Pathological Mechanism of Diabetes Based on NF-κB Signaling Pathway and Traditional Chinese Medicine Intervention： A Review / 中国实验方剂学杂志

As people's living standards improve， the development trend of diabetes has gradually become severe. Diabetes is a chronic inflammatory disease associated with abnormal expression of nuclear factor-kappa B （NF-κB） in patients. NF-κB exists in various tissue cells and participates in the regulation of a variety of genes related to immune function and inflammation. Varieties of factors can activate NF-κB when the body is stimulated by external factors， so as to produce inflammation and other reactions. Previous studies on NF-κB mainly focus on cancer， and the pathological mechanism of the treatment of diabetes by related signaling pathways and the progress of traditional Chinese medicine （TCM） treatment have not been systematically elaborated on. By referring to the relevant literature in China and abroad， it was found that NF-κB is not isolated in the development and progression of diabetes but is associated with signal molecules related to inflammation， oxidative stress， and energy metabolism， and it is involved in mediating inflammation， pancreatic β cell apoptosis， insulin signal transduction， and other physiological functions. Therefore， blocking the transmission of NF-κB signaling pathway is beneficial to the treatment of diabetes. At present， Western medicine for the treatment of diabetes mainly includes oral hypoglycemic drugs and insulin injections， but the adverse reactions are obvious. TCM has been characterized by multi-target， extensive action， and excellent curative effects in the treatment of diabetes. TCM and its compounds with functions of tonifying Qi and promoting blood circulation， regulating qi and eliminating phlegm， clearing heat and detoxifying， and nourishing Yin and moistening dryness can effectively intervene in the abnormal expression of NF-κB signaling pathway in vivo through anti-inflammatory effects. In this paper， the association between NF-κB signaling pathway and diabetes was summarized， and the modern research progress of TCM intervention of NF-κB signaling pathway in the treatment of diabetes in the past five years was reviewed， so as to lay a laboratory foundation for the study of a new pathological mechanism of diabetes based on NF-κB signaling pathway and provide new targets and research direction for the prevention and treatment of diabetes and development of related TCM.

Effect of Zuogui Jiangtang Tongmai Prescription on Inflammatory Injury of Human Umbilical Vein Endothelial Cells Induced by High Glucose and LPS Based on GPR43/β-arrestin-2/IκBα/NF-κB Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effects and mechanism of Zuogui Jiangtang Tongmai prescription （ZJTP） on human umbilical vein endothelial cells （HUVECs） damaged by high glucose combined with lipopolysaccharide （LPS）. MethodThe survival rate of cells was determined by cell counting kit-8 （CCK-8）， and the level of tumor necrosis factor-α （TNF-α） was determined by enzyme-linked immunosorbent assay （ELISA） to determine the optimal injury concentration and action time of LPS， as well as the optimal action concentration of ZJTP drug-containing serum. HUVECs were divided into a blank control group， a model group， a ZJTP drug-containing serum group， and an SCFA mixed liquid group. ELISA was used to detect the level of endothelin-1 （ET-1）， nitric oxide （NO）， interleukin-1β （IL-1β）， interleukin-6 （IL-6）， and TNF-α. Western blot was performed to detect the protein expression of G protein-coupled receptor43 （GPR43）， β-suppressor protein-2 （β-arrestin-2）， nuclear factor-κB suppressor α （IκBα）， and nuclear factor κB p65 （NF-κB p65）. The nucleation of NF-κB p65 was observed by immunofluorescence staining （IF）. The role of GPR43 in the regulation of inflammatory injury was observed by means of small interfering ribonucleic acid （siRNA）. The cells after intervention were divided into an empty carrier group， a ZJTP drug-containing serum group， a Si-GPR43 group， and a Si-GPR43 + ZJTP drug-containing serum group. The content of IL-1β， IL-6， and TNF-α was detected by ELISA. The protein expression of pathways was detected by Western blot. IF was used to observe the nucleation of NF-κB p65. ResultThe optimal molding condition was 1 mg·L-1 LPS for 24 h. The optimal drug intervention condition was 5% ZJTP drug-containing serum for 24 h. Compared with the blank control group， the content of ET-1 in the model group was significantly increased， and the content of NO was significantly decreased （P<0.01）. The levels of inflammatory factors were significantly increased （P<0.01）. The expressions of GPR43 and IκBα were significantly decreased， while the protein expressions of β-arrestin-2 and NF-κB p65 were significantly increased （P<0.01）. NF-κB p65 protein was transferred from the extranuclear to the intranuclear （P<0.01）. Compared with the model group， the content of ET-1 in the ZJTP drug-containing serum group was decreased， and the content of NO was increased （P<0.05）. The levels of inflammatory factors decreased （P<0.05）. The protein expressions of GPR43 and IκBα were increased， while the expressions of β-arrestin-2 and NF-κB p65 were decreased （P<0.05）. The amount of NF-κB p65 transferred from the intranuclear to the extranuclear decreased （P<0.01）. The mechanism study showed that compared with the Si-GPR43 group， the content of IL-1β， IL-6， and TNF-α were significantly decreased after treatment with ZJTP drug-containing serum （P<0.01）. The protein expressions of GPR43 and IκBα were significantly increased （P<0.01）， while the protein expressions of β-arrestin-2 and NF-κB p65 were significantly decreased （P<0.01）. The amount of NF-κB p65 transferred from the extranuclear to the intranuclear decreased （P<0.01）. ConclusionZJTP has a protective effect on HUVECs with high glucose and LPS-induced inflammatory injury， which may be related to the regulation of GPR43/β-arrestin-2/IκBα/NF-κB pathway.

Expression of TUBB4B in mouse primary spermatocyte GC-2 cells and its regulatory effect on NF-κB and MAPK signaling pathway / 南方医科大学学报

OBJECTIVE@#To explore the interaction between Tubulin beta 4B class IVb (TUBB4B) and Agtpbp1/cytosolic carboxypeptidase- like1 (CCP1) in mouse primary spermatocytes (GC-2 cells) and the role of TUBB4B in regulating the development of GC-2 cells.@*METHODS@#Lentiviral vectors were used to infect GC-2 cells to construct TUBB4B knockdown and negative control (NC-KD) cells. The stable cell lines with TUBB4B overexpression (Tubb4b-OE) and the negative control (NC-OE) cells were screened using purinomycin. RT-qPCR and Western blotting were used to verify successful cell modeling and explore the relationship between TUBB4B and CCP1 expressions in GC-2 cells. The effects of TUBB4B silencing and overexpression on the proliferation and cell cycle of GC-2 cells were evaluated using CCK8 assay and flow cytometry. The signaling pathway proteins showing significant changes in response to TUBB4B silencing or overexpression were identified using Western blotting and immunofluorescence assay and then labeled for verification at the cellular level.@*RESULTS@#Both TUBB4B silencing and overexpression in GC-2 cells caused consistent changes in the mRNA and protein expressions of CCP1 (P < 0.05). Similarly, TUBB4B expression also showed consistent changes at the mRNA and protein after CCP1 knockdown and restoration (P < 0.05). TUBB4B knockdown and overexpression had no significant effect on proliferation rate or cell cycle of GC-2 cells, but caused significant changes in the key proteins of the nuclear factor kappa-B (NF-κB) signaling pathway (p65 and p-p65) and the mitogen-activated protein kinase (MAPK) signaling pathway (ErK1/2 and p-Erk1/2) (P < 0.05); CCP1 knockdown induced significant changes in PolyE expression in GC-2 cells (P < 0.05).@*CONCLUSIONS@#TUBB4B and CCP1 interact via a mutual positive regulation mechanism in GC-2 cells. CCP-1 can deglutamize TUBB4B, and the latter is involved in the regulation of NF-κB and MAPK signaling pathways in primary spermatocytes.

Activity and mechanism of palbinone against hepatic fibrosis and inflammation / 药学学报

The study aims to investigate the anti-hepatic fibrosis and anti-inflammatory activities of palbinone, and to explore the internal regulatory mechanism, so as to lay an active foundation for its development as an anti-non-alcoholic steatohepatitis (NASH) candidate. First, sulforhodamine B (SRB) method was used to detect the effect of palbinone on the proliferation of human hepatic stellate cells LX-2 and rat hepatic stellate cells HSC-T6. Following, in the in vitro hepatic fibrosis cell model that activated by transforming growth factor beta 1 (TGF-β1), quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the inhibitory effect of different concentrations of palbinone on the transcription level and protein expression level of hepatic fibrosis markers. And the regulating mechanism of palbinone on fibrosis-related genes was analyzed at the same time. In addition, in the inflammatory cell model that induced by lipopolysaccharide (LPS) and nigericin, ELISA was used to detect the effect of palbinone on the released interleukin-1β (IL-1β) level. At the same time, Western blot was used to detect the effect of palbinone on the related proteins of inflammatory pathway. The results showed that palbinone could significantly inhibit the proliferation activity of LX-2 and HSC-T6, and their half maximal inhibitory concentration (IC50) values ​​were (375.11 ± 55.45) and (260.27 ± 36.81) nmol·L-1, respectively. In addition, palbinone showed a dose-dependent inhibitory effect on the expression levels of TGF-β1-induced fibrosis-related genes, including collagen type Ⅰ α 1 (COL1A1), TGF-β1, α-smooth muscle actin (α-SMA) and tissue inhibitor of metalloproteinase 1 (TIMP1). Mechanism study showed that palbinone may decrease the expression level of Yes-associated protein (YAP), thereby weakening its activation effect on the downstream fibrosis pathway. In addition, palbinone also exerted an anti-inflammatory effect by inhibiting the activity of nuclear factor kappa-B (NF-κB) signaling pathway and reducing inflammatory factors cysteinyl aspartate specific proteinase-1 (caspase-1) and IL-1β release. In conclusion, palbinone can not only inhibit the proliferation and activation of hepatic stellate cells by inhibiting the expression of YAP, but also inhibit the expression and release of inflammatory factors at the same time. All these studies provide theoretical support for the development of palbinone as an anti-nonalcoholic steatohepatitis drug.

Network Pharmacology and in vitro Experimental Verification on Intervention of Quercetin, Present in Chinese Medicine Yishen Qutong Granules, on Esophageal Cancer / 中国结合医学杂志

OBJECTIVE@#To explore the potential mechanism of Yishen Qutong Granules (YSQTG) for the treatment of esophageal cancer using network pharmacology and experimental research.@*METHODS@#The effective components and molecular mechanism of YSQTG in treating esophageal cancer were expounded based on network pharmacology and molecular docking. The key compound was identified by high-performance liquid chromatography and mass spectrometry (HPLC-MS) to verify the malignant phenotype of the key compounds in the treatment of esophageal cancer. Then, the interaction proteins of key compounds were screened by pull-down assay combined with mass spectrometry. RNA-seq was used to screen the differential genes in the treatment of esophageal cancer by key compounds, and the potential mechanism of key compounds on the main therapeutic targets was verified.@*RESULTS@#Totally 76 effective compounds of YSQTG were found, as well as 309 related targets, and 102 drug and disease interaction targets. The drug-compound-target network of YSQTG was constructed, suggesting that quercetin, luteolin, wogonin, kaempferol and baicalein may be the most important compounds, while quercetin had higher degree value and degree centrality, which might be the key compound in YSQTG. The HPLC-MS results also showed the stable presence of quercetin in YSQTG. By establishing a protein interaction network, the main therapeutic targets of YSQTG in treating esophageal cancer were Jun proto-oncogene, interleukin-6, tumor necrosis factor, and RELA proto-oncogene. The results of cell function experiments in vitro showed that quercetin could inhibit proliferation, invasion, and clonal formation of esophageal carcinoma cells. Quercetin mainly affected the biological processes of esophageal cancer cells, such as proliferation, cell cycle, and cell metastasis. A total of 357 quercetin interacting proteins were screened, and 531 genes were significantly changed. Further pathway enrichment analysis showed that quercetin mainly affects the metabolic pathway, MAPK signaling pathway, and nuclear factor kappa B (NF- κ B) signaling pathway, etc. Quercetin, the key compound of YSQTG, had stronger binding activity by molecular docking. Pull-down assay confirmed that NF- κ B was a quercetin-specific interaction protein, and quercetin could significantly reduce the protein level of NF- κ B, the main therapeutic target.@*CONCLUSION@#YSQTG can be multi-component, multi-target, multi-channel treatment of esophageal cancer, it is a potential drug for the treatment of esophageal cancer.

Traditional Chinese Medicine Intervention in Colorectal Cancer Based on NF-κB Signaling Pathway： A Review / 中国实验方剂学杂志

Colorectal cancer （CRC） is one of the leading causes of cancer-related deaths worldwide， with increasing incidence and mortality rates. The disease often develops covertly and lacks specific symptoms in its early stages， leading to late-stage diagnoses in most patients. It has become a prominent research topic in the field of digestive system tumors. The exact mechanisms underlying CRC are not yet clear and involve factors such as genetics， gene mutations， inflammatory responses， and aberrant activation of tumor-related signaling pathways. Nuclear factor-kappa B （NF-κB） is a crucial transcription factor that participates in various biological processes， including inflammatory responses， immune responses， cell proliferation， and apoptosis. Research suggests that NF-κB， serving as a molecular link between inflammation and cancer， is highly expressed in CRC. It promotes the occurrence and development of CRC by regulating the activity of target genes such as cell pro-inflammatory factors， chemokines， angiogenic factors， metastasis factors， and anti-apoptotic proteins. Currently， common treatments for CRC include surgery， radiation therapy， and chemotherapy drugs like 5-fluorouracil. However， these treatments have limitations such as significant adverse reactions， high metastasis rates， and the development of drug resistance. Therefore， the search for effective， low-adverse-reaction drugs to replace or supplement current treatments is essential. In recent years， traditional Chinese medicine （TCM） has shown some effectiveness in preventing and treating CRC. TCM has been found to inhibit the growth of CRC cells by modulating the NF-κB signaling pathway， playing a positive role in the occurrence and progression of CRC. Based on the asthenia in origin and sthenia in superficiality and deficiency-excess in complexity in CRC， this article summarized and analyzed the mechanisms and effects of TCM interventions targeting the NF-κB signaling pathway in CRC， and reviewed advances of 10 Chinese medicinal compound formulas and 37 Chinese medicinal monomer components of different types， including flavonoids， phenols， alkaloids， glycosides， and terpenoids with the effects of dispelling pathogenic factors， reinforcing healthy qi， and removing toxins in the prevention and treatment of CRC by targeting the NF-κB pathway. It is found that Chinese medicine can inhibit CRC cell proliferation， invasion， metastasis， angiogenesis， and inflammation by modulating the NF-κB signaling pathway， induce cell apoptosis， restore drug and radiation sensitivity， and counteract CRC. This article is expected to provide insights and references for the in-depth exploration and treatment of CRC mechanisms.

Geju Hugan Tablets Ameliorate Alcohol-induced Liver Injury in Mice by Regulating NF-κB and Bcl-2/Bax Signaling Pathways / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of Geju Hugan tablets on the liver of mice with alcohol-induced liver injury， and explore the underlying mechanism based on nuclear factor-κB p65 （NF-κB p65） and B-cell lymphoma-2 （Bcl-2）/Bcl-2-associated X protein （Bax） signaling pathways. MethodAccording to the body weight， 60 SPF-grade male ICR mice were randomized into normal， model， Compound Yiganling tablets （0.16 g·kg-1）， and low-， medium-， and high-dose （0.2， 0.4， 0.8 g·kg-1， respectively） Geju Hugan tablets groups. The drugs were administrated at the corresponding doses by gavage， and the normal and model groups with equal volume of pure water once a day for 28 consecutive days. On day 29， the mice in other groups except the normal group were administrated with liquor （53% Vol） by gavage twice a day at the doses of 20， 10 mL·kg-1 and with the interval of 6 h. Samples were harvested on day 30. The histopathological changes in the liver were observed by hematoxylin-eosin （HE） staining， and the ultrastructural changes in hepatocytes were observed by transmission electron microscopy. The enzyme-linked immunosorbent assay was employed to measure the levels of malonaldehyde （MDA）， reduced glutathione （GSH）， and triglycerides （TG） in the liver tissue and the levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in the serum. Western blotting was employed to determine the protein levels of NF-κB p65， phosphorylated p-inhibitor kappa B alpha （p-IκBα）， Bcl-2， and Bax in the liver tissue. ResultCompared with the normal group， the model group showed increases in the ALT， AST， MDA， and TG levels， a decrease in the GSH level， and increases in the liver injury scores evaluated based on the HE， oil red O， and transmission electron microscopy （P<0.01）. Moreover， the model group showed up-regulated expression of NF-κB， p-IκBα， and Bax （P<0.05， P<0.01） and down-regulated expression of Bcl-2 （P<0.05） in the liver tissue. Compared with the model group， Geju Hugan tablets of all the doses lowered the ALT， AST， MDA， and TG levels and elevated the GSH level （P<0.01）. The liver injury scores assessed based on HE staining and transmission electron microscopy in the medium- and high-dose Geju Hugan tablets groups were lower than those in the model group （P<0.01）. Compared with the model group， medium- and high-dose Geju Hugan tablets down-regulated the protein levels of NF-κB， p-IκBα， and Bax （P<0.01） and all doses of Geju Hugan tablets up-regulated the protein level of Bcl-2 （P<0.01）. ConclusionGeju Hugan tablets protect mice from alcohol-induced liver injury by down-regulating NF-κB signaling pathway to alleviate inflammation in the liver tissue and down-regulating the expression of Bax and up-regulating the expression of Bcl-2 to inhibit hepatocyte apoptosis.

The preventive effect and mechanism of Dayuanyin in hypoxic pulmonary hypertension through NF-κB signaling pathway / 药学学报

Dayuanyin (DYY) has been shown to reduce lung inflammation in both coronavirus disease 2019 (COVID-19) and lung injury. This experiment was designed to investigate the efficacy and mechanism of action of DYY against hypoxic pulmonary hypertension (HPH) and to evaluate the effect of DYY on the protection of lung function. Animal welfare and experimental procedures are approved and in accordance with the provision of the Animal Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Science. Male C57/BL6J mice were randomly divided into 4 groups: control group, model group, DYY group (800 mg·kg-1), and positive control sildenafil group (100 mg·kg-1). The animals were given control solvents or drugs by gavage three days in advance. On day 4, the animals in the model group, DYY group and sildenafil group were kept in a hypoxic chamber containing 10% ± 0.5% oxygen, and the animals in the control group were kept in a normal environment, and the control solvent or drugs continued to be given continuously for 14 days. The right ventricular systolic pressure, right ventricular hypertrophy index, organ indices and other metrics were measured in the experimental endpoints. Meantime, the expression levels of the inflammatory factors in mice lung tissues were measured. The potential therapeutic targets of DYY on pulmonary hypertension were predicted using network pharmacology, the expression of nuclear factor kappa B (NF-κB) signaling pathway-related proteins were measured by Western blot assay. It was found that DYY significantly reduced the right ventricular systolic pressure, attenuated lung injury and decreased the expression of inflammatory factors in mice. It can also inhibit hypoxia-induced activation of NF-κB signaling pathway. DYY has a protective effect on lung function, as demonstrated by DYY has good efficacy in HPH, and preventive administration can slow down the disease progression, and its mechanism may be related to inhibit the activation of NF-κB and signal transducer and activator of transcription 3 (STAT3) by DYY.

Mechanism of Alisma plantago-aquatica Linn. improving chronic glomerulonephritis based on network pharmacology and experimental verification / 药学学报

This study aims to explore the improvement and the mechanism of the Alisma plantago-aquatica Linn. (ApL) on chronic glomerulonephritis (CGN). All animal experiments were followed the regulation of the Experimental Animal Ethical Committee of Shanghai University of Traditional Chinese Medicine. CGN mouse model was established by a single tail-vein injection of doxorubicin (Dox) (20 mg·kg-1). One week after Dox administration, the mice received water extract of ApL (85 and 255 mg·kg-1) by gavage once a day for 14 days. At the end of experiment, the urine albumin-to-creatinine ratio (ACR), serum albumin (ALB), blood urea nitrogen (BUN) and serum creatinine (SCr) were detected, kidney histopathological H&E staining was analyzed. Active ingredients and action targets of ApL were collected from TCMSP database, and CGN-related targets were obtained from Genecards database. STRING platform was employed to perform protein-protein interaction (PPI), and Metascape platform was used for KEGG pathway and GO enrichment analysis. The results of experiments demonstrated that ApL (85 and 255 mg·kg-1) could reduce the ACR and the content of SCr and BUN, and increase the content of ALB in mice. Network pharmacology results predicted that nuclear factor kappa-B (NF-κB)-related pathway and biological process of oxidoreductase activity regulation may be involved in the ApL-provided amelioration on CGN. The verification results showed that ApL could inhibit the activation of NF-κB and the expression of inflammatory factors in mice, and reduce the activity of renal myeloperoxidase (MPO). Meanwhile, ApL promoted the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and increased the expression of its downstream gene mRNA, and reduced the level of renal malondialdehyde (MDA) and reactive oxygen species (ROS), and further elevated renal glutathione (GSH) level. Based on network pharmacology combined experiments, this study found that ApL may improve CGN in mice through multiple targets and multiple pathways, in which the inhibition of NF-κB signaling and the activation of Nrf2 signaling may be important mechanisms involved.

Mechanism of NF-κB Signaling Pathway in Regulating Lung Cancer and Intervention of Traditional Chinese Medicine： A Review / 中国实验方剂学杂志

Lung cancer is the leading cause of cancer-related deaths， and standard treatments for lung cancer， including surgery， radiotherapy， chemotherapy， targeted therapy， and immunotherapy， have shown significant clinical effects. However， current available treatment strategies are still unable to cure the disease. Since the majority of lung cancer patients are diagnosed at an advanced stage， surgical options are often lost， and the primary approach is typically a combination of radiotherapy and chemotherapy. However， the adverse reactions associated with these treatments limit their effectiveness and application， and the damage caused to normal tissues is often more severe than that inflicted on the tumor. Currently， traditional Chinese medicine （TCM） has been used as part of combination therapy for cancer treatment due to its unique system of syndrome differentiation， flexible compatibility， and safety and efficacy. TCM prescriptions and single drugs with multiple components and targets can simultaneously regulate multiple pathways. As reported， among numerous pathways involved in the regulation of lung cancer， the nuclear factor-κB （NF-κB） signaling pathway plays a key role in inducing cell transcription and is one of the main pathways involved in the occurrence and development of lung cancer. It can specifically regulate inflammatory responses， cell proliferation， apoptosis， invasion and metastasis， angiogenesis， and multidrug resistance in lung cancer. TCM prescriptions and single drugs can inhibit lung cancer cell proliferation， invasion， and metastasis， induce apoptosis and autophagy in lung cancer cells， suppress angiogenesis， regulate immune function， and treat multidrug resistance by regulating the NF-κB signaling pathway. Therefore， they play a role in intervening in lung cancer. However， there is currently a lack of systematic literature research that comprehensively summarizes and elucidates these aspects in China and abroad. Therefore， it is important to provide a systematic elucidation of the mechanism underlying the regulation of the NF-κB signaling pathway in lung cancer and review TCM interventions in lung cancer based on the NF-κB signaling pathway. This study is expected to provide references for the clinical application of lung cancer therapeutic drugs and the development of new drugs.

Effects of flurochloridone on apoptosis and Nrf2/HO-1 and NFκB signaling pathways in mouse testis and TM4 cells / 环境与职业医学

Background Flurochloridone (FLC) is toxic to male reproduction and can induce apoptosis of testicular tissue and supporting cells under oxidative stress. Of particular concern is whether nuclear factor-erythrocyte 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling pathway and nuclear factor kappa B (NFκB) signaling pathway participate this process. Objective To observe apoptosis of testicular tissue and sertoli TM4 cells and alterations of Nrf2/HO-1 and NFκB signaling pathways in mice treated with FLC in vivo/in vitro. Methods (1) Animal experiment. Testis samples were harvested from male C57BL/6 mice after 28-day FLC (0, 3, 15, 75, and 375 mg·kg−1 per day) exposure via oral route. Malondialdehyde (MDA) and superoxide dismutase (SOD) in homogenate of testicular tissue were measured by colorimetry. Apoptosis of testicular tissue was evaluated by TUNEL staining. Expression and distribution of Nrf2 and NFκB were detected by immunohistochemistry. Protein expression levels of Nrf2, HO-1, NAD(P)H: quinone oxidoreductase 1 (NQO1), NFκB, inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), and phosphorylated recombinant inhibitory subunit of nuclear factor kappa-B alpha (P-IκBα) in testicular tissue homogenate were determined by Western blotting. (2) Cell experiment. TM4 cell lines were treated with 40, 80, 120, 160, and 200 μmol·L−1 FLC for 6 h, and cell viability was detected by CCK-8. After 6 h exposure to 40, 80, and 160 μmol·L−1 FLC, the apoptosis rate was detected by flow cytometry, and the protein expression levels of Nrf2, HO-1, NQO1, NFκB, IKKβ, and IκBα were detected by Western blotting. Results (1) Animal experiment. Apoptosis occurred in the interstitial and basal parts of spermatogenic tubules in male C57BL/6 mice after 28 days of oral FLC exposure. Compared with the control group, the MDA level in testicular tissue of the 375 mg·kg−1 FLC-treated group was significantly increased (P<0.05), and the SOD activity was significantly decreased (P<0.05). After 375 mg·kg−1 FLC exposure, apoptosis occurred in the interstitial and basal parts of spermatogenic tubules. The results of immunohistochemistry showed the expression of Nrf2 and NFκB in the interstitium and basal part of spermatogenic tubules of the treated groups. Compared with the control group, the protein levels of Nrf2, NQO1, P-IκBα, NFκB, and IKKβ in the 15, 75, and 375 mg·kg-1 groups were significantly increased (P<0.001), and the HO-1 protein level was significantly increased in the 375 mg·kg−1 group (P<0.001). (2) Cell experiment. Compared with the control group, the TM4 cell viabilities in the 40, 80, 120, 160, and 200 μmol·L−1 FLC-treated groups significantly decreased (P<0.01). The apoptosis rates were significantly increased (P<0.05), and the apoptosis rates increased from 5.7% in the control group to 7.4%, 9.4%, and 11.7% in the 40, 80, and 160 μmol·L−1, respectively. The Nrf2 protein level in the 40 μmol·L−1 group was significantly increased (P<0.01), while the levels significantly decreased in the 80 and 160 μmol·L−1 groups (P<0.01). The HO-1 protein levels in the 40, 80, and 160 μmol·L−1 groups were significantly increased (P<0.01). The level of NQO1 protein in the 40 μmol·L−1 group was significantly increased (P<0.01). The NFκB protein levels were significantly increased in the 80 and 160 μmol·L−1 groups (P<0.001). The IκBα protein levels were significantly decreased in all treated groups (P<0.001). The IKKβ protein had no significant change. Conclusion FLC induces testicular tissue apoptosis, and the process affects Nrf2/HO-1 signaling pathway and NFκB signaling pathway. The in vitro study confirms that FLC could induce apoptosis of TM4 cells and activate Nrf2/HO-1 and NFκB signaling pathways.

Protective effect of luteolin on acute lung injury induced by extracorporeal circulation and its mechanism / 中国胸心血管外科临床杂志

@#Objective    To investigate the protective effect of atomized inhalation of nano-luteolin preparation on acute lung injury caused by extracorporeal circulation, and to explore the anti-inflammatory mechanism of luteolin, so as to provide study basis for clinical application. Methods    Thirty male SD rats aged 5-6 weeks and weighting 160-190 g, were randomly divided into a preoperative baseline (BL) group, arteriovenous partial diversion (ECC) group, luteolin atomization pretreatment for 1 h group, 2 h group, and 3 h group by random number method, with 6 rats in each group. In the BL group, lung tissue samples were collected directly without any treatment. The ECC group received mechanical ventilation, and the whole body was heparinized after the jugular arteriovenous intubation. The flow was transferred for 30 minutes, followed by observation for 60 minutes, then lung tissue samples were collected. Subjects in the 1 h, 2 h and 3 h groups were placed in a small animal atomizer 1 h, 2 h and 3 h before flow transfer respectively, and the subsequent operation was the same as that in the ECC group. The inflammatory level of lung tissue was detected to evaluate the degree of pathological injury of lung tissue. Western blotting (WB) was used to detect the contents of p65, IKKα, IKKβ and IKKγ in the cytoplasm of lung tissue samples of each group. Results    Compared with the ECC group, the levels of IL-6 and TNF-α in lung tissues and the degree of pathological injury in the 1 h, 2 h and 3 h groups decreased, and the difference between the 3 h group and the ECC group was statistically different (P<0.05). WB results showed that compared with the ECC group, the levels of p65 in lung tissue of the 1 h, 2 h and 3 h groups decreased; the levels of IKKβ in the lung tissue increased in the 1 h, 2 h and 3 h groups, and the difference of the 3 h group was statistically different from the ECC group (P<0.05). Conclusion    Luteolin has a protective effect on acute lung injury induced by ECC, and atomization 3 h in advance has the best protective effect on lung. The mechanism plays a protective role in ECC-induced acute lung injury, may be through inhibition of IKKβ phosphorylation, thereby inhibiting the classical NF-κB signaling pathway.

High mobility group box-1 inhibition reduces expression of matrix metalloproteinase-9 in astrocytes in rats after spinal cord injury / 中华创伤骨科杂志

Objective:To investigate the role and underlying mechanisms of inhibiting high mobility group box-1 (HMGB1) in the expression of matrix metalloproteinase-9 (MMP-9) in spinal cord astrocytes (AS) in rats after spinal cord injury (SCI).Methods:After an SCI model was established in Sprague-Dawley (SD) rats using a modified Allen's Weight-Dropping method and ethyl pyruvate (EP) or glycyrrhizin (GL) was used to inhibit the effect of HMGB1, the rats were divided into a sham group, an SCI group, an SCI+EP (50 mg/kg) group, and an SCI+GL (100 mg/kg) group. The expression levels of glial fibrillary acid protein (GFAP) and MMP-9 in spinal cord AS were observed. After the spinal cord AS in SD rats was cultured and incubated by the oxygen-glucose deprivation/reoxygenation (OGD/R) procedure, the expression of MMP-9 protein was detected at 6 h/R 6 h, 12 h, 24 h, and 48 h after OGD. The time point with the highest expression was chosen in the subsequent experiments as an OGD/R group. HMGB1 was inhibited by HMGB1 shRNA or EP to observe the effect of HMGB1 on the expression of MMP-9 protein in AS treated with OGD/R. Then, toll-like receptor 4 (TLR4) inhibitor, TIR-domain-containing adaptor inducing interferon- β (TRIF) inhibitor, and nuclear factor-kappa B (NF- κB) inhibitor were used to investigate the effects of TLR4/TRIF/NF- κB signaling pathway during the regulation of HMGB1 on MMP-9 in vitro. Results:Western blot showed that the expression of MMP-9 protein in the spinal cord was significantly increased in rats at 1 d after SCI, and the expression of MMP-9 protein in the SCI+EP group and the SCI+GL group was significantly lower than that in the SCI group ( P<0.001). Immunofluorescence showed that GFAP and MMP-9 proteins were co-localized in the spinal cord after SCI, and the expression of GFAP and MMP-9 proteins in the SCI+EP and SCI+GL groups was significantly lower than that in the SCI group ( P<0.05). Since the expression of MMP-9 protein in the spinal cord AS cultured in vitro was significantly higher in the OGD 6h/R 12h group than that in the normal group and the OGD 6h/R 6h, 24, and 48 h groups, the OGD 6h/R 12h was taken as the OGD/R group. The MMP-9 protein expression in AS in the OGD/R+HMGB1 shRNA group and the OGD/R+EP group was significantly lower than that in the OGD/R group ( P<0.001). In the cultured AS, moreover, inhibiting TLR4, TRIF, and NF- κB reduced MMP-9 protein expression after OGD 6 h/R 12 h when compared with that in the OGD/R group ( P<0.001). Conclusions:HMGB1 inhibition may result in a reduction in MMP-9 expression both in the spinal cord AS in SCI rats and in AS after OGD/R treatment in vitro. HMGB1 may regulate MMP-9 protein expression in AS after OGD/R treatment via the TLR4/TRIF/NF- κB signal pathway.

Duchenne muscular dystrophy: RANK/RANKL/OPG (receptor activator of nuclear factor-kB/RANK ligand/osteoprotegerin) system and glucocorticoids / Distrofia muscular de Duchenne: el sistema RANK/RANKL/OPG (receptor activador del factor nuclear-kB/ligando de RANK/osteoprotegerina) y glucocorticoides

Abstract Duchenne muscular dystrophy (DMD) is an X-linked inherited disorder. Patients present with decreased bone mineral density (BMD) due to glucocorticoid therapy and progressive muscle weakness. Bone remodeling allows bone volume and structure to be maintained and controlled by local and systemic factors. These include the receptor activator of the nuclear factor-kB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system, a determining pathway in the balance between bone formation and resorption. Disruptions in this complex, caused by factors such as glucocorticoids, can affect bone metabolism. The extensive action of the RANK/RANKL/OPG pathway suggests an influence on dystrophic muscle pathophysiology. This review aimed to highlight some aspects of the RANK/RANKL/OPG system, the effect of glucocorticoids on this pathway, and the pathophysiology of the patient with DMD.

Resumen La distrofia muscular de Duchenne (DMD) es un trastorno hereditario ligado al cromosoma X. Los pacientes presentan una disminución de la densidad mineral ósea (DMO) debido a los efectos adversos del tratamiento con glucocorticoides y a la debilidad muscular progresiva. El remodelado óseo permite mantener el volumen y la estructura ósea, proceso controlado por factores locales y sistémicos. Entre ellos destaca el sistema del receptor activador del factor nuclear-kB (RANK), su ligando natural RANKL (RANKL) y la osteoprotegerina (OPG), una vía determinante en el equilibrio entre la resorción y formación ósea. Las alteraciones en este complejo, originadas por factores como los glucocorticoides, pueden afectar el metabolismo óseo. La amplia acción de RANKL y OPG ha sugerido una influencia en la fisiopatología de la DMD. El objetivo de esta revisión fue destacar algunos aspectos del sistema RANK/RANKL/OPG, el efecto de los glucocorticoides en esta vía y la fisiopatología del paciente con DMD.

Progress of the Nod-like receptor family pyrin domain-containing protein 12 in autoinflammatory diseases / 中华实用儿科临床杂志

The Nod-like receptor family pyrin domain-containing protein 12(NLRP12), a newly discovered member of the Nod-like receptor family, is one of the important pattern recognition receptors.It recognizes a variety of pathogens, initiates downstream immune responses, and participates in the regulation of multiple inflammatory responses.NLRP12 is related to the occurrence and progression of inflammatory diseases.In this review, the structure and function of NLRP12 as well as NLRP12-associated autoinflammatory diseases were discussed, so as to provide new insights for the understanding, exploration and treatment of NLRP12-associated autoinflammatory diseases.

Rhamnus crenata leaf extracts exhibit anti-inflammatory activity via modulating the Nrf2/HO-1 and NF-κB/MAPK signaling pathways

Objective: To elucidate the potential anti-inflammatory mechanisms of Rhamnus crenata leaf extracts using RAW264.7 cells. Methods: We used 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay to measure cell viability. Nitric oxide (NO) production was measured using Griess reagent. Western blotting and RT-PCR assays were carried out for analyzing the protein and gene expressions of pro-inflammatory mediators, respectively. Moreover, PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), and BAY11-7082 (NF-κB inhibitor) were used to evaluate the anti-inflammatory mechanism of Rhamnus crenata leaf extract. Results: Rhamnus crenata leaf extracts significantly inhibited the production of the pro-inflammatory mediators such as NO, iNOS, COX-2, IL-1β, and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Rhamnus crenata leaf extracts also suppressed LPS-induced degradation of IκB-α and nuclear accumulation of p65, which resulted in the inhibition of NF-κB activation in RAW264.7 cells. Additionally, the extracts attenuated the phosphorylation of p38, ERK1/2, and JNK in LPS-stimulated RAW264.7 cells. Moreover, HO-1 expression induced by Rhamnus crenata leaf extracts was significantly downregulated by SB230580, PD98059, SP600125 and BAY11-7082. Conclusions: Rhamnus crenata leaf extract may upregulate HO-1 expression through inhibition of p38, ERK1/2, and NF-κB activation, which may contribute to the anti-inflammatory activity of the extracts. Rhamnus crenata leaf extracts may have great potential for the development of anti-inflammatory drugs to treat acute and chronic inflammatory diseases.

Diabetic nephropathy patients show hyper-responsiveness to N6-carboxymethyllysine

The aim of this study was to evaluate the impact of N6-carboxymethyllysine (CML) on NF-κB gene expression and tumor necrosis factor (TNF) production in diabetic nephropathy. This was an observational study comprised of three groups: diabetic nephropathy (n=30), type II diabetes mellitus (n=28), and healthy volunteers (n=30). Blood samples collected from the study participants were cultured for 24 h in the presence of CML or an appropriate control. After incubation, the cultures were centrifuged to separate the cells from the conditioned media. cDNA was prepared from the cell pellet and used to quantify NF-κB gene expression by quantitative real-time polymerase chain reaction (PCR). The conditioned media were used to measure TNF production by enzyme-linked immunosorbent assay (ELISA). The CML-induced fold change in NF-κB gene expression was significantly different among the study groups (P=5.4×10-5). Also, the CML-induced fold change in TNF levels was significantly different among the three groups (P=4.3×10-8). These results imply that patients with diabetic nephropathy and type II diabetes mellitus showed an elevated response to CML.

The study on inflammatory mechanism of cognitive dysfunction induced by 1-bromopropane in rats / 中华行为医学与脑科学杂志

Objective:To observe the role of neuroinflammation in cognitive dysfunction induced by 1-bromopropane (1-BP) in rats.Methods:Male Wistar rats were randomly divided into control group, 1-BP group, pyrrolidine dithiocarbamate(PDTC)+ 1-BP group and PDTC group, with 15 rats in each group. Rats in 1-BP group and PDTC+ 1-BP group were given 800 mg / kg 1-BP by gavage, and rats in control group and PDTC group were given equal volume corn oil once a day for 12 days; rats in PDTC group and PDTC+ 1-BP group were intraperitoneally injected with 100 mg / kg PDTC 30 minutes after gavage, while rats in control group and 1-BP group were injected with equal volume of normal saline once a day for 12 days.From the 7th to 12th day of the experiment, ten rats in each group were randomly selected and subjected to Morris water maze test for detect the cognitive function. In the positioning navigation test, the learning ability of rats was evaluated by the escape latency and total swimming distance respectively. In the space exploration experiment, the memory ability of experimental animals was evaluated by the number of times crossing the target platform. After the experiment, ten rats were sacrificed, the cerebral prefrontal cortex was harvested. The cytosolic and nuclear NF-κB expression and phosphorylation were detected by Western blot, the mRNA levels of TNF-α and IL-1β were detected by qRT-PCR. After cardiac perfusion fixation, the brains of 5 rats were taken to make frozen sections for immunohistochemical staining and Nissl staining. SPSS 20.0 software was used for statistical analysis, repetitive measurement deviation analysis was used for the analysis of the swimming distance and the escape latency in positioning navigation test, One-way ANOVA was used for the analysis of the number of times crossed the target platform in spatial probe test and other data. Tukey's test was used for Post hoc comparison.Results:The results of Morris water maze showed that there was significant interaction between group and training time in the total swimming distance of rats in the four groups ( F=3.762, P<0.05). Simple effect analysis showed that the total swimming distance of 1-BP group in 1-4 days were longer than those of control group (all P<0.05), while the total swimming distance of PDTC+ 1-BP group in 1-4 days were shorter than those of 1-BP group (all P<0.05). There was significant interaction between group and training time in the escape latency among the four groups ( F=6.541, P<0.01). The escape latencies of 1-BP group in 1-4 days were longer than those of control group (all P<0.05), while the escape latencies of PDTC+ 1-BP group in 1-4 days were shorter than those of 1-BP group (all P<0.05). The results of space exploration experiment showed that there was significant difference in the number of crossing the platform among the four groups ( F=75.333, P<0.01). The number of crossing the platform (1.08±0.29) in 1-BP group was lower than that in the control (3.35±0.05) ( P<0.01). The number of crossing the platform (1.95±0.26) in PDTC+ 1-BP group was higher than that in 1-BP group ( P<0.01). It had significant difference both in the cytoplasm and in the nucleus of the NF-κB protein level in prefrontal cortex among rats of the four groups ( F=20.865, 23.877, both P<0.01). The levels of NF-κB in cytoplasm and nucleus of rats in 1-BP group were both higher than those in control group (cytoplasm: (177.3±32.1)%, (100.0±8.4)%, P<0.01; nucleus: ( 173.2±27.1)%, (100.0±8.4)%, P<0.01). While the levels of NF-κB in cytoplasm and nucleus of 1-BP+ PDTC group were both lower than those of 1-BP group (cytoplasm: (148.7±22.0)%, (177.3±32.1)%, P<0.01; nucleus: (149.7±18.8)%, (173.2±27.1)%, P<0.01). The results of qRT-PCR showed that there were significant differences in the mRNA levels of TNF-α and IL-1β in the prefrontal cortex among the four groups ( F=17.464, 17.382, both P<0.01). The levels of TNF-α and IL-1β mRNA in 1-BP group were higher than those in control group (both P<0.05), and the levels of TNF-α and IL-1β mRNA in PDTC+ 1-BP group were both lower than those in 1-BP group (both P<0.05). The results of immunohistochemistry showed that compared with the control group, the number of microglia and astrocytes in the 1-BP group increased (microglia: (158.30±9.68), (110.20±16.30), P<0.05; astrocytes: (122.76±4.35), (80.24±6.96), P<0.05), and the morphology was also activated, with light staining and reduced number of Nissl bodies in neurons.The number of microglia and astrocytes in PDTC + 1-BP group was lower than that in 1-BP group (microglia: (131.70±14.67), (158.30±9.68), P<0.05; astrocytes: (101.54±4.55), (122.76±4.35), P<0.05), and the Nissl body staining of neurons was significantly deepened. Conclusion:NF-κB signaling pathway might be the key mechanism of 1-BP neurotoxicity. PDTC intervention could significantly improve the neuroinflammatory response and behavioral disorders of experimental animals intoxicated with 1-BP.

Therapeutic effect and mechanism of Erhuang quzhi granules on non-alcoholic fatty liver disease in mice / 药学学报

Erhuang quzhi compounds is one of the protecting liver and inhibiting toxin prescriptions series summarized by Jinqi Yuan and other famous doctors of traditional Chinese medicine during the long-term clinical practice. It is very effective for non-alcoholic fatty liver disease (NAFLD), but its mechanism is not clear. This research investigated mechanism of Erhuang quzhi granules (EQG) in the treatment of NAFLD. All the animal welfare and experimental procedures are in accordance with the regulations of the Animal Ethics Committee of the First Affiliated Hospital of Shihezi University. Mouse models of NAFLD were established by feeding with methionine and choline deficient diet (MCDD) for five weeks. While feeding MCDD, the treatment groups were given EQG (16.25 g·kg-1·d-1) and atorvastatin (ATO, 7.20 mg·kg-1·d-1) by gavage. The effects of EQG on serum biochemical indices, liver pathological changes, and inflammatory cytokines in mice of NAFLD were investigated. Quantitative real-time PCR (qPCR), immunocytochemistry (ICH) and Western blot assays were used to detect the levels of mRNA and protein associated with nuclear factor kappa B/Nod-like receptor protein 3 (NF-κB/NLRP3) in liver. The results showed that EQG significantly reduced the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and improved the level of low-density lipoprotein cholesterol (LDL-C). The result of hematoxylin-eosin (HE) staining showed that EQG reduced lipid deposition in livers of mice. Meanwhile, EQG notably decreased the levels of interleukin (IL)-1β, IL-6, IL-18 and tumor necrosis factor-α (TNF-α), and mRNA levels of NF-κB, NLRP3, IL-1β, TNF-α, down-regulated the expression of F4/80, IκB kinase β (IKKβ), NLRP3 and apoptosis-associated speck-like protein containing a CARD (ASC) and inhibited the activation of NF-κB and cysteinyl aspartate specific proteinase-1 (caspase-1). These findings announced that EQG could improve NAFLD via NF-κB/NLRP3 pathway possibly, which provides a theoretical basis for the further development and utilization of EQG in clinic.

Phillygenin Mitigates LPS/ATP-induced L02 Cell Inflammation by Regulating P2X7R/NF-κB/NLRP3 Inflammasome Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the effects of phillygenin （PHI） on the inflammation in L02 cells induced by lipopolysaccharide （LPS） and adenosine triphosphate （ATP） and the expression of purinergic 2X7 receptor （P2X7R）， NOD-like receptor family pyrin domain containing 3 （NLRP3）， and nuclear factor kappa B （NF-κB） expression. MethodIn this study， the inflammation model was induced in L02 cells by 100 μg·L-1 LPS treatment for 24 h and 5 mmoL·L-1 ATP treatment for 5 h. The cells in the PHI groups were cultured with PHI （100， 50， 25 mg·L-1） for 6 h in the LPS treatment period， followed by LPS treatment for another 18 h. After ATP treatment for 5 h， the mRNA and protein expression of interleukin-1β （IL-1β）， interleukin-18 （IL-18）， P2X7R， NLRP3， Caspase-1 precursor （pro-Caspase-1）， cleaved Caspase-1， NF-κB， and NF-κB inhibitor protein α （IκBα） in L02 cells was detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. Molecular docking was used to predict whether P2X7R could bind to PHI， and DCFH-DA was employed to detect the accumulation of reactive oxygen species （ROS） in cells. P2X7R was silenced by small interfering ribonucleic acid （siRNA）， and then the mRNA expression of IL-1β， IL-18， P2X7R， NLRP3， Caspase-1， NF-κB， and IκBα was detected by Real-time PCR. ResultReal-time PCR and Western blot showed that compared with the normal group， the model group showed increased expression of IL-1β and IL-18 （P<0.05）， and compared with the model group， the PHI groups showed down-regulated IL-1β， IL-18 mRNA and protein expression （P<0.05）. Molecular docking suggested a good binding effect of PHI to P2X7R. Real-time PCR and Western blot analysis showed that the expression of P2X7R in the model group was significantly up-regulated compared with that in the normal group （P<0.05）， and compared with the model group， the PHI groups showed down-regulated mRNA and protein expression of P2X7R （P<0.05）. DCFH-DA results showed that compared with the normal group， the model group showed increased content of ROS （P<0.05）， and compared with the model group， the PHI groups decreased the accumulation of ROS （P<0.05）. As demonstrated by Real-time PCR and Western blot， compared with the normal group， the model group showed increased expression of NLRP3 inflammasome and NF-κB （P<0.05）， and compared with the model group， the PHI groups significantly decreased the mRNA and protein expression of NLRP3 and cleaved Caspase-1， and up-regulated the mRNA and protein expression of NF-κB and IκBα （P<0.05）. Real-time PCR analysis showed that compared with the results in the model group， after silencing P2X7R by siRNA， the mRNA expression of IL-1β， IL-18， P2X7R， NLRP3， Caspase-1， NF-κB， and IκBα was decreased （P<0.05）. PHI exerted the same effect， and the mRNA expression was further reduced after the combination of them. ConclusionPHI is presumed to suppress the expression of the NLRP3/NF-κB signaling pathway by down-regulating upstream P2X7R to alleviate the LPS/ATP-induced inflammation in L02 cells， suggesting that P2X7R may be the target of PHI against inflammation.