophorae Tonkinensis Radix et Rhizoma, pharmacological mechanism, and side effects, active ingredients, etc. Among them, the research on the efficacy and toxicity of the active ingredients of Sophorae Tonkinensis Radix et Rhizoma has become a hot trend.

[Knowledge map analysis of research progress of Strychni Semen based on CiteSpace].

To analyze the study advance of Strychni Semen, a kind of traditional Chinese medicine, this study systematically retrieved the related Chinese literatures about Strychni Semen from CNKI database platforms and the core database of Web of Science, and used bibliometrics and CiteSpace 5.6.R5 software to visually display the authors, research institutions, keywords and other contents. A total of 1 895 Chinese literatures and 1 599 English literatures were included in the study. The analysis of Chinese and English literature authors showed that CAI Bao-chang and CHEN Jun had the most publications on Strychni Semen, and CAI Bao-chang's team was the core research team. According to the analysis of publishing institutions, Nanjing University of Traditional Chinese Medicine and Chinese Academy of Science were the research institutions with the largest number of Chinese and English literatures, respectively. But there was less cooperation between Chinese and English study institutions. The analysis of keywords in Chinese and English literatures showed that the research contents of Strychni Semen mainly focused on component analysis, research methods, receptor targets, clinical application, synergistic and attenuation measures. Break analysis showed that the apoptosis induced by Strychni Semen was a hot research topic, and research on components, toxicity and pharmacokinetics will be the research hotspot in future. The research on Strychni Semen is still in the developing period. This study has provided reference for the rapid grasp of the research contents and the judgment of research hotspots.

[Effect of Tripterygium Glycosides Tablets on reproductive toxicity in male rats with â ¡ type collagen induced arthritis].

The aim of this paper was to observe the toxic effect of Tripterygium Glycosides Tablets on the reproductive system of Ⅱ type collagen induced arthritis(CIA) male rats, and to explore the toxic mechanism preliminarily. Fifty SD rats were randomly divided into normal control group(Con), model group(CIA), Tripterygium Glycosides Tablets clinical equivalent dose groups of 1, 2, 4 times(9, 18, 36 mg·kg~(-1)), 10 rats in each group, and were given by gavage once a day for 42 days after the first immunization. The organ index of testis and epididymis were calculated on days 21 and 42. Histopathological and morphological changes of testis and epididymis were observed under optical microscope. Sperm count, sperm malformation rate and sperm kinetic parameters in epididymal tissues were observed by computer assisted sperm analysis(CASA). The concentration of testosterone(T), nitric oxide synthase(NOS) and aromatase(CYP19 A1) in serum were detected by ELISA. Immunohistochemistry was used to observe the expression of Bax and Bcl-2 related proteins in the apoptosis pathway of testis and epididymis. The results showed that, compared with Con group, CIA group significantly increased the rate of testicular spermatogenic tubule lesion and sperm malformation, decreased the average path speed, and no significant changes were observed in other groups. Tripterygium Glycosides Tablets at 4 times clinical equivalent dose can significantly reduce the testis index(P<0.01), each dose group can reduce the epididymis index(P<0.05). Each dose group of Tripterygium Glycosides Tablets could cause different degrees of damage to the testis and epididymis, the proportion of testicular histopathology lesions increased, the number of spermatogenic cells in the seminiferous tubules decreased, and so on. It could reduce the number of sperm, increase the rate of sperm deformity, make the parameters of sperm dynamics abnormal, and so on. Tripterygium Glycosides Tablets at 4 times dose could significantly reduce the content of serum sex hormone T and key enzyme of androgen synthesis(P<0.05 or P<0.01), but had no effect on CYP19 A1. The expression of Bax and Bcl-2 in testis and epididymis were increased by 2 and 4 times doses of Tripterygium Glycosides Tablets(P<0.05, P<0.01 or P<0.01). The results showed that 21 d administration of Tripterygium Glycosides Tablets at equal or higher doses could induce obvious toxic effect to the reproductive organs of CIA male rats, and lower the level of serum sex hormone T and the key enzyme of androgen synthesis, NOS. The mechanism of abnormal changes of Bax and Bcl-2 in Testis and epididymis is still to be elucidated.

[Overview of hepatotoxicity studies on Tripterygium wilfordii in recent 20 years].

Tripterygium wilfordii is widely used in the treatment of rheumatism with curative effect. However,its toxicity and adverse reactions,especially the hepatotoxicity,rank the first in the herbs induced liver injury,is the key factors hindering its clinical application. This paper reviewed the literatures related to the hepatotoxicity of T. wilfordii in recent 20 years,and summarized the characteristic of hepatotoxicity induced by T. wilfordii,the factors causing liver injury,the mechanism of toxicity,and the measures to reduce toxicity. In animal experiments,the T. wilfordii induced-hepatotoxicity in physiological state was more serious than pathological state. The T. wilfordii induced-hepatotoxicity is related to various toxic components contained in it,but alkaloids are the most toxic one.Overdose and cumulative overdose are the lead causing of hepatotoxicity induced by T. wilfordii. The theory of oxidative stress is still an important mechanism of T. wilfordii induced-hepatotoxicity,and Nrf2,as a key regulatory enzyme of oxidative stress,has become an important target for drugs to against T. wilfordii induced-hepatotoxicity. Mitochondrial autophagy and liver hypersensitivity are new mechanisms of liver injury induced by T. wilfordii. The measures such as dosage control,drug compatibility and dosage form variations can help to reduce the hepatotoxicity induced by T. wilfordii. This paper clarified the current situation and shortcomings of safety research on T. wilfordii,so as to propose new research strategies and provide ideas for rational evaluation of safety and clinical safe drug use of T. wilfordii.