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Drug repositioning provides new clinical indications for existing drugs. The imbalance between body's "immune-inflammation" regulation is one of the important factors in the occurrence and development of diabetic nephropathy (DN). Chinese patent medicine Kunxian capsule is clinically used for treating rheumatoid arthritis with satisfying immune-modulatory and anti-inflammatory actions. Notably, accumulating clinical evidence based on small cohorts had shown that Kunxian capsule may be used to treat DN. But the underlying pharmacological mechanisms remain unclear. Therefore, this study integrated "drug target-disease gene-biological pathway-function module" multi-level associated network analysis, and in vivo and in vitro experiments, to verify the pharmacological effects of Kunxian capsules in DN and to elucidate its molecular mechanisms. The experimental protocol was reviewed by the Laboratory Animal Welfare and Ethics Committee of China Academy of Chinese Medical Sciences, and it complies with the relevant regulations on laboratory animal welfare and ethics. As a result, the network analysis showed that the candidate targets of Kunxian capsule against DN were significantly involved into various functional modules which were related to modulation of immune-inflammation system, basement membrane lesion, abnormal hemorheology, energy metabolism and hormone metabolism, and the number of targets enriched by PI3K/AKT/NF-κB pathway is the largest. In addition, both in vivo and in vitro experiments demonstrated that Kunxian capsule by gavage effectively reduced blood glucose, improved insulin resistance, reduced blood lipid, inhibited renal extracellular matrix protein production and renal inflammation, improved renal function and pathological damages, and inhibited the activity of PI3K/AKT/NF-κB/TNF-α/IL-1β pathway in diabetic nephropathy rats. Collectively, these findings suggest the therapeutic potentials of Kunxian capsule to alleviate DN by regulating the imbalance of immune-inflammation system.
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OBJECTIVE To study the protective effect and mechanism of Shiyifang medicinal wine （SYF） on knee osteoarthritis（KOA）of rabbit induced by papain . METHODS Thirty-five rabbits were randomly divided into blank group ，model group，positive group （Diclofenac diethylamine emulsion 200 mg/kg），SYF high -dose group （386 mg/kg）and SYF low -dose group（97 mg/kg），with 7 rabbits in each group （all had 4 males and 3 females）. Except for the blank group ，the other groups ’ rabbits were injected 0.5 mL papain mixture （containing 2% papain and 0.03 mol/L L -cysteine）into the right knee cavity on day 1， 4 and 7，to replicate KOA model . Blank group was given constant volume of normal saline . From the 15th day ，drugs were applied to right hind knee joints of rabbits in each group ，twice a day for 20 days. At the same time ，the diameter of right knee joints of rabbits was measured by vernier calipers at day 0，8，14 and 35 to calculate swelling degree . After the experiment ，the levels of IL-1β，TNF-α and PGE 2 in synovial tissue were determined by enzyme -linked immunosorbent assay . Hematoxylin-eosin（HE） staining was used to prepare the sections of synovial tissue ，and the pathological changes were observed . The relative mRNA expressions of TLR 4，MyD88 and NF - кB p 65 in the TLR 4/MyD88/NF- кB signaling pathway were detected by real -time quantitative polymerase chain reaction . The relative protein expressions of TLR 4，MyD88，NF-кB p 65 and p -NF-кB p 65 were detect by Western blot . RESULTS Compared with blank group，the degree of knee swelling could be increased in model group ，the pathological damage of synovial tissue was more serious ，and the levels of IL -1β，TNF-α and PGE 2 were increased significantly in synovial tissue （P＜0.05）. The relative expression levels of TLR 4，MyD88，NF-кB p 65 mRNAs and TLR 4，MyD88，p-NF-кB p 65 proteins were significantly increased（P＜0.05）. Compared with model group ，swelling degree of right hind knee and the pathological injury degree of synovial tissue were significantly improved in each treatment group ，while the levels of IL -1β，TNF-α and PGE 2 in synovial tissue were significantly decreased （P＜0.05）. The relative mRNA expressions of TLR 4，MyD88 and NF -кB p 65 and relative protein expressions of TLR 4，MyD88（except for SYF low -dose group ）and p -NF-кB p 65 were all significantly decreased （P＜0.05）. CONCLUSIONS SYF shows protective effect on KOA induced by papain ，the mechanism of which is associated with decreasing the levels of IL -1β，TNF-α and PGE 2 and down -regulating TLR 4/MyD88/NF-кB signaling pathway .
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OBJECTIVE： To study the improvement effect of Shenrong bunao capsule on learning and memory ability of Alzheimer’s disease （AD） model mice， and to investigate its mechanism. METHODS： Totally 72 mice were randomly divided into blank control group （normal saline）， model group （normal saline）， Piracetam tablets group （positive control，0.80 g/kg），Shenrong bunao capsule high-dose，middle-dose and low-dose groups （1.92， 0.96， 0.48 g/kg）， with 12 mice in each group. Except that blank control group was given constant volume of normal saline subcutaneously. Other groups were given D-galactose （150 mg/kg） subcutaneously and sodium nitrite （50 mg/kg） intraperitoneally every day to induce AD model. At the same time，they were given relevant medicine intragastrically，once a day， for consecutive 60 d. 1 h after last administration， Morris water maze test was used to measure escape latency and times of crossing the platform within 90 s. HE staining was used to observe pathological changes of cerebral cortex in mice. Immunohistochemistry was used to detect the expressions of TNF-α， NF-κB p65， PI3K and Akt in cerebral cortex of mice. RESULTS： Compared with blank control group， escape latency was prolonged significantly （P＜0.01）， and the times of crossing the platform within 90 s was decreased significantly in model group （P＜0.01）. The neurons in cerebral cortex was damaged obviously， and the number of intact neurons was decreased significantly （P＜0.01）. The protein expressions of TNF-α， NF-κB p65， PI3K and Akt in cerebral cortex were increased significantly （P＜0.01）. Compared with model group， except that there was no statistical significance in escape latency， protein expressions of TNF-α， NF-кB p65， PI3K and Akt in Shenrong bunao capsule low-dose group （P＞0.05）， above indexes of other administration groups were improved significantly （P＜0.05 or P＜0.01）. CONCLUSIONS： Shenrong bunao capsule can improve the learning and memory ability of AD model mice， and its mechanism may be related to the inhibiting the protein expressions of TNF-α，NF-κB p65， PI3K and Akt in cerebral cortex region and relieving inflammation injury so as to protect cranial nerve.
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Objective To explore the effect of araloside on the proliferation and apoptosis of HeLa cells.Methods Human cervical cancer cell line HeLa was cultured in vitro,the experiment was divided into 4 groups as follows:blank group,araloside trea-ted groups(50,100,200 μg/mL).Normal saline and araloside (50,100,200 μg/mL)were gave,respectively.24,48 and 72 hours lat-er,the cells were collected.Cell proliferation was detected by MTT,cell apoptosis was determined by flow cytometry,the expression of pAkt1,pIкBα,NF-κB (p65),Bcl-2 and Caspase-3 were evaluated by western blot.Results Araloside obviously inhibited the pro-liferation and increased the apoptosis level of HeLa cells in a time-dose dependent manner.Moreover,Araloside significantly de-creased the phosphorylation of Akt1 and IκBα,reduced the expression of NF-κB(p65)and Bcl-2,whereas obviously increased Caspase-3 content.Conclusion Araloside could inhibit the proliferation and promote the apoptosis of HeLa cells via suppressing Akt/NF-кB signaling pathway.
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Background and purpose:Ovarian cancer-associated ifbroblasts (CAF) are known to promote epithelial malignancy. The chemoattractant cytokine growth-regulated oncogene alpha (Gro-α) secreted from CAF has been reported to mediate the stroma-epithelia interaction in tumor microenvironment, leading to the development of epithelial ovarian cancer, however, the detailed mechanism is unknown.This study was to determine whether Gro-αcould promote ovarian tumorigenesis through activating NF-кB nuclear translocation and VEGF expression in stromal ifbroblasts. Methods:ELISA was used to measure the levels of Gro-αin two cancer-associated ifbroblasts (CAF) and normal ifbroblasts (NF) isolated from high-grade serous ovarian cancer or normal ovarian tissues. CAF conditioned medium (CM) or Gro-αwas used to treat NF, while PS1145, the inhibitor of NF-кB, was used as control. NF-кB subunit p65 and vascular endothelial growth factor (VEGF) were detected by Western blot in cells after treatment. Xenograft tumors from nude mice were generated by injection of CAF, NF, or OVCA429 alone or OVCA429 mixed with CAF or NF, and by injection of OVCA429 mixed with NF cells that were treated with or without CAF-CM or Gro-α, or with NF cells that were treated with CAF-CM or Gro-αplus PS1145. The tumor growth curve was measured and the blood vessel density in xenograft tumor tissues was examined by histopathological analysis. Results:The levels of Gro-αwere 5-6 folds higher in CAF than in NF. Treatment of NF with CAF-CM or Gro-αstimulated the nuclear translocation of NF-кB subunit p65, and the expression of VEGF, but suppressed the expression of thrombospondin 1, the anti-angiogenesis factor, compared with control cells. However, treatment of NF with the NF-кB inhibitor PS1145 reversed these results. The animal assay revealed that CAF stimulated tumor growth stronger than NF, and NF treated with CAF-CM or Gro-α, but not along with PS1145, enhanced xenograft tumor growth through promoting angiogenesis. Conclusion:Ovarian CAF promotes the nuclear translocation of NF-кB and the expression of VEGF through Gro-αautocrine in tumor microenvironment to facilitate angiogenesis and ovarian cancer development.
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Objective To detect the expression of orphan nuclear receptor RORC gene and the level of serum interleukin-17 in acute lung injury (ALI) rats, and to explore the effects and possible regulation mechanisms of helper T lymphocyte 17 (Th17) in the ALI.Methods Twenty four SD male rats were randomly assigned to three groups, control group, model group, and pyrrolidine dithiocarbamates (PDTC) prevention group.The rats in model group and PDTC prevention group were intravenously injected LPS (6 mg/kg) for inducing ALI.The PDTC prevention group had been intraperitoneally injected PDTC (120 mg/kg) thirty minutes before LPS injection.All rats were killed twelve hours after LPS injecttion.The lung wet/dry weight ratio was measured.Lung pathologic tissue scored after hematoxylin-eosin (HE) stain.The expression of alveolar macrophages (AM) nuclear factor-KBP65 (NF-κBP65) was detected by immunohistochemistry.The level of serum IL-17 was detected by ELISA method.The expression of AM orphan nuclear receptor RORC gene was determined by SYBR Green Ⅰ real-time polymerase chain reaction.Results Compared with control group.the lung wet/dry weight ratio.lung pathologic tissue score, the expression of AM NF-κBP65.the level of serum IL-17 and the expression of AM orphan nuclear receptor RORC gene were significantly increased (P < 0.05).However, compared with model group, these changes were prevented in PDTC prevention group.Conclusions Th17 might participate in the pathological process of ALI.Activation of NF-кB might influence RORC gene expression, induce Th17 differentiation, and elevate the level of serum IL-17.
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Both viral diseases and cancer account for a large proportion of serious health problems.Viral infection and cancer are biologically and medically correlated and in many ways share common cellular pathways that lead to disease development or progression.Better understanding how these signaling events are specifically activated by different pathogenic stimuli and how they activate different downstream transcriptions in response to these stimuli at high specificity and efficiency will provide a new molecular basis for the development of novel disease biomarkers and therapeutic or preventive targets against both classes of diseases.Research in our laboratory has been prompted to investigate the regulation and modes of action of these pathways,with a more intensive focus on the NF-кB signaling,in the settings of severe or oncogenic viral infection as well as cancer development.It is hoped that our research will lead to eventual clinical application of biomarkers derived from these signaling pathways.
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In this study,the effects of pirrolidine dithiocarbamate (PDTC) plus leflunomide (Lef) models were investigated.NIH mice and Wistar rats served as donors and recipients respectively.Mouse-to-rat cardiac xenotransplantation was performed.The recipients were divided into 5 groups:group A (the control group),group B (PDTC group),group C (PDTC plus CsA group),group D (PDTC plus Lef group) and group E (PDTC plus Lef and CsA group).The expressions of IKKα/β,by immunohistochemistry and Western blot as well as electrophoretic mobility shift assay (EMSA).The median survival time of cardiac xenografts in the control group,PDTC group,PDTC plus CsA group,PDTC plus Lef group and PDTC plus Lef and CsA group was (2.17±0.41),(2.33±0.52),(4.67±1.21),(7.00±1.79) and (9.00±1.41) days respectively.The survival time of xenografts in the PDTC plus Lef and CsA group was significantly longer than that in other four groups (P<0.05 for all),that in the PDTC plus Lef group longer than that in the control group,PDTC group and PDTC plus CsA group (P<0.05 for all),that in PDTC plus CsA group longer than the control group and PDTC binding activity were notably increased in mouse-to-rat cardiac xenografts.The expressions were decreased in the control group,PDTC group,PDTC plus CsA group,PDTC plus Lef and PDTC plus Lef and CsA group in turn.It was concluded that PDTC plus Lef and CsA can significantly suppress prolonging the survival of the cardiac xenografts.
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Objective To continually observe the monocyte chemoattraetant protein-1(MCP-1) expression in rats with anilateral ureteml obstruction, and to explore the relationship between MCP-1 and p38MAPK, and as well as the nuclear transcription faetor-кB (NF-кB). Method Thirty-six rots were randomly assigned to sham op-eration group (contorl group) and unilateral ureteral obstruction group (UUO group). Renal tissues were examined under light microscope 8 h,24 h and 72 h after operation, Immunhistochemistry was used to measure the expression of MCP-1 and NF-кB. RT-PCR and Western bot were employed to determine MCP-1 mRNA and p38MAPK pro-tein,respectively.Serum creatinine and ure nitrogen were examined with biochemistry methods. Results Com-pared with control group, the expression of MCP-1 mRNA in UUO group dramatically increased (control group:0.401±0.039;UUO group 8 h:0.894±0.137;24 h: 1.416±0.135;72 h: 1.894±0.14, P<0.05).The explosion of MCP-1 protein in renal interstitium of UUO group also markedly increased (control group: 50.08±3.210. UUO group 8 h:108.25±4.325;24 h: 179.34±3.237;72 h: 230.12±3.026, P<0.05),and the expressions of NF-кB and p38MAPK in UUO group were also stimulated (p38MAPK: control group: 110.65±9.734. UUO group 8 h:200.15±8.326; 24 h:272.74±7.244;72 h:549.11±9.544, P<0.05). There were positive correlations between MCP-1 and p38MAPK as well as NF-кB(r=0.74,r=0.81,P<0.01).Conclusions Tne increased expression of MCP-1 may participate in the injury mechanisms of renal tubulointesti-tinm in UUO rats. The increased expression of p38MAPK may induce NF-кB expression in the tubules, and then NF-кB promotes the expression of MCP-1.
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The aim of our study was to gain insight into the molecular and cellular mechanisms of post-angioplasty restenosis using balloon catheter-induced injury model in the rat carotid artery. SD rats were subjected balloon catheterization at one side carotid artery as study group and another side as control group. Six rats were killed on the 6 h, and 3rd, 7th, 14th, 28th day after balloon-induced injury respectively. The intimal thickness and the expression of NF-кB and I-кB were detected by HE-staining, gel electrophoretic mobility shift assay (EMSA) and Western-blot methods. The results showed that: (1) The thickening of intima was observed on the 3rd day after balloon-induced injury,and it became more significant on the 7th, 14th and 28th day. The area ratio of intima/media was in-creased significantly (P＜0.05); (2) The expression of NF-кB was not detectable in the control group,however, in study group, the expression of NF-кB was detected on the 6th h after balloon-induced injury, reached the peak on the 14th day, and on 28th day, strong expression of NF-кB was observed;(3) The expression of I-кB protein was reduced after balloon-induced injury, and there were signifi-cant differences between the study group and the control group (P＜0.05). It was concluded that the alteration of NF-кB/I-кB system might play an important role in aberrant proliferation within the in-tima and vascular remodeling following vascular injury. To block NF-кB activation and its role in ar-terial restenosis initiation may potentially provide a novel therapeutic tool for the treatment and pre- vention of arterial restenosis.
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The relationship between intracelluar trypsinogen activation and NF-r,B activation in rat pancreatic acinar cells induced by M3 cholinergic receptor agonist (carbachoi) hyperstimulation was studied. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active pro- tease inhibitor (pefabloc) and NF-кB inhibitor (PDTC) in vitro. Intracelluar trypsin activity was measured by using a fluorogenie substrate. The activity of NF-кB was monitored by using electro- phoretic mobility shift assay. The results showed that after pretreatment with 2 mmol/L pefabloc, the activities of trypsin and NF-кB in pancreatic acinar cells treated with high concertrations of carbachol (10-3 mol/L) in vitro was significantly decreased as compared with control group (P＜0.01). The addi- tion of 10-2mol/L PDTC resulted in a significant decrease of NF-кB activities in pancreatic acinar cells after treated with high concertrations of carbachol (10-3 mol/L) in vitro, but the intracelluar trypsinogen activity was not obviously inhibited (P＞0.05). It was concluded that intracelluar trypsi- nogen activation is likely involved in the regulation of high concertrations of carbachol-induced NF-кB activation in pancreatic acinar cells in vitro. NF-кB activation is likely not necessary for high concertrations of carbachol-induced trypsinogen activation in pancreatic acinar cells in vitro.
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Objective To investigate the mechanisms of action of adiponectin on receptor activator of NF-Kb ligand(Rankl) and osteoprotegerin (OPG)expressions in human osteoblasts.Methods Real-time PCR was used to detect the expressions of RANKL and OPG mRNA in cultured human osteoblasts. The phosphorylations of JNK, p38 mitogen-activated protein kinase (MAPK) , ERK1/2 were assayed by Western blot. RNA interference for adiponectin receptor, MAPK inhibitors SB203580 and SP600125 were used for elucidating the mechanism of the action of adiponectin in regulating OPG and RANKL expressions. Results Suppression of adiponectin receptor-1 (AdR1) expression with siRNA abolished the adiponectin-regulated expressions of OPG and RANKL mRNA in human osteoblasts. Furthermore, pretreatment of osteoblasts with MAPK inhibitor SB203580 abolished the expressions of adiponectin-regulated RANKL and OPG mRNA, but SP600125 did not show the effect. Conclusion Adiponectin induces the expression of RANKL and inhibits the expression of OPG in human osteoblasts through AdR1/p38 MAPK pathways.