1.Influencing factors of significant corneal astigmatism in pterygium patients during the perioperative period
Shiru CHAI ; Xiaofen ZHENG ; Hua YU ; Zhen LI ; Yuguo KANG
International Eye Science 2026;26(4):683-686
AIM: To explore the factors associated with significant corneal astigmatism during the perioperative period in patients with pterygium. METHODS: Patients with primary pterygium presenting at Shanxi Eye Hospital between February and June 2025 were enrolled. All patients underwent medical history collection. Pre- and postoperative data were obtained using Pentacam, anterior segment photography, Image J software, and anterior segment optical coherence tomography(AS-OCT). All patients underwent pterygium excision combined with autologous bulbar conjunctival flap transplantation under local infiltration anesthesia. RESULTS: A total of 76 patients(76 eyes)with pterygium were finally enrolled(30 males, 46 females)with a mean age of 62.2±8.2 y. The mean length of corneal invasion by pterygium was 3.61±0.89 mm, the mean depth of invasion into the anterior corneal surface was 0.15±0.09 mm, and the median area of corneal invasion was 10.25(6.90, 18.75)mm2. The median preoperative corneal astigmatism was 1.50(0.70, 5.45)D. Median astigmatism was 0.8(0.40, 1.28)D at 2 wk postoperatively and 0.60(0.30, 1.15)D at 1 mo postoperatively. Patient age showed a positive correlation with preoperative astigmatism, and with residual astigmatism at 2 wk and 1 mo postoperatively(all P<0.05). The length of corneal invasion was positively correlated with preoperative astigmatism and residual astigmatism at both postoperative timepoints(P<0.01). The depth of invasion showed no significant linear correlation with astigmatism at any stage(P=0.250, 0.761, 0.686). The area of corneal invasion was positively correlated with astigmatism at all stages(P<0.01). Patients were grouped based on significant astigmatism(≥1.0 D)and non-significant astigmatism(<1.0 D), after adjusting for other variables, age(P=0.031)and the area of corneal invasion(P=0.004)were identified as risk factors for significant astigmatism. Pterygium invasion length was not significant factors(P>0.05). Receiver operating characteristic(ROC)analysis showed the highest area under the curve(AUC)for the invasion area(AUC=0.915). CONCLUSION: Significant preoperative corneal astigmatism in pterygium patients is correlated with patient age, the length of corneal invasion, and the area of invasion. The area of pterygium invasion into the cornea is the strongest predictor of significant preoperative corneal astigmatism.
2.Construction of An Automated Segmentation Visual Foundation Model for Pathological Images of Hemorrhoids and Its Application in Traditional Chinese Medicine Clinical Syndrome Analysis
Shijie ZHANG ; Ao ZHANG ; Kang WANG ; Bin KANG ; Xiaofan YU ; Xujing FENG ; Jinyu CAO ; Wenzhen HUANG ; Kang DING
Journal of Traditional Chinese Medicine 2026;67(7):764-769
This paper proposes a two-stage method integrating visual foundation models (VFM) and diffusion models. The segment anything model (SAM) as VFM is combined with the SegRefiner diffusion model to construct the SAM-SegRefiner framework for automated segmentation of edema, inflammation, and thrombus regions in histopathological images of hemorrhoidal tissue, providing a reproducible technical tool for the objective quantification of pathological morphology and its application in traditional Chinese medicine (TCM) syndrome research. Trained and validated on multi-center retrospective data, the SAM-SegRefiner model achieved an average pixel accuracy of 95.32% and a mean intersection over union (mIoU) of 66.81% on an independent test set, significantly outperfor-ming comparative models such as U-Net, MixU-Net, and SAM-Med2D, and also demonstrating robust cross-center generalization capability. Furthermore, by correlating the quantitatively segmented results from the model with the patients' TCM syndrome types, the potential associations between pathomorphological features and TCM syndrome differentiation have been explored. The analysis revealed no statistically significant differences in the degree of inflammatory infiltration and thrombus formation among different syndrome types, suggesting a complex relationship between local pathological changes and systemic syndrome manifestations.
3.Mechanism of Different Dosage Forms of Kaixinsan in Improving Mitochondrial Function for Prevention and Treatment of Cognitive Disorder Based on AMPK/PGC-1α/SIRT3 Pathway
Shuyue KANG ; Yanzi YU ; Jiaqun SUN ; Wenxuan CHEN ; Yaqin YANG ; Qi WANG ; Weirong LI ; Limei YAO
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):15-24
ObjectiveTo explore the effects of different dosage forms of Kaixinsan (KXS) on the morphology and function of mitochondria in rat models of Alzheimer's disease (AD) and potential mechanisms of action. MethodsMale SD rats were randomly assigned to a sham group, model group, treatment groups receiving KXS decoction, powders, and granules (3.08 g·kg-1), as well as donepezil group (0.51×10-3 g·kg-1), with 10 rats in each group. AD model was created using intracerebroventricular injection of streptozocin (STZ). After 30 days of administration, behavioral assessments were conducted, and mitochondrial morphology was observed using transmission electron microscopy. Mitochondrial respiratory chain complex content was measured via enzyme-linked immunosorbent assay (ELISA). Changes in mitochondrial membrane potential were measured via JC-1 staining, and superoxide dismutase (SOD) activity and reactive oxygen species (ROS) levels were measured via biochemical assays. The mRNA expression of adenosine 5'-monophosphate-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), and silent information regulator 3 (SIRT3) was detected by real-time fluorescent quantitative polymerase chain reaction (Real-time PCR), and Western blot was used to examine the protein expression levels of optic atrophy protein1 (OPA1), mitochondrial fission protein 1 (FIS1), AMPK, p-AMPK, PGC-1α, and SIRT3. ResultsCompared with the sham group, rats in the model group had significantly lower recognition index, spontaneous alternation rate, escape latency, number of platform crossings, time spent in the target quadrant, and percentage of distance traveled in the target quadrant distance (P<0.05, P<0.01). Significant mitochondrial damage was observed in the hippocampal tissue, with a marked decrease in mitochondrial respiratory chain complex content (P<0.01) and reduced mitochondrial membrane potential (P<0.05). Additionally, the SOD activity was reduced, while ROS levels were elevated (P<0.01). The mRNA expression of PGC-1α and SIRT3 was significantly downregulated (P<0.01), along with decreased protein expression levels of OPA1, p-AMPK/AMPK, PGC-1α, and SIRT3, whereas FIS1 protein expression was significantly upregulated (P<0.05, P<0.01). Compared with the model group, rats in KXS-treated groups (various dosage forms) showed significant improvement in behavioral indexes (P<0.05, P<0.01), reduced hippocampal mitochondrial damage, and more organized mitochondrial cristae. Mitochondrial respiratory chain complex content was significantly increased (P<0.05, P<0.01), and mitochondrial membrane potentials were elevated (P<0.05). SOD activity was elevated, and ROS levels were significantly reduced (P<0.05, P<0.01). Furthermore, the mRNA expression of PGC-1α and SIRT3 was upregulated, with increased protein levels of OPA1, p-AMPK/AMPK, PGC-1α, and SIRT3, while FIS1 protein expression levels were significantly reduced (P<0.05, P<0.01). Across the KXS-treated groups, the granule group showed a higher spontaneous alternation rate than the decoction and powder groups (P<0.05). ConclusionKXS decoction, powders, and granules can improve the learning and memory ability of rats, with granules being the most effective. The mechanism of action may involve activation of the AMPK/PGC-1α/SIRT3 signaling pathway, improvement of the mitochondrial function, and subsequent amelioration of the brain energy metabolism disorders.
4.Effect of Shenxiong Huanglian Jiedu Decoction on Neuronal Damage and Aβ Clearance in Mice Model of Alzheimer's Disease
Jing LIU ; Kang CHEN ; Yushun ZHOU ; Zhezuo ZHANG ; Guran YU ; Hao LI
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):43-52
ObjectiveTo investigate the effects of Shenxiong Huanglian Jiedu decoction on the clearance of amyloid β-protein (Aβ) and neuronal damage in the mouse model of Alzheimer's disease (AD). MethodsA total of 36 SPF-grade 2-month-old C57BL/6J mice were used in this study, and the modeling was performed by bilateral hippocampal injection of Aβ oligomers in C57BL/6J mice. The experiment was conducted with a blank group, a sham operation group, a model group, low- and high-dose (3.27,6.54 g·kg-1, respectively) Shenxiong Huanglian Jiedu decoction groups, and a positive control (donepezil hydrochloride, 0.65 mg·kg-1) group. At the end of the drug intervention, the learning and memory abilities and the activities of mice were evaluated by the Morris water maze and open field tests. Brain histopathology was examined by hematoxylin-eosin and Nissl staining. Additionally, in vivo imaging was employed to measure the metabolism of fluorescent Aβ in the cerebrospinal fluid, and staining of ionized calcium-binding adapter molecule-1 (Iba-1) was employed to assess microglial activation in the hippocampal tissue. Additionally, neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) levels in the brain tissue and serum were determined by the immunofluorescence assay and enzyme-linked immunosorbent assay. Western blot was conducted to determine the expression of inflammation and pathway-related proteins in the hippocampal tissue. ResultsCompared with the blank group and the sham operation group, the escape latency of the mice in the model group was prolonged, the platform residence time was shortened, the hippocampal tissue showed pathological manifestations such as neuronal pyknosis, Nissl body dissolution, and microglia activation. The metabolic rate of fluorescent Aβ through cerebrospinal fluid was slowed down, and the expression levels of BDNF, NT-3, and interleukin-10 (IL-10) in the hippocampus were significantly decreased (P<0.01). The expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and phosphorylated nuclear transcription factor-κB (p-NF-κB p65) in hippocampus were significantly increased (P<0.05, P<0.01). Compared with the model group, the escape latency of mice in the low and high dose groups of Chinese medicine and donepezil group was shortened, and the platform residence time was prolonged. Neuronal karyopyknosis, Nissl body dissolution and microglia activation in hippocampus were improved. Fluorescence Aβ was metabolized faster by cerebrospinal fluid. The expression of BDNF and NT-3 in hippocampus was increased (P<0.01), and the expression of TLR4, MyD88 and p-NF-κB p65 was significantly decreased (P<0.05, P<0.01). The expression of TNF-α in the hippocampus of the high-dose group was significantly decreased (P<0.05), and the expression of IL-10 was significantly increased (P<0.05). The expression of TNF-α, IL-6 and IL-1β in the hippocampus of the donepezil group was significantly decreased (P<0.05, P<0.01). ConclusionShenxiong Huanglian Jiedu decoction may mitigate neuronal damage and enhance cerebrospinal fluid flow in the mouse model of AD, thereby promoting the clearance of Aβ and improving the learning and memory abilities. These beneficial effects are likely mediated through the inhibition of microglial activation, reduction of inflammation, and modulation of the TLR4/MyD88/NF-κB signaling pathway.
5.Mechanism of Different Dosage Forms of Kaixinsan in Improving Mitochondrial Function for Prevention and Treatment of Cognitive Disorder Based on AMPK/PGC-1α/SIRT3 Pathway
Shuyue KANG ; Yanzi YU ; Jiaqun SUN ; Wenxuan CHEN ; Yaqin YANG ; Qi WANG ; Weirong LI ; Limei YAO
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):15-24
ObjectiveTo explore the effects of different dosage forms of Kaixinsan (KXS) on the morphology and function of mitochondria in rat models of Alzheimer's disease (AD) and potential mechanisms of action. MethodsMale SD rats were randomly assigned to a sham group, model group, treatment groups receiving KXS decoction, powders, and granules (3.08 g·kg-1), as well as donepezil group (0.51×10-3 g·kg-1), with 10 rats in each group. AD model was created using intracerebroventricular injection of streptozocin (STZ). After 30 days of administration, behavioral assessments were conducted, and mitochondrial morphology was observed using transmission electron microscopy. Mitochondrial respiratory chain complex content was measured via enzyme-linked immunosorbent assay (ELISA). Changes in mitochondrial membrane potential were measured via JC-1 staining, and superoxide dismutase (SOD) activity and reactive oxygen species (ROS) levels were measured via biochemical assays. The mRNA expression of adenosine 5'-monophosphate-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), and silent information regulator 3 (SIRT3) was detected by real-time fluorescent quantitative polymerase chain reaction (Real-time PCR), and Western blot was used to examine the protein expression levels of optic atrophy protein1 (OPA1), mitochondrial fission protein 1 (FIS1), AMPK, p-AMPK, PGC-1α, and SIRT3. ResultsCompared with the sham group, rats in the model group had significantly lower recognition index, spontaneous alternation rate, escape latency, number of platform crossings, time spent in the target quadrant, and percentage of distance traveled in the target quadrant distance (P<0.05, P<0.01). Significant mitochondrial damage was observed in the hippocampal tissue, with a marked decrease in mitochondrial respiratory chain complex content (P<0.01) and reduced mitochondrial membrane potential (P<0.05). Additionally, the SOD activity was reduced, while ROS levels were elevated (P<0.01). The mRNA expression of PGC-1α and SIRT3 was significantly downregulated (P<0.01), along with decreased protein expression levels of OPA1, p-AMPK/AMPK, PGC-1α, and SIRT3, whereas FIS1 protein expression was significantly upregulated (P<0.05, P<0.01). Compared with the model group, rats in KXS-treated groups (various dosage forms) showed significant improvement in behavioral indexes (P<0.05, P<0.01), reduced hippocampal mitochondrial damage, and more organized mitochondrial cristae. Mitochondrial respiratory chain complex content was significantly increased (P<0.05, P<0.01), and mitochondrial membrane potentials were elevated (P<0.05). SOD activity was elevated, and ROS levels were significantly reduced (P<0.05, P<0.01). Furthermore, the mRNA expression of PGC-1α and SIRT3 was upregulated, with increased protein levels of OPA1, p-AMPK/AMPK, PGC-1α, and SIRT3, while FIS1 protein expression levels were significantly reduced (P<0.05, P<0.01). Across the KXS-treated groups, the granule group showed a higher spontaneous alternation rate than the decoction and powder groups (P<0.05). ConclusionKXS decoction, powders, and granules can improve the learning and memory ability of rats, with granules being the most effective. The mechanism of action may involve activation of the AMPK/PGC-1α/SIRT3 signaling pathway, improvement of the mitochondrial function, and subsequent amelioration of the brain energy metabolism disorders.
6.Effect of Shenxiong Huanglian Jiedu Decoction on Neuronal Damage and Aβ Clearance in Mice Model of Alzheimer's Disease
Jing LIU ; Kang CHEN ; Yushun ZHOU ; Zhezuo ZHANG ; Guran YU ; Hao LI
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):43-52
ObjectiveTo investigate the effects of Shenxiong Huanglian Jiedu decoction on the clearance of amyloid β-protein (Aβ) and neuronal damage in the mouse model of Alzheimer's disease (AD). MethodsA total of 36 SPF-grade 2-month-old C57BL/6J mice were used in this study, and the modeling was performed by bilateral hippocampal injection of Aβ oligomers in C57BL/6J mice. The experiment was conducted with a blank group, a sham operation group, a model group, low- and high-dose (3.27,6.54 g·kg-1, respectively) Shenxiong Huanglian Jiedu decoction groups, and a positive control (donepezil hydrochloride, 0.65 mg·kg-1) group. At the end of the drug intervention, the learning and memory abilities and the activities of mice were evaluated by the Morris water maze and open field tests. Brain histopathology was examined by hematoxylin-eosin and Nissl staining. Additionally, in vivo imaging was employed to measure the metabolism of fluorescent Aβ in the cerebrospinal fluid, and staining of ionized calcium-binding adapter molecule-1 (Iba-1) was employed to assess microglial activation in the hippocampal tissue. Additionally, neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) levels in the brain tissue and serum were determined by the immunofluorescence assay and enzyme-linked immunosorbent assay. Western blot was conducted to determine the expression of inflammation and pathway-related proteins in the hippocampal tissue. ResultsCompared with the blank group and the sham operation group, the escape latency of the mice in the model group was prolonged, the platform residence time was shortened, the hippocampal tissue showed pathological manifestations such as neuronal pyknosis, Nissl body dissolution, and microglia activation. The metabolic rate of fluorescent Aβ through cerebrospinal fluid was slowed down, and the expression levels of BDNF, NT-3, and interleukin-10 (IL-10) in the hippocampus were significantly decreased (P<0.01). The expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and phosphorylated nuclear transcription factor-κB (p-NF-κB p65) in hippocampus were significantly increased (P<0.05, P<0.01). Compared with the model group, the escape latency of mice in the low and high dose groups of Chinese medicine and donepezil group was shortened, and the platform residence time was prolonged. Neuronal karyopyknosis, Nissl body dissolution and microglia activation in hippocampus were improved. Fluorescence Aβ was metabolized faster by cerebrospinal fluid. The expression of BDNF and NT-3 in hippocampus was increased (P<0.01), and the expression of TLR4, MyD88 and p-NF-κB p65 was significantly decreased (P<0.05, P<0.01). The expression of TNF-α in the hippocampus of the high-dose group was significantly decreased (P<0.05), and the expression of IL-10 was significantly increased (P<0.05). The expression of TNF-α, IL-6 and IL-1β in the hippocampus of the donepezil group was significantly decreased (P<0.05, P<0.01). ConclusionShenxiong Huanglian Jiedu decoction may mitigate neuronal damage and enhance cerebrospinal fluid flow in the mouse model of AD, thereby promoting the clearance of Aβ and improving the learning and memory abilities. These beneficial effects are likely mediated through the inhibition of microglial activation, reduction of inflammation, and modulation of the TLR4/MyD88/NF-κB signaling pathway.
7.Research advances in the disease burden of viral hepatitis in China
Jian LI ; Fuzhen WANG ; Zhongdan CHEN ; Jinlei QI ; Ailing WANG ; Fanghui ZHAO ; Yuanyuan KONG ; Jing SUN ; Jiaqi KANG ; Zundong YIN ; Zhongfu LIU ; Jidong JIA ; Yu WANG
Journal of Clinical Hepatology 2025;41(2):221-227
Over the past three decades, China has made significant progress in the prevention and control of viral hepatitis, and the incidence rates of new-onset pediatric hepatitis B virus infections and acute viral hepatitis in the population have reduced to a relatively low level; however, there is still a heavy disease burden of chronic viral hepatitis in China, which severely affects the health status of the population. This study systematically summarizes the achievements of viral hepatitis prevention and control in China, analyzes existing problems and challenges, and proposes comprehensive prevention and control strategies and measures to eliminate viral hepatitis as a public health threat based on the national conditions of China, in order to provide a reference for related departments in China on how to achieve the action targets for eliminating viral hepatitis as a public health threat by 2030.
8.Anti-osteoporosis Effect of Isorhamnetin: A Review
Shilong MENG ; Xu ZHANG ; Yawei XU ; Yang YU ; Wei LI ; Yanguang CAO ; Xiaolin SHI ; Wei ZHANG ; Kang LIU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(5):347-352
Osteoporosis is a common senile bone metabolism disease, clinically characterized by decreased bone mass, destruction of bone microstructure, increased bone fragility, and easy fracture. It tends to occur in the elderly and postmenopausal women, seriously threatening the quality of life and physical and mental health of the elderly. At present, the treatment of osteoporosis is mainly based on oral western medicines, such as calcium, Vitamin D, and bisphosphonates. Still, there are drawbacks such as a long medication cycle and many adverse reactions. In recent years, due to the advantages of multi-component, multi-pathway, and multi-target, some traditional Chinese medicines and effective ingredients can regulate the osteogenic and osteoclastic differentiation process in both directions and are widely used in the prevention and treatment of osteoporosis. Hippophae rhamnoides is a commonly used herbal medicine, and its fruits are rich in flavonoids, polyphenols, fatty acids, vitamins, and trace elements, which have been proven to have a good anti-osteoporosis effect. Isorhamnetin is the main effective ingredient of Hippophae rhamnoides fruits, which has many pharmacological effects such as anti-inflammation, anti-oxidative stress, anti-aging, and anti-tumor. Studies have shown that isorhamnetin can participate in the regulation of bone metabolism and has a good anti-osteoporosis effect. However, the pharmacological effects and related mechanisms of isorhamnetin against osteoporosis have not been systematically summarized. Therefore, this paper reviewed the pharmacological effects and related mechanisms of isorhamnetin against osteoporosis by referring to relevant literature to provide more basis for the development and application of isorhamnetin.
9.Palpitations, Shortness of Breath, Weakness in Limbs, Edema, and Dyspnea: A Rare Inflammatory Myopathy with Positive Aniti-mitochondrial Antibodies and Cardiac Involvement
Chunsu LIANG ; Xuchang ZHANG ; Ning ZHANG ; Lin KANG ; Xiaohong LIU ; Jiaqi YU ; Yingxian LIU ; Lin QIAO ; Yanli YANG ; Xiaoyi ZHAO ; Ruijie ZHAO ; Na NIU ; Xuelian YAN
Medical Journal of Peking Union Medical College Hospital 2025;16(1):248-255
This article presents a case study of a patient who visited the Geriatric Department of Peking Union Medical College Hospital due to "palpitations, shortness of breath for more than 2 years, limb weakness for 6 months, edema, and nocturnal dyspnea for 2 months". The patient exhibited decreased muscle strength in the limbs and involvement of swallowing and respiratory muscles, alongside complications of heart failure and various arrhythmias which were predominantly atrial. Laboratory tests revealed the presence of multiple autoantibodies and notably anti-mitochondrial antibodies. Following a comprehensive multidisciplinary evaluation, the patient was diagnosed with anti-mitochondrial antibody-associated inflammatory myopathy. Treatment involved a combination of glucocorticoids and immunosuppressants, along with resistance exercises for muscle strength and rehabilitation training for lung function, resulting in significant improvement of clinical symptoms. The case underscores the importance of collaborative multidisciplinary approaches in diagnosing and treating rare diseases in elderly patients, where careful consideration of clinical manifestations and subtle abnormal clinical data can lead to effective interventions.
10.In situ Analytical Techniques for Membrane Protein Interactions
Zi-Yuan KANG ; Tong YU ; Chao LI ; Xue-Hua ZHANG ; Jun-Hui GUO ; Qi-Chang LI ; Jing-Xing GUO ; Hao XIE
Progress in Biochemistry and Biophysics 2025;52(5):1206-1218
Membrane proteins are integral components of cellular membranes, accounting for approximately 30% of the mammalian proteome and serving as targets for 60% of FDA-approved drugs. They are critical to both physiological functions and disease mechanisms. Their functional protein-protein interactions form the basis for many physiological processes, such as signal transduction, material transport, and cell communication. Membrane protein interactions are characterized by membrane environment dependence, spatial asymmetry, weak interaction strength, high dynamics, and a variety of interaction sites. Therefore, in situ analysis is essential for revealing the structural basis and kinetics of these proteins. This paper introduces currently available in situ analytical techniques for studying membrane protein interactions and evaluates the characteristics of each. These techniques are divided into two categories: label-based techniques (e.g., co-immunoprecipitation, proximity ligation assay, bimolecular fluorescence complementation, resonance energy transfer, and proximity labeling) and label-free techniques (e.g., cryo-electron tomography, in situ cross-linking mass spectrometry, Raman spectroscopy, electron paramagnetic resonance, nuclear magnetic resonance, and structure prediction tools). Each technique is critically assessed in terms of its historical development, strengths, and limitations. Based on the authors’ relevant research, the paper further discusses the key issues and trends in the application of these techniques, providing valuable references for the field of membrane protein research. Label-based techniques rely on molecular tags or antibodies to detect proximity or interactions, offering high specificity and adaptability for dynamic studies. For instance, proximity ligation assay combines the specificity of antibodies with the sensitivity of PCR amplification, while proximity labeling enables spatial mapping of interactomes. Conversely, label-free techniques, such as cryo-electron tomography, provide near-native structural insights, and Raman spectroscopy directly probes molecular interactions without perturbing the membrane environment. Despite advancements, these methods face several universal challenges: (1) indirect detection, relying on proximity or tagged proxies rather than direct interaction measurement; (2) limited capacity for continuous dynamic monitoring in live cells; and (3) potential artificial influences introduced by labeling or sample preparation, which may alter native conformations. Emerging trends emphasize the multimodal integration of complementary techniques to overcome individual limitations. For example, combining in situ cross-linking mass spectrometry with proximity labeling enhances both spatial resolution and interaction coverage, enabling high-throughput subcellular interactome mapping. Similarly, coupling fluorescence resonance energy transfer with nuclear magnetic resonance and artificial intelligence (AI) simulations integrates dynamic structural data, atomic-level details, and predictive modeling for holistic insights. Advances in AI, exemplified by AlphaFold’s ability to predict interaction interfaces, further augment experimental data, accelerating structure-function analyses. Future developments in cryo-electron microscopy, super-resolution imaging, and machine learning are poised to refine spatiotemporal resolution and scalability. In conclusion, in situ analysis of membrane protein interactions remains indispensable for deciphering their roles in health and disease. While current technologies have significantly advanced our understanding, persistent gaps highlight the need for innovative, integrative approaches. By synergizing experimental and computational tools, researchers can achieve multiscale, real-time, and perturbation-free analyses, ultimately unraveling the dynamic complexity of membrane protein networks and driving therapeutic discovery.

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