Discovery and characterization of a novel cGAS covalent inhibitor for the treatment of inflammatory bowel disease.

Cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, acts as a nucleotidyl transferase that catalyzes ATP and GTP to form cyclic GMP-AMP (cGAMP) and plays a critical role in innate immunity. Hyperactivation of cGAS-STING signaling contributes to hyperinflammatory responses. Therefore, cGAS is considered a promising target for the treatment of inflammatory diseases. Herein, we report the discovery and identification of several novel types of cGAS inhibitors by pyrophosphatase (PPiase)-coupled activity assays. Among these inhibitors, 1-(1-phenyl-3,4-dihydro-1H-pyrrolo[1,2-a]pyrazin-2-yl)prop-2-yn-1-one (compound 3) displayed the highest potency and selectivity at the cellular level. Compound 3 exhibited better inhibitory activity and pathway selectivity than RU.521, which is a selective cGAS inhibitor with anti-inflammatory effects in vitro and in vivo. Thermostability analysis, nuclear magnetic resonance and isothermal titration calorimetry assays confirmed that compound 3 directly binds to the cGAS protein. Mass spectrometry and mutation analysis revealed that compound 3 covalently binds to Cys419 of cGAS. Notably, compound 3 demonstrated promising therapeutic efficacy in a dextran sulfate sodium (DSS)-induced mouse colitis model. These results collectively suggest that compound 3 will be useful for understanding the biological function of cGAS and has the potential to be further developed for inflammatory disease therapies.

[A clinical study of thunder-fire moxibustion combined with meibomian gland massage in treatment of meibomian gand dysfunction].

OBJECTIVE: To observe the effect of thunder-fire moxibustion combined with meibomian gland massage in improving meibomian gland dysfunction (MGD) and explore its mechanism. METHODS: Seventy-two MGD patients with 144 eyes in the Jinhua Hospital of Traditional Chinese Medicine from February 2019 to January 2021 were selected and randomly divided into an experimental group (n=36,72 eyes) and a control group (n=36, 72 eyes). Patients in the control group received 0.1% fluo-rometholone eye drops and 0.1% sodium hyaluronate eye drops, 1-2 drops per time, four times per day, and the meibomian glands were massaged once per day. Patients in the experimental group received additional thunder-fire moxibustion on the basis of the treatment of the control group, 10 cones per time, once per day. One month after treatment, meibomian gland function was assessed, and the levels of interleukin-6 (IL-6) and prostaglandin E2(PGE2) in tears were detected. RESULTS: After treatment, the scores of Ocular Surface Disease Index, meibomian hyperemia, meibomian gland opening, meibomian gland loss, and meibomian gland secretion function were lower than those before treatment in the two groups, and the scores of the experimental group were lower than those of the control group (P<0.05). After treatment, the tear break-up time and tear meniscus height were higher than those before treatment in the two groups, which were higher in the experimental group than those in the control group (P<0.05). The post-treatment levels of IL-6 and PGE2 were lower than those before treatment in the two groups, and the levels in the experimental group were lower than those in the control group (P<0.05). CONCLUSION: Thunder-fire moxibustion combined with meibomian gland massage can significantly improve the function of the meibomian glands and lower the levels of IL-6 and PGE2 in tears.

Effects of hypoxia on DNA hydroxymethylase Tet methylcytosine dioxygenase 2 in a KG-1 human acute myeloid leukemia cell line and its mechanism.

Hypoxia is involved in the epigenetic modification of leukemia. As an important DNA hydroxymethylase and a tumor suppressor gene, the expression regulating mechanism of Tet methylcytosine dioxygenase 2 (TET2) remains unclear. The aim of the present study was to explore whether hypoxia and hypoxia-inducible factor 1α (HIF-1α) regulate TET2 gene expression and its demethylation function in acute myeloid leukemia (AML). The human AML cell line KG-1 was used in the present study. The results demonstrated that hypoxia could increase proliferation, enhance metabolism and inhibit apoptosis in KG-1 cells, as detected by the cell counting kit-8 assay, lactate dehydrogenase assay and Annexin V-FITC/propidium iodide staining, respectively. Hypoxia reduced the genome methylation status in KG-1 cells detected using 5-methylcytosine and 5-hydroxymethylcytosine detection kits. In addition, HIF-1α overexpression increased TET2 expression, 5-hmC level and cyclin-dependent kinase inhibitor 2B [p15(INK4B)] gene demethylation compared with the HIF-1α non-overexpression group in KG-1 cells detected by reverse transcription-quantitative PCR, western blotting, 5-hydroxymethylcytosine detection kits and methylation-specific PCR, respectively. The inhibition of HIF-1α by inhibitor YC-1 reduced demethylation in KG-1 cells by decreasing TET2 expression. It was also revealed that HIF-1α could enhance TET2 transcriptional activity by binding to the hypoxia response element of the TET2 gene promoter region using chromatin immunoprecipitation and luciferase reporter gene assays. TET2 may be a potential target gene regulated by HIF-1α. Hypoxia was demonstrated to regulate the expression of TET2 by HIF-1α, which in turn affected the methylation and expression of downstream target genes and served a role in the occurrence and progression of leukemia. In the present study, the association between hypoxia metabolism and epigenetic regulation in AML was investigated and the findings provided a new idea and experimental basis for the diagnosis and treatment of hematologic malignancies.

[Effect on the pain and tear film stability in patients after pterygium excision treated with intradermal needling].

OBJECTIVE: To analyze the effects of intradermal needling for pain and tear film stability in patients after pterygium excision. METHODS: A total of 76 patients (98 affected eyes) with primary pterygium were randomly divided into an observation group (38 cases, 53 affected eyes) and a control group (38 cases, 45 affected eyes).In the control group, only pterygium resection was performed, in the observation group, intradermal needling after pterygium resection was applied at Cuanzhu (BL 2), Yuyao (EX-HN 4), Taiyang (EX-HN 5), Sibai (ST 2), Hegu (LI 4), removed after 24 h and changed three times a week. The pain level of 3 days after surgery, dry eye symptoms, the basic tear secretion test (Schirmer-Ⅰ), and the tear-break time (BUT) changes before surgery, 2 weeks after surgery and 4 weeks after surgery were compared between the two groups, and the clinical efficacy was evaluated. RESULTS: The pain level of 3 days after surgery in the observation group was significantly lower than that in the control group (P<0.05). The dry eye symptom scores at 2 weeks and 4 weeks after surgery in the two groups were significantly lower than those before surgery (all P<0.05), and the dry eye symptom scores in the observation group were significantly lower than those in the control group (both P<0.05). The Schirmer-Ⅰ test at 2 weeks and 4 weeks after surgery was significantly prolonged than that before surgery(all P<0.05), and the Schirmer-Ⅰ test in the observation group was significantly longer than that in the control group (both P<0.05). The BUT at 2 weeks and 4 weeks after surgery in the two groups was significantly longer than that before surgery (all P<0.05), and the BUT in the observation group was significantly longer than that in the control group (both P<0.05). The total effective rate in the observation group was 89.5% (34/38), which was higher than 71.1% (27/38) in the control group (P<0.05). CONCLUSION: Intradermal needling can effectively reduce the pain level of patients after pterygium resection, improve dry eye symptoms, promote the secretion of tears and improve the tear film stability.

[Establishment and verification of dose effect relation curves between T cell receptor gene mutation frequency and ionizing radiation dose].

OBJECTIVE: To establish the dose-effect curve between TCR MF and ionizing radiation. METHODS: Peripheral lymphocytes were collected from 8 healthy adults (4 males and 4 females) and cultured in vitro with 12 well culture plates. They were stimulated by PHA-P and IL-2 after exposed to different doses of irradiation (0.00 - 8.00 Gy) and cultured for 7 d. The dose-effect curve was established after measuring TCR MF using flow cytometry. Also, using the same method, we separated and cultured the peripheral lymphocytes collected from 16 radiotherapy cancer patients, whose radiation styles and doses were different, and then measured TCR MF to estimate the whole equivalent dose of radiotherapy patients through the dose-effect curve. Peripheral blood was collected and cultured, chromosome aberration (dicentric and ring) was determined under microscope to estimate irradiation dose. RESULTS: The relationship of dose-effect between the TCR MF and ionizing radiation (0.00 - 8.00 Gy) was well, the curve of large dose group (2.00 - 8.00 Gy), low dose group (0.00 - 1.00 Gy) and 0.00 - 8.00 Gy dose group were met with the quadratic polynomial model, the equation was TCR MF = -32.8579 + 20.5436D + 0.6341D(2), TCR MF = 1.796 + 0.017D + 5.155D(2) and TCR MF = -0.6229 + 6.305D + 0.6919D(2), respectively. D was the radiation dose (Gy). Using the established curve and the chromosome aberration method to estimate the systemic exposure dosage, the average relative deviation was 16.8%. CONCLUSION: The curve established by the TCR gene mutation analysis technology can be applied to exposure dose estimation of victims in ionization radiation accidents.

Time-dependent changes of lipid peroxidation and antioxidative status in nerve tissues of hens treated with tri-ortho-cresyl phosphate (TOCP).

Tri-ortho-cresyl phosphate (TOCP) could induce a delayed neurodegenerative condition known as organophosphorus easter-induced delayed neurotoxicity (OPIDN) in human beings and sensitive animals. However, the mechanisms of OPIDN remain unknown. This study investigated the time-dependent changes of the lipid peroxidation (malondialdehyde, MDA) and antioxidative status (glutathione, GSH; glutathione peroxidase, GSH-Px; glutathione reductase, GR; superoxide dismutase, SOD and anti-reactive oxygen species, anti-ROS) in nerve tissues for elucidating the mechanism of OPIDN induced by TOCP. Adult hens were treated with TOCP by gavage at a single dosage of 750 mg/kg. TOCP was dissolved in corn oil and administered at 0.65 ml/kg. The control hens received an equivalent volume of corn oil by gavage. Hens were sacrificed after 0, 5, 10, 15 and 21 days of treatment and the cerebrum, spinal cord, sciatic nerve were dissected, homogenized and used for the determination of lipid peroxidation and antioxidative status. The results showed that treatment with TOCP increased lipid peroxidation and reduced the antioxidative status in cerebrum, spinal cord and sciatic nerve. The levels of MDA increased by 33% (P<0.01) in cerebrum on 5th day after TOCP treatment and at clinical sign score of 1-2, and increased respectively by 32% and 15% (P<0.01) in spinal cord and sciatic nerve on 10th day after TOCP treatment and at clinical sign score of 3-4. Further changes of MDA were also observed after 15 and 21 days post-dosing and at clinical sign score of 5-6 and 7-8. There is a decrease in the activities of SOD, GSH-Px, GR, anti-ROS, and GSH content in cerebrum, spinal cord and sciatic nerve of hens after 5, 10, 15 and 21 days post-dosing and at clinical sign score of 1-2, 3-4, 5-6 and 7-8. Thus, OPIDN induced by TOCP was associated with elevation of lipid peroxidation and reduction of antioxidative status, and the time-dependent changes of these indexes in hens nerve tissues occurred. Sciatic nerve was the main target tissue and MDA was most sensitive among all indexes. The time-dependent and tissue specific changes of lipid peroxidation and antioxidative status in cerebrum, spinal cord and sciatic nerve suggest that ROS and concomitant lipid peroxidation, at least in part, are involved in the toxic effects of TOCP on nerve tissues and that oxidative stress may play a role in the occurrence and development of OPIDN induced by TOCP.

[Alterations of microtubule and microfilament expression in spinal cord of carbon disulfide intoxicated rats].

OBJECTIVE: To investigate whether the alterations of microtubule and microfilament expression are responsible for the neurotoxicity of carbon disulfide. METHODS: Wistar rats were administered with carbon disulfide by gavage at a dosage of 300 or 500 mg/kg for continuous 12 weeks (five times per week). Spinal cords of carbon disulfide-intoxicated rats and their age-matched controls were Triton-extracted and ultracentrifuged to yield a pellet and a corresponding supernatant fraction. Then, the contents of alpha-tubulin, beta-tubulin, and beta-actin in both fractions were determined by immunoblotting. In the meantime, their mRNA levels in spinal cords were quantified using reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: In the supernatant fraction, the contents of beta-tubulin and beta-actin in both treated groups increased significantly (P < 0.01) the content of beta-tubulin increased by 141% and 158% respectively, and the content of beta-actin increased by 19% and 32% respectively. In the pellet fraction, the content of beta-tubulin in both groups increased by 107%(P < 0.01) and 118%(P < 0.01) respectively, and the others keep unaffected. In the meantime, the levels of of mRNA expression of beta-tubulin and beta-actin gene were elevated consistently in CS(2)-treated groups (P < 0.01) the levels of mRNA expression of beta-tubulin increased by 207% and 212% respectively, and the levels of mRNA expression of beta-actin increased by 94% and 91% respectively. CONCLUSION: Carbon disulfide intoxication results in alternations of microtubule and microfilament expression, and the alternations might be related to its neurotoxicity.