ObjectiveTo investigate the material basis and mechanism of action of Tuoli Xiaodu San in treating ulcerative colitis （UC） by integrating transcriptomics， network pharmacology， and experimental validation. MethodsNetwork pharmacology was initially employed to screen the active components and potential mechanisms of Tuoli Xiaodu San for treating UC. A UC mouse model was established by dextran sulfate sodium （DSS） induction. The mice were divided into the following groups： normal， model， high-dose （11.3 g·kg^-1） Tuoli Xiaodu San， low-dose （5.7 g·kg^-1） Tuoli Xiaodu San， and positive control （mesalazine， 0.4 g·kg^-1）. Intragastric administration commenced on day 1 of modeling and continued for 7 consecutive days. The disease activity index （DAI） was assessed daily. Hematoxylin-eosin （HE） staining was used to observe colonic pathological changes. Serum levels of tumor necrosis factor-alpha （TNF-α）， interleukin-1 beta （IL-1β） and interleukin-6 （IL-6） were measured by enzyme-linked immunosorbent assay （ELISA）. Transcriptome sequencing was performed on mouse colonic tissues， and the results were integrated with network pharmacology findings for in-depth analysis of Tuoli Xiaodu San's potential mechanisms in treating UC. Finally， the expression of key genes and proteins in the identified signaling pathways were detected using Western blot and Real-time polymerase chain reaction （Real-time PCR）. ResultsThe combined analysis of network pharmacology and transcriptomics results showed that the multi-pathway network with phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） signaling pathway as its core was the key mechanism of Tuoli Xiaodu San in the treatment of UC. Tuoli Xiaodu San administration significantly ameliorated weight loss， diarrhea， and bloody stools in UC mice， reduced the DAI scores （P<0.05， P<0.01）， lowered the colonic histopathological scores （P<0.01）， alleviated colon shortening （P<0.01）， and downregulated serum levels of TNF-α， IL-1β， and IL-6 （P<0.05， P<0.01）. Molecular biology experiments confirmed that Tuoli Xiaodu San significantly inhibited the mRNA and protein expression， as well as the phosphorylation levels， of PI3K， Akt， and p65 in colonic tissues （P<0.05， P<0.01）. ConclusionTuoli Xiaodu San can regulate the multi-pathway network with PI3K/Akt as its core through multi-component synergy， thereby reducing colonic inflammatory damage and exerting a therapeutic effect on UC.

Insulin-like growth factor-1 receptor (IGF-1R) has been made an attractive anticancer target due to its overexpression in cancers. However, targeting it has often produced the disappointing results as the role played by cross talk with numerous downstream signalings. Here, we report a disobliging IGF-1R signaling which promotes growth of cancer through triggering the E3 ubiquitin ligase MEX3A-mediated degradation of RIG-I. The active β-arrestin-2 scaffolds this disobliging signaling to talk with MEX3A. In response to ligands, IGF-1Rβ activated the basal βarr2 into its active state by phosphorylating the interdomain domain on Tyr64 and Tyr250, opening the middle loop (Leu130‒Cys141) to the RING domain of MEX3A through the conformational changes of βarr2. The models of βarr2/IGF-1Rβ and βarr2/MEX3A could interpret the mechanism of the activated-IGF-1R in triggering degradation of RIG-I. The assay of the mutants βarr2^Y64A and βarr2^Y250A further confirmed the role of these two Tyr residues of the interlobe in mediating the talk between IGF-1Rβ and the RING domain of MEX3A. The truncated-βarr2 and the peptide ATQAIRIF, which mimicked the RING domain of MEX3A could prevent the formation of βarr2/IGF-1Rβ and βarr2/MEX3A complexes, thus blocking the IGF-1R-triggered RIG-I degradation. Degradation of RIG-I resulted in the suppression of the IFN-I-associated immune cells in the TME due to the blockade of the RIG-I-MAVS-IFN-I pathway. Poly(I:C) could reverse anti-PD-L1 insensitivity by recovery of RIG-I. In summary, we revealed a disobliging IGF-1R signaling by which IGF-1Rβ promoted cancer growth through triggering the MEX3A-mediated degradation of RIG-I.

The well-known insulin-like growth factor 1 (IGF1)/IGF-1 receptor (IGF-1R) signaling pathway is overexpressed in many tumors, and is thus an attractive target for cancer treatment. However, results have often been disappointing due to crosstalk with other signals. Here, we report that IGF-1R signaling stimulates the growth of hepatocellular carcinoma (HCC) cells through the translocation of IGF-1R into the ER to enhance sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) activity. In response to ligand binding, IGF-1Rβ is translocated into the ER by β-arrestin2 (β-arr2). Mass spectrometry analysis identified SERCA2 as a target of ER IGF-1Rβ. SERCA2 activity is heavily dependent on the increase in ER IGF-1Rβ levels. ER IGF-1Rβ phosphorylates SERCA2 on Tyr⁹⁹⁰ to enhance its activity. Mutation of SERCA2-Tyr⁹⁹⁰ disrupted the interaction of ER IGF-1Rβ with SERCA2, and therefore ER IGF-1Rβ failed to promote SERCA2 activity. The enhancement of SERCA2 activity triggered Ca²⁺_ER perturbation, leading to an increase in autophagy. Thapsigargin blocked the interaction between SERCA2 and ER IGF-1Rβ and therefore SERCA2 activity, resulting in inhibition of HCC growth. In conclusion, the translocation of IGF-1R into the ER triggers Ca²⁺_ER perturbation by enhancing SERCA2 activity through phosphorylating Tyr⁹⁹⁰ in HCC.

Objective]To investigate the change of spinal pro?inflammatory cytokines in a rat model of fentanyl induced acute hyperalgesia.[Methods]64 male SD rats were divided into 2 groups(n=32),fentanyl group and NS group. The rats were subcutaneously injected with fentanyl (60 μg/kg) or normal saline (1.2 mL/kg) 4 times with 15?minute intervals. Mechanical nociceptive thresholds and thermal nociceptive latency were measured via the tail pressure test(Tail flick thresholds,TFT) and paw withdrawal test(Paw withdrawal latency,PWL)on the day before,at 1,2,3,and 4 hour and on 1~5 day after injection. 4 rats were killed concomitantly and the lumber spinal cord were harvested to analysis the expression of NF-κB,PGE2,TNF-α,IL-1β,and IL-6.[Results]There were no significant changes of TFT,PWL and the expression of spinal inflammatory cytokines such as NF-κB, PGE2,IL-1β,and TNF-αcompared to baseline of rats treated with normal saline. The value of TFT ,PWL in fentanyl group raised to the highest(above the baseline)at the 1st hour after fentanyl injections and decreased thereafter,reached the lowest at the 1st day, raised increasinglyand up to baseline on the 3 day after injection. NF-κB,PGE2,IL-1β,and TNF-αincreased at the 4th hour,on 1 and 2 day and IL-6 increased at the 4 hour and onthe 1 day after fentanyl injections.[Conclusion]Subcutaneously injection of fentanyl induced significant mechanical and thermal hyperalgesia and increased spinal pro?inflammatory cytokines parallelly , indicated that fentanyl induced acute hyperalgesia is associated with spinal inflammatory reaction in rats.

OBJECTIVE:To study the analgesic effect and mechanism of Gutongtie paste on model rats with formaldehyde-in-duced pain. METHODS:60 SD rats were randomly divided into blank group,model group,Gutongtie paste low-dose,medi-um-dose,high-dose groups(0.594,1.188. 2.376 g/paste,containing crude drug 0.48,0.96,1.92 g)and prednisone acetate group (ig,0.0054 g/kg,external bonding matrix). Model rats with pain was induced by formaldehyde method and immediately adminis-trated after modeling. Electronic tenderness instrument was adopted to determine the pain threshold of rats'ankle joint after adminis-tration of 1,2,3,4,6 h. After 6 h,blood sample 0.3 mL was taken from abdominal aorta then rats were sacrificed. Enzyme linked immunosorbent assay (ELISA) was conducted to determine the β-endorphin (β-EP),prostaglandin E2 (PGE2) contents;spectrophotometry was used to determine nitric oxide(NO)content in rats'serum and inflammatory tissue;and radioimmunoassay was adopted to detect the substance P content in rats'serum,inflammatory tissue and brain tissue. RESULTS:Compared with be-fore modeling,pain thresholds in model group at each period were significantly decreased (P<0.05 or P<0.01). Compared with blank group,PGE2,NO of rats,substance P content in inflammatory tissue and brain tissue in model group were significantly in-creased (P<0.05 or P<0.01). Compared with model group,pain thresholds in Gutongtie paste groups at corresponding time points were increased,PGE2 and substance P contents in inflammatory tissue and brain tissue were decreased (P<0.05 or P<0.01);β-EP and NO contents in serum in Gutongtie paste medium-dose,high-dose groups(P<0.05 or P<0.01),NO contents in serum in Gutongtie paste high-dose group were decreased(P<0.05). CONCLUSIONS:Gutongtie paste has a certain analgesic and anti-inflammatory effect,and the mechanism may be related to reducing PGE2, NO, substance P contents, increasing β-EP content.

Objective To investigate the inhibitant effects of parecoxib, a cyclooxygenase-2 (COX-2) inhibitor, in acute fentanyl induced hyperalgesia in rats. Methods (1) Thirty SD rats (n=6 for each group) were subcutaneously injected with fentanyl (40 μg/kg × 4 times with a 15 min-interval) or saline to establish acute fentanyl induced hyperalgesia model, andparecoxib (5, 10 mg/kg) was administrated intraperitoneally in parecoxib group. Mechanical nociceptive thresholds were measured by the tail pressure test every hour during 1~4 hours and once a day during 1~5 days. (2) 24 SD rats (n = 6 foreach group) were subcutaneously injected with fentanyl as above described and randomly administrated intraperitoneally with parecoxib in 10 mg/kg in 15 min before and at the 4th hour and the 1st day after fentanyl injection except rats in the control group, mechanical nociceptive thresholds were measured by the tail pressure test at time points as above described. Results (1)Acute high dose fentanyl injection induced mechanical hyperalgesia and parecoxib (at 5 or 10 mg/kg)inhibited fentanyl induced hyperalgesia in rats. (2)Parecoxib inhibited fentanyl induced hyperalgesia at 15 min before and at the 4th hour after, but not on the 1st day after fentanyl injection. Conclusions This study provides the first evidence that subanalgesia doses of parecoxib had inhibitory effects on acute fentanyl induced hyperalgesia in time-dependent manners in rats.