Currently, researchers have identified several mutated genes associated with hereditary eye diseases; however, effective therapeutic options remain scarce. The emergence of clustered regularly interspaced short palindromic repeats(CRISPR)and its associated proteins(CRISPR-associated proteins, Cas)offers a promising approach for treating these diseases. CRISPR/Cas9 enables precise targeting and modification of specific genetic sequences, allowing for the correction of mutated genes, as well as knockout or replacement of pathogenic genes to achieve therapeutic effects. In ophthalmology, CRISPR/Cas9 has been applied to various hereditary eye disorders, including corneal dystrophy, congenital cataracts, glaucoma, and retinitis pigmentosa. Additionally, significant progress has been made to utilize CRISPR/Cas9 to develop disease models. Therefore, it has great potential for clinical applications. However, challenges such as delivery efficiency and off-target effects remain. This review summarizes the mechanism of CRISPR/Cas9, its applications in genetic eye diseases and disease models, as well as the existing challenges, aiming to provide new insights for treatment.

BACKGROUND:Copper is an essential trace element and plays a key role in series of physiological activities in the body.Metabolic disturbance of copper is closely associated with multiple diseases.Copper metabolism is mainly involved in the absorption,transport,storage and excretion of copper ions,and all the above processes regulate copper homeostasis in the body.In recent years,many studies have confirmed that copper homeostasis disorder severely affects the metabolic activities of the body and cause diseases in various systems.Besides,the role of copper in oral diseases has been of great interest.OBJECTIVE:To explore the role of copper in occurrence,development,and treatment of oral diseases,and provide a comprehensive overview of research advances in this field.METHODS:The first author searched relevant studies on copper in oral diseases using a computer in PubMed,Web of Science,and CNKI.The key words were"Cu,copper,copper metabolism,oral diseases,oral squamous cell carcinoma,periodontitis,oral submucous fibrosis,oral lichen planus,recurrent oral ulceration,pultitis"in English and Chinese.After screening,78 articles were included for further review and analysis.RESULTS AND CONCLUSION:(1)Patients with oral squamous cell carcinoma have elevated concentrations of copper in serum and saliva,and elevated copper promotes cancer progression through oxidative stress and promoting angiogenesis.Excessive elevation or reduction of copper concentration in tumor cells can inhibit the growth of tumor cells.The combination of copper and anticancer drugs can significantly improve the efficacy of drugs.(2)The concentration of copper in the serum of patients with periodontitis is increased,and excessive copper can aggravate periodontitis through promoting oxidative stress.Combination of copper and drugs can promote periodontal bone regeneration and periodontal tissue healing.(3)The level of copper is positively correlated with degree of oral mucosa fibrosis.Copper that enters the oral mucosa promotes fibrosis of oral mucosa by enhancing activity of lysyloxidase to increase production of collagen.(4)Copper levels are elevated in patients with oral lichen planus,and elevated copper may promote the progression of oral lichen planus by modulating immune cell function.(5)In patients with recurrent oral ulcers,serum copper level is significantly increased,and utilization of copper becomes disordered,which could decrease copper-containing enzyme activity,thus affecting the healing of ulcers.Copper is closely associated with multiple oral diseases and therapies targeting at copper could obviously enhance the therapeutic effect of drugs.But further studies are still needed to uncover its mechanisms to lay foundation for the better treatment of oral diseases.

AIM:This study aims to investigate how nuclear factor E2-related factor 2(Nrf2)influences the proliferation of primary rat pulmonary artery smooth muscle cells(PASMCs)under CoCl2-induced hypoxic conditions,spe-cifically through the aerobic glycolysis pathway,and to elucidate its potential mechanisms of action.METHODS:Prima-ry Sprague-Dawley(SD)rat PASMCs were isolated via enzymatic digestion and exposed to 200 μmol/L CoCl2 to establish a chemical hypoxia cell model.Experiments were conducted at various time points(0,6,24 and 48 h)to assess changes in the protein expression of key aerobic glycolysis enzymes[pyruvate kinase M2(PKM2),lactate dehydrogenase A(LDHA)and monocarboxylate transporter 4(MCT4)]and proliferating cell nuclear antigen(PCNA)in PASMCs subjected to CoCl2 stimulation.The optimal concentrations of the Nrf2 activator dimethyl fumarate(DMF)and the inhibitor ML385 were de-termined using Western blot analysis.Subsequently,the cells were divided into four groups:normoxic control(NC)group,CoCl2 group,CoCl2+DMF group,and NC+ML385 group.The proliferation and migration abilities,along with the protein expression levels of HIF-1α,Nrf2,and key aerobic glycolysis enzymes,were assessed in PASMCs from each group.RESULTS:In the hypoxic model of rat PASMCs induced by CoCl2,Nrf2 protein levels significantly decreased over time(P<0.05),while the levels of key enzymes and PCNA protein significantly increased(P<0.05).Furthermore,the migratory capability of PASMCs was markedly enhanced(P<0.05).Under DMF intervention,the protein expression of key aerobic glycolysis enzymes decreased significantly,along with a reduction in the proliferation and migration abilities of rat PASMCs(P<0.05).Conversely,the experimental results showed opposite trends when Nrf2 expression was inhibit-ed by ML385(P<0.05).CONCLUSION:Nrf2 plays a crucial role in mitigating the proliferation and migration of chemi-cally hypoxic primary rat PASMCs by downregulating aerobic glycolysis.

Neovascular fundus diseases mainly include neovascular age-related macular degeneration(nAMD)and dia-betic retinopathy(DR).Pathological neovascular leakage and the subsequent retinal detachment are the main causes of se-vere visual impairment.Anti-vascular endothelial growth factor(VEGF)is the first-line treatment for neovascular fundus diseases,but it has shortcomings,such as the need for frequent intravitreal injections and poor patient compliance.With the annually increasing incidence of acquired neovascular fundus diseases like nAMD and DR,there is an urgent need for safer and more long-lasting treatment options.In recent years,the field of gene therapy has advanced rapidly,with thera-peutic strategies mainly involving gene supplementation and editing.The mechanism underlying gene therapy can be suc-cinctly described as the correction of pathological alterations induced by defective genes.This is achieved either by the in-troduction of exogenous functional genes to restore normal cellular processes or by directly editing aberrant genes at the ge-nomic level.Extensive basic and clinical research has demonstrated that gene therapy is both safe and effective.There are dozens of clinical trials on retinal gene therapy being carried out currently,focusing not only on inherited retinal diseases but also on neovascular fundus diseases.In this article,the application of the gene supplementation,clustered regularly in-terspaced short palindromic repeats(CRISPR)and CRISPR-associated protein 9(Cas9)system in the treatment of nAMD and DR is summarized.

ObjectiveBased on the adenosine 5'-monophosphate （AMP）-activated protein kinase/protein kinase B/nuclear factor erythroid 2-related factor 2 （AMPK/Akt/Nrf2） pathway， this study aims to explore the mechanism by which modified Guishenwan improves glucose and lipid metabolism and oxidative stress in polycystic ovary syndrome （PCOS） rats. MethodsA PCOS rat model was established by continuous oral administration of letrozole （1 mg·kg^-1·d^-1） for 21 days. Successfully modeled rats were randomly divided into a model group， a metformin group （0.25 g·kg^-1）， and low-， medium-， and high-dose modified Guishenwan groups （4.01， 8.02， and 16.04 g·kg^-1·d^-1）， with 8 rats in each group. Ten normal rats were assigned to the normal group. The drug groups were given their respective doses， while the normal and model groups were given an equal volume of normal saline. Intervention lasted for 4 weeks. Testosterone （T）， estradiol （E₂）， follicle-stimulating hormone （FSH）， and luteinizing hormone （LH） were measured by enzyme-linked immunosorbent assay （ELISA）， and the LH/FSH ratio was calculated. Fasting blood glucose （FPG）， fasting insulin （FINS）， triglyceride （TG）， and total cholesterol （TC） levels were measured using an automatic biochemical analyzer， and the insulin resistance index （HOMA-IR） and insulin sensitivity index （HOMA-ISI） were calculated. Oral glucose tolerance test （OGTT） and insulin tolerance test （ITT） were conducted. Malondialdehyde （MDA）， advanced glycation end products （AGEs）， and superoxide dismutase （SOD） levels in serum and ovarian tissue were measured using a chemical fluorescence method. Hematoxylin-eosin （HE） staining was used to assess ovarian tissue pathology. Real-time quantitative fluorescent polymerase chain reaction （Real-time PCR） and Western blot were used to measure the expression of AMPK/Akt/Nrf2 pathway-related genes and proteins in ovarian tissue. ResultsCompared with the normal group， the model group exhibited significantly increased levels of T， LH， LH/FSH， FPG， FINS， TG， TC， and HOMA-IR， while FSH， E₂， and HOMA-ISI were significantly decreased （P<0.05， P<0.01）. MDA and AGEs levels were significantly higher in both serum and ovarian tissue， and SOD levels were significantly reduced （P<0.05）. AMPK， Akt， and Nrf2 mRNA and protein expression in ovarian tissue was also significantly reduced （P<0.05）. The OGTT and ITT results showed significantly higher blood glucose levels at each time point （P<0.05， P<0.01）， with impaired glucose and insulin tolerance. Ovarian follicles showed polycystic changes， reduced corpus luteum， and sparse granulosa cell layers. Compared with the model group， the metformin group and the high-dose modified Guishenwan group showed significant decreases in T， LH， LH/FSH， FPG， FINS， TG， TC， and HOMA-IR， while FSH， E₂， and HOMA-ISI were significantly increased （P<0.05， P<0.01）. In the high-dose modified Guishenwan group， MDA and AGEs levels in serum and ovarian tissue were significantly reduced， and SOD levels were significantly increased （P<0.05）. The mRNA and protein expression of AMPK， Akt， and Nrf2 in ovarian tissue was significantly increased （P<0.05）. OGTT and ITT results showed that blood glucose levels in rats decreased significantly at each time point （P<0.05， P<0.01）. No obvious abnormalities were observed in ovarian tissue. Compared with the low-dose modified Guishenwan group， the high-dose group showed significant decreases in T， LH， LH/FSH， FPG， FINS， TG， TC， and HOMA-IR， while FSH， E₂， and HOMA-ISI were significantly increased （P<0.05）. OGTT and ITT results indicated that the high-dose modified Guishenwan group significantly improved glucose and insulin tolerance in rats. No significant abnormalities were observed in ovarian tissue. ConclusionModified Guishenwan effectively improves glucose and lipid metabolism abnormalities and inhibits oxidative stress in PCOS rats， potentially through regulation of the AMPK/Akt/Nrf2 pathway.

ObjectiveTo explore the mechanism by which modified Guishenwan alleviates inflammation in the rat model of polycystic ovary syndrome （PCOS） by regulating the mitogen-activated protein kinase （MAPK）/nuclear factor kappa B （NF-κB） pathway. MethodsAccording to the random number table method， 60 SPF female SD rats were randomized into a normal group （n=10） and a modeling group （n=50）. The normal group received routine feeding， while the modeling group was administrated with letrozole （1 mg·kg^-1·d^-1） by gavage for 21 days for the modeling of PCOS. The successfully modeled rats were randomized into model， diane-35 （0.2 g·kg^-1·d^-1）， high- （16.04 g·kg^-1·d^-1）， medium- （8.02 g·kg^-1·d^-1）， low- （4.01 g·kg^-1·d^-1） dose modified Guishenwan groups. The drug intervention groups were administrated with modified Guishenwan at corresponding doses by gavage， and the normal group and model group were given equal volumes of normal saline. All the groups were continuously treated for 28 days. After treatment， Gram staining of vaginal smears was employed to observe the estrous cycle in each group. Enzyme-linked immunosorbent assay was employed to determine the levels of follicle-stimulating hormone （FSH）， estradiol （E₂）， luteinizing hormone （LH）， testosterone （T）， and progesterone （PROG） in the plasma， as well as interleukin-1 beta （IL-1β）， tumor necrosis factor-alpha （TNF-α）， and interleukin-10 （IL-10） in the plasma and ovarian tissue. The LH/FSH ratio was calculated. The morphological changes in the ovarian tissue were observed by hematoxylin-eosin （HE） staining. Western blot was employed to determine the protein levels of extracellular-regulated protein kinase （ERK）， c-Jun N-terminal kinase （JNK）， p38 MAPK， NF-κB p65， IκBα， p-JNK， p-ERK， p-p38 MAPK， p-NF-κB p65， and p-IκBα in the ovarian tissue. Real-time quantitative polymerase chain reaction was used to determine the mRNA levels of ERK， JNK， p38 MAPK， NF-κB p65， and IκBα in the ovarian tissue. ResultsCompared with the normal group， the model group was in the estrus phase， with an increase in the number of ovarian vesicles and decreases in granulosa cells and corpus luteum formation （P<0.05）， and lowered levels of FSH and E₂ and elevated levels of LH， T， and LH/FSH in the plasma （P<0.05）. Compared with the model group， high-， medium-， and low-dose modified Guishenwan recovered the estrous cycle， increased the generation of granulosa cells and corpus luteum， reduced the number of vesicles， elevated the levels of FSH and E₂， and lowered the levels LH， T， and LH/FSH （P<0.05， P<0.01） in a dose-dependent manner. High-dose modified Guishenwan demonstrated the best therapeutic effect. Therefore， subsequent experiments for exploring the treatment mechanism were conducted in the normal group， model group， and high-dose modified Guishenwan group. The results showed that compared with the model group， high-dose modified Guishenwan lowered the levels of IL-1β， TNF-α， and IL-10 and elevated the level of IL-10 in the plasma and ovarian tissue （P<0.05， P<0.01）， down-regulated the protein levels of p-ERK， p-JNK， p-p38 MAPK， p-NF-κB p65， and p-IκBα， while up-regulating the protein level of IκBα （P<0.01）. At the same time， the mRNA levels of ERK， JNK， p38 MAPK， and NF-κB p65 in the high-dose modified Guishenwan group were down-regulated （P<0.05， P<0.01）. ConclusionModified Guishenwan can improve the ovarian function in rat model of PCOS induced by letrozole and has anti-inflammatory effects， which may be related to inhibition of the MAPK/NF-κB pathway.

Sepsis-acquired weakness (SAW) is a common complication in critically ill patients, yet significant gaps remain in both mechanistic understanding and therapeutic interventions for this condition. SAW not only prolongs the duration of mechanical ventilation and hospitalization but is also closely associated with increased mortality. Even if these SAW patients survive, they often experience long-term physical dysfunction after hospital discharge, leading to diminished quality of life. Emerging evidence suggests that sustained mitochondrial dysfunction may constitute a pivotal pathophysiological basis for the development and progression of SAW, primarily encompassing five key aspects: dysregulated mitochondrial quality control (MtQC), impaired oxidative phosphorylation (OXPHOS), exacerbated oxidative stress, disrupted Ca²⁺; homeostasis, and their mediation of diverse myofiber injuries. This article systematically elucidates the central role of mitochondrial dysfunction in the pathogenesis of SAW. Furthermore, we explore potential therapeutic strategies targeting mitochondrial function, including mitigating mitochondrial oxidative stress, optimizing nutritional support, and supplementing with muscle-derived mesenchymal stem cells. These insights provide a critical theoretical framework for understanding SAW mechanisms and developing clinical interventions, with particular emphasis on the translational value of mitochondrial-targeted therapies in improving outcomes for septic patients.
Humans ; Sepsis/metabolism* ; Mitochondria/metabolism* ; Muscle Weakness/etiology* ; Oxidative Stress ; Oxidative Phosphorylation

Objective To investigate the role of the prefrontal cortex in the generation and adaptation of chro-nic subjective tinnitus using resting-state functional magnetic resonance imaging(rs-fMRI).Methods Resting-state functional magnetic resonance imaging scan were acquired from 20 patients with chronic subjective tinnitus and 20 healthy controls.Fractional amplitude of low-frequency fluctuations(fALFF)and seed-based whole-brain functional connectivity(FC)methods were used to detect abnormal prefrontal cortex activity in tinnitus patients and to investi-gate interactions between prefrontal cortex activity and brain regions associated tinnitus perception.The analysis aimed to assess the relationship between prefrontal cortex spontaneous neural activity,atypical functional connectivi-ty across various brain regions,and clinical characteristics of tinnitus.Results Compared with healthy controls,pa-tients with chronic tinnitus showed a significant reduction in fALFF values in some specific brain areas of prefrontal cortex,including the left/right medial superior frontal gyrus and the left/right middle frontal gyrus.Functional con-nectivity values were notably enhanced between the left medial superior frontal gyrus and the left anterior insula,as well as between the right medial superior frontal gyrus and the left superior temporal gyrus.Furthermore,increased functional connectivity was observed between the left middle frontal gyrus and the right middle temporal gyrus,as well as between the right middle frontal gyrus and the left parahippocampal gyrus,left superior parietal lobule,and left supplementary motor area.Importantly,the functional connectivity between the left middle frontal gyrus and the right superior temporal gyrus exhibited a negative correlation with tinnitus handicap inventory scores(r=-0.627,P=0.003)and visual analogue scale scores(r=-0.596,P=0.005).Conclusion There are abnormal brain function changes in medial prefrontal cortex and dorsolateral prefrontal cortex in patients with chronic subjec-tive tinnitus,accompanied by changes in the intensity of functional connections with the salience and auditory net-works.These abnormalities are highly related to the severity of tinnitus.The prefrontal cortex may play an impor-tant role in the sensory prediction and auditory regulation of tinnitus.

Objective To investigate the protective effects and mechanism of Shuilian Fengkui Formula(SFF)of Yao medicine on sequelae pelvic inflammatory disease(SPID)in rats.Methods The SPID rat model was established by mechanical injury coupled with bacterial infection,and then the rats were randomly divided into:the SPID group,the positive drug(Fukeqianjin tablets)group,SFF groups at low,medium and high doses,and the sham-operation group,with 8 rats in each group;SPID group,SFF groups,TLR4 agonist CRX-527(CRX-527)group,SFF plus CRX-527 group,with 8 rats in each group.Corresponding drug intervention was given for 21 consecutive days.The indexes of uterus,spleen,thymus and other organs were calculated.The histopathological changes in rat uterus were observed by HE staining.The levels of interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),immunoglobulin G(IgG),immunoglobulin M(IgM)in serum were detected by ELISA;The protein expression levels of Toll receptor 4(TLR4),myeloid differentiation factor(MyD88),nuclear factor kappa B p65(NF-κB p65),and phosphorylated nuclear factor kappa B p65(p-NF-κB p65)in rat uterine tissues were detected by Western blot.Results Compared with the sham operation group,the uterine index of SPID group significantly increased(P<0.01),the spleen index and thymus index significantly decreased(P<0.01),the pathological damage of uterine tissue was serious,the contents of IL-1β,IL-6,TNF-α in serum significantly increased(P<0.01),the contents of IgG and IgM significantly decreased(P<0.01),the TLR4,MyD88 protein and the ratio of p-NF-κB p65/NF-κB p65 protein in uterine tissue significantly increased(P<0.01).Compared with the SPID group,the uterine index of the positive drug group and the SFF groups significantly decreased(P<0.05),the spleen index and thymus index significantly increased(P<0.05),the pathological damage of uterine tissue significantly improved,the contents of IL-1β,IL-6 and TNF-α in serum were significantly lowered(P<0.05),the contents of IgG and IgM significantly increased(P<0.05),and the TLR4,MyD88 protein and the ratio of p-NF-κB p65/NF-κB p65 protein in uterine tissue significantly downregulated(P<0.01).However,CRX-527 significantly reversed the effects of SFF on SPID in rats.Conclusion SFF could improve SPID in rats by impeding the inflammatory response,and its mechanism might be attributed to inhibition of TLR4/MyD88/NF-κB signaling pathway.

This study aims to screen epitope antigens targeting the receptor binding domain(RBD)of porcine epidemic diarrhea virus(PEDV)based on its amino acid sequence(GenBank accession number:AKN45969.1),prepare PEDV RBD polyclonal antibody,and perform their identification.Bioinformatics analysis software was used to predict the potential antigenic epitopes of PEDV RBD and sequence comparison with porcine coronavirus strains was performed,the selected dominant antigen epitopes were then conjugated with keyhole limpet hemocyanin(KLH),to synthesize pep-tides directly and immunize mice to generate specific antibody,Western blot technique and indirect immunofluorescence assay were utilized to identify the specificity of the antibodies,and indirect ELISA method was further applied to determine the antibody potency.Results showed the selected PEDV RBD dominant epitope sequence shared 100％similarity with 18 other PEDV strains,while exhibiting low sequence similarity with 11 TGEV strains(27.8％—29.3％)and 16 PDCoV strains(10.5％—13.4％),indicating good epitope conservation.Western blot showed that the specificity of the prepared peptide antibody specifically recognized the PEDV RED protein overexpressed in Ex-pi293F cells and overexpressed in baculovirus system,and at the same time,the antibody was still able to detect the PEDV S protein expressed in PEDV-infected Vero cells at a 1∶2 000 dilution,while it did not react with TGEV-and PDCoV-infected ST cells,indicating that the good specificity of the peptide antibody.ELISA revealed that the potency of specific antibodies in mouse serum could reach up to 1∶25 600.The above results indicate that bioinformatics techniques were suc-cessfully utilized to predict antigenic epitopes of PEDV RBD protein,and specific PEDV RBD pep-tide antibodies were prepared.