Objective:To investigate the clinical features, molecular etiology, and treatment of a family with Treacher Collins Syndrome 2 (TCS2).Methods:Information of the proband (female, 8 years old) including medical history and family history was collected. Physical examination and examinations concerning laboratory, audiology, and radiology were performed on the proband. Physical examination was also performed on the family members. Genomic DNA of proband was extracted for whole exome sequencing, and then the genomic DNA of family members was extracted for Sanger sequencing. POLR1D and TCS2 related literatures published before August 31,2023 were searched and sifted in PubMed and CKNI databases. The clinical characteristics of TCS2 were summarized. Results:The proband had poor hearing since childhood, with pure tone audiometry indicating conductive hearing loss. She had a smaller jaw, bilateral preauricular fistulas and cup-shaped ear deformities. Temporal bone CT scan revealed deformities in the left external ear canal, bilateral middle ear and inner ear. A bone-conduction hearing aid device was surgically implanted, resulting in restoration of almost normal hearing levels. The proband′s mother also had a slightly smaller jaw. Genetic analysis revealed a novel heterozygous variant NM_015972.4:c.38_47del in the POLR1D gene in the proband, which was inherited from her mother. A review of the literature revealed no clear evidence of genotype-phenotype correlation in TCS2. Conclusions:Molecular diagnosis plays a vital role in the diagnosis of TCS2. Patients with normal facial phenotype may be carriers of pathogenic variants in the POLR1D gene and have the risk of passing it to the offsprings with complete penetrance. Proper bone conductive hearing devices can improve the quality of life of TCS2 patients.

AIM:To investigate the effect of gastrodin(GAS)on microglia-mediated inflammatory response after hypoxic-ischemic brain damage(HIBD)neonatal mice by regulating the expression of JAK1/STAT1 pathway through C-C chemokine recepeor 5(CCR5).METHODS:Forty-eight C57BL/6J mice at about 10 days after birth were randomly divided into sham group,HIBD model group and HIBD+GAS group.BV-2 microglia were divided into control(Con)group,oxygen glucose deprivation(OGD)group,oxygen glucose deprivation with gastrodin intervention(OGD+GAS)group,GAS group,Maraviroc(MVC)group,OGD+MVC group,and OGD+MVC+GAS group.The mRNA expression of CCL4 and CCR5 were detected by RT-qPCR.The protein expression of CCR5,p-JAK1,p-STAT1,tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were detected by Western blot.The expression of CCR5,p-JAK1 and p-STAT1 in cells were observed by immunofluorescence staining.RESULTS:(1)Compared with sham group,the expression levels of CCL4 and CCR5 mRNA,and CCR5,p-JAK1 and p-STAT1 proteins were significantly higher in the ischemic side of the corpus callosum in HIBD group(P＜0.05).(2)Compared with Con group,the protein levels of CCR5,p-JAK1 and p-STAT1 significantly increased in BV-2 cells of OGD group(P＜0.05).The protein levels of CCR5,p-JAK1 and p-STAT1 in BV-2 cells of OGD+GAS group were significantly lower than those of OGD group(P＜0.05).(3)Maraviroc did not cause significant BV-2 cell death in the 0～80 μmol/L range.The p-JAK1 and p-STAT1 protein levels in MVC+OGD group were significantly lowered compared with OGD group(P＜0.05),but no significant difference was found between MVC+ OGD and OGD+MVC+GAS groups.CONCLUSION:Gastrodin can exert neuroprotective effects via CCR5/JAK1/STAT1 signaling pathway.

Objective To investigate the mechanism of gastrodin for inhibiting microglia-mediated inflammation after hypoxic-ischemic brain damage(HIBD)in neonatal rats.Methods Thirty-nine 3-day-old SD rats were randomly divided into sham group,HIBD group and gastrodin treatment group.Western blotting was used to detect the expressions of TNF-α,IL-1β,IL-10 and TGF-β1 in the corpus callosum of the rats.The potential targets of gastrodin for treatment of HIBD were screened by network pharmacology analysis.The expressions of PI3K/AKT signaling pathway proteins following HIBD-induced microglial activation in the rats and in cultured microglial BV-2 cells with oxygen-glucose deprivation(OGD)were detected with Western blotting.The effects of LY294002(a specific inhibitor of the PI3K/AKT pathway)and gastrodin on TNF-α and TGF-β1 mRNA levels in BV-2 cells with OGD was detected with RT-qPCR.Results In the neonatal rats with HIBD,gastrodin treatment significantly decreased TNF-α and IL-1β expressions and enhanced IL-10 and TGF-β1 expressions in the ischemic corpus callosum.Network pharmacology analysis showed significant enrichment of the PI3K/AKT signaling pathway and a strong binding between gastrodin and PI3K.Gastrodin significantly promoted PI3K and AKT phosphorylation in neonatal rats with HIBD and in BV-2 cells exposed to OGD.In BV-2 cells with OGD,gastrodin obviously suppressed OGD-induced increase of TNF-α and reduction of TGF-β1 mRNA expressions,and this effect was strongly attenuated by LY294002 treatment.Conclusion Gastrodin can inhibit microglia-mediated inflammation in neonatal rats with HIBD by regulating the PI3K/AKT signaling pathway.

Objective To investigate the mechanism behind the protective effects of gastrodin against microglia-mediated inflammatory responses following hypoxic-ischemic brain damage(HIBD)in neonatal mice.Methods Thirty-six 10-day-old C57BL/6J mice were randomized into sham-operated group,HIBD(induced by ligation of the left common carotid artery followed by hypoxia for 40 min)group,and HIBD with gastrodin treatment groups(n=12).In gastrodin treatment group,100 mg/kg gastrodin was injected intraperitoneally 1 h before and at 2 and 12 h after hypoxia.After the treatments,the expressions of CCR5,AKT,p-AKT,and TNF-α and the co-expression of IBA1 and CCR5 in the corpus callosum of the mice were detected with Western blotting and immunofluorescence double staining.In a BV2 microglial cell model of oxygen-glucose deprivation(OGD),the effects of pretreatment with gastrodin and Maraviroc(an CCR5 antagonist)on protein expressions of CCR5,AKT,p-AKT,TNF-α and IL-1β were evaluated using Western blotting and immunofluorescence double staining.Results The neonatal mice with HIBD showed significantly increased expressions of CCR5 and TNF-α with lowered p-AKT expression in the brain tissues,and GAS treatment obviously reversed these changes.HIBD also significantly increased the co-expression of IBA1 and CCR5 in the corpus callosum of the mice,which was obviously lowered by gastrodin treatment.In BV2 cells,OGD significantly increased the expressions of CCR5,TNF-α,and IL-1β and decreased the expression of p-AKT,and these changes were inhibited by treatment with gastrodin,Maraviroc or their combination;the inhibitory effect of the combined treatment did not differ significantly from that of gastrodin or Maraviroc alone.Conclusion Gastrodin can produce neuroprotective effects in neonatal mice with HIBD by inhibiting inflammatory cytokine production and activate AKT phosphorylation via inhibiting CCR5.

Objective:To investigate the effect of gastrodin(GAS)on the sex-determining region Y-box2(SOX2)/β-catenin pathway in microglia induced by lipopolysaccharide(LPS).Methods:BV2 microglia was cultured in vitro and divided into the following groups:Control group(Control),LPS group(LPS),LPS+0.17 mmol/L gastrodin treatment group(LPS+GAS-L),LPS+0.34 mmol/L gastrodin treatment group(LPS+GAS-H),SOX2 inhibitor pronethalolgroup(PR),LPS+PR group(LPS+PR),and LPS+PR+GAS group(LPS+PR+GAS).Effect of PR on BV2 microglia viability was detected by CCK-8.The expression of SOX2,β-catenin,mannose receptor(CD206)and tumor necrosis factor-α(TNF-α)was assessed using Western Blot and immunofluorescence double staining.Results:PR did not induce significant BV2 cell death in the 0～40 μmol/L range.After LPS treatment,the expression levels of SOX2,β-catenin,and TNF-α significantly increased in the LPS group,while CD206 decreased(P＜0.05).Following GAS treatment,the expression levels of SOX2,β-catenin,and TNF-α significantly decreased,while CD206 increased(P＜0.05).Compared to the LPS group,the expression levels of β-catenin and TNF-α significantly de-creased in the PR group(P＜0.05),but no significant difference was observed between the LPS+GAS and LPS+PR+GAS group.Conclusion:GAS significantly inhibits LPS-induced microglia activation potentially through the inhibi-tion of the SOX2/β-catenin signaling pathway,and exerts anti-inflammatory effects.

Objective To investigate the mechanism of gastrodin for inhibiting microglia-mediated inflammation after hypoxic-ischemic brain damage(HIBD)in neonatal rats.Methods Thirty-nine 3-day-old SD rats were randomly divided into sham group,HIBD group and gastrodin treatment group.Western blotting was used to detect the expressions of TNF-α,IL-1β,IL-10 and TGF-β1 in the corpus callosum of the rats.The potential targets of gastrodin for treatment of HIBD were screened by network pharmacology analysis.The expressions of PI3K/AKT signaling pathway proteins following HIBD-induced microglial activation in the rats and in cultured microglial BV-2 cells with oxygen-glucose deprivation(OGD)were detected with Western blotting.The effects of LY294002(a specific inhibitor of the PI3K/AKT pathway)and gastrodin on TNF-α and TGF-β1 mRNA levels in BV-2 cells with OGD was detected with RT-qPCR.Results In the neonatal rats with HIBD,gastrodin treatment significantly decreased TNF-α and IL-1β expressions and enhanced IL-10 and TGF-β1 expressions in the ischemic corpus callosum.Network pharmacology analysis showed significant enrichment of the PI3K/AKT signaling pathway and a strong binding between gastrodin and PI3K.Gastrodin significantly promoted PI3K and AKT phosphorylation in neonatal rats with HIBD and in BV-2 cells exposed to OGD.In BV-2 cells with OGD,gastrodin obviously suppressed OGD-induced increase of TNF-α and reduction of TGF-β1 mRNA expressions,and this effect was strongly attenuated by LY294002 treatment.Conclusion Gastrodin can inhibit microglia-mediated inflammation in neonatal rats with HIBD by regulating the PI3K/AKT signaling pathway.

Objective To investigate the mechanism behind the protective effects of gastrodin against microglia-mediated inflammatory responses following hypoxic-ischemic brain damage(HIBD)in neonatal mice.Methods Thirty-six 10-day-old C57BL/6J mice were randomized into sham-operated group,HIBD(induced by ligation of the left common carotid artery followed by hypoxia for 40 min)group,and HIBD with gastrodin treatment groups(n=12).In gastrodin treatment group,100 mg/kg gastrodin was injected intraperitoneally 1 h before and at 2 and 12 h after hypoxia.After the treatments,the expressions of CCR5,AKT,p-AKT,and TNF-α and the co-expression of IBA1 and CCR5 in the corpus callosum of the mice were detected with Western blotting and immunofluorescence double staining.In a BV2 microglial cell model of oxygen-glucose deprivation(OGD),the effects of pretreatment with gastrodin and Maraviroc(an CCR5 antagonist)on protein expressions of CCR5,AKT,p-AKT,TNF-α and IL-1β were evaluated using Western blotting and immunofluorescence double staining.Results The neonatal mice with HIBD showed significantly increased expressions of CCR5 and TNF-α with lowered p-AKT expression in the brain tissues,and GAS treatment obviously reversed these changes.HIBD also significantly increased the co-expression of IBA1 and CCR5 in the corpus callosum of the mice,which was obviously lowered by gastrodin treatment.In BV2 cells,OGD significantly increased the expressions of CCR5,TNF-α,and IL-1β and decreased the expression of p-AKT,and these changes were inhibited by treatment with gastrodin,Maraviroc or their combination;the inhibitory effect of the combined treatment did not differ significantly from that of gastrodin or Maraviroc alone.Conclusion Gastrodin can produce neuroprotective effects in neonatal mice with HIBD by inhibiting inflammatory cytokine production and activate AKT phosphorylation via inhibiting CCR5.