目的：本研究旨在探究水飞蓟宾治疗HIV感染免疫无应答者（Immunological non-responder，INR）的临床疗效及潜在作用机制。重点关注水飞蓟宾（Silibinin，SIL）能否通过调节肠道菌群改善HIV感染INR的慢性炎症和异常免疫激活等紊乱状态，最终提高CD4⁺ T细胞计数，并阐明SIL促进免疫重建的分子机制，为INR的临床治疗策略提供理论和应用依据。

方法：

1、受试者招募和药物干预：本研究纳入53名在中国医科大学附属第一医院关爱门诊接受抗病毒治疗且具有肝功能损伤表现的HIV感染INR。INR定义为接受抗病毒治疗2年以上，病毒载量小于50拷贝/mL且CD4⁺ T细胞计数<500 cells/μL。所有参与者均为男男同性性行为者（Men who have sex with men，MSM）。开展为期12周的水飞蓟宾干预，并随访至48周。

2、临床指标收集和检测：首先，通过医院LIS系统收集肝功能等生化指标（ALT、AST、ALP、GGT，直接胆红素、间接胆红素等），评价SIL对其临床适应症肝功能保护作用。其次，通过流式细胞技术检测SIL治疗前后T细胞计数及外周血免疫细胞表型；通过Olink蛋白组学技术检测SIL治疗前后92种炎症相关蛋白（Inflammation-related proteins, IRP）的变化，确定SIL治疗HIV免疫不应答的临床疗效。最后，对差异的炎症相关蛋白进行 KEGG分析，探索SIL治疗HIV感染免疫不应答的潜在通路和靶点。

3、肠道菌群和代谢物分析：通过16S rDNA技术分析SIL治疗前后粪便细菌组成变化和功能差异；通过液相色谱-串联质谱法分析SIL治疗前后粪便代谢物组成变化和功能差异，筛选SIL通过肠道菌群调节HIV免疫不应答的潜在通路。进一步通过液相色谱-串联质谱法检测SIL治疗前后粪便和血浆中氨基酸和水溶性维生素。利用网络药理学手段分析SIL治疗前后差异菌、差异代谢产物与HIV感染免疫耗竭相关的共同基因靶点，通过STRING建立PPI蛋白互作网络，使用Cytoscape的MCODE插件寻找高度互联的网络，利用DAVID数据库进行KEGG和GO分析。

4、NAD+补救合成途径检测：利用ELISA检测正常体检人群（Healthy Control，HC）、HIV感染免疫应答者（immunological responder，IR）和免疫不应答者NAD+补救合成途径的关键分子水平和（或）活性，明确INR组NAD+补救合成途径的异常；利用ELISA检测SIL治疗前后NAD+补救合成途径的关键分子水平和（或）活性，证明SIL通过纠正NAD+补救合成途径改善免疫紊乱的潜在功能。

5、潜在有益菌与NAD+补救合成途径关联分析：在NCBI Genome数据库检索SIL治疗后显著富集的Blautia菌和Eubacterium hallii菌全基因组序列，分析细菌基因组，确定是否含有烟酰胺磷酸核糖转移酶（NAMPT）特征结构域；将Blautia wexlerae菌与烟酰胺(nicotinamide，NAM)、NAMPT特异性抑制剂FK866共培养，证明Blautia菌是否编码并表达NAMPT；通过Autodock Vina分子对接分析，解析SIL与NAMPT-NAM复合体的相互作用模式。用NAM灌胃无菌小鼠（GF）和无特殊病原体小鼠（SPF），收集肠组织，进行NAMPT免疫组化染色，并检测血清NAM水平，证明肠道菌群对NAM的代谢作用。

6、体外验证水飞蓟宾和NAD+对T细胞功能的影响：CCK-8检测不同剂量SIL对细胞的毒性作用；用不同浓度SIL或NAD+处理HIV感染INR的原代外周血单个核细胞，利用流式细胞技术检测细胞活化和耗竭。采用JC-1荧光探针技术，评估NAD+对细胞线粒体膜电位的影响，并测定ATP含量。

结果：

1、SIL改善INR的细胞免疫状态，降低炎症水平，且具有临床安全性

经12周SIL治疗后，受试者CD4⁺T细胞和CD8⁺ T细胞计数均显著提升（P < 0.0001），表明水飞蓟宾可促进INR免疫重建。水飞蓟宾治疗后CD4⁺T细胞和CD8⁺T细胞活化水平均显著降低（P < 0.001），但调节性T细胞（Treg）、Th17细胞、记忆性T细胞及T细胞衰老水平未见显著变化，提示SIL可能通过选择性抑制免疫活化发挥治疗作用。Olink多重炎症蛋白分析显示SIL显著改变了INR的炎症相关蛋白谱：上调的蛋白多与免疫调节和组织修复相关；下调的蛋白主要富集于病毒与细胞因子相互作用、IL-17信号通路和NF-κB信号通路。SIL治疗后INR的ALT和AST水平明显下降，表明SIL发挥了肝脏保护作用；SIL治疗不影响血脂和离子代谢，证实其具有良好的临床安全性。

2、SIL治疗后肠道菌群组成和相关代谢物发生显著改变

α多样性分析显示SIL治疗后的粪便细菌丰富度显著降低（P < 0.0001），β多样性分析显示菌群发生系统性重构，即主坐标分析（PCoA）和非度量多维尺度分析（NMDS）中治疗前后细菌菌明显分区（P < 0.001）。在属水平，水飞蓟宾治疗显著增加了多个有益菌属的丰度，如双歧杆菌属（Bifidobacterium），布劳特氏菌属（Blautia）和真杆菌（Eubacterium hallii group）。相关性分析识别出54个与免疫指标显著相关的菌属（P < 0.05），这些菌属呈现两种截然不同的调控模式：以Blautia属、Eubacterium hallii group属为代表的促进性菌群与T细胞计数呈正相关，同时与T细胞活化呈负相关；以Ruminococcus gnavus group为代表的抑制性菌群与T细胞计数呈负相关，与T细胞活化呈正相关。

功能预测表明SIL治疗后高度富集的菌与氨基酸、碳水化合物和维生素代谢相关，特别是维生素B3（烟酸和烟酰胺）代谢通路。靶向代谢组学发现，治疗后粪便和血浆中的NAM水平显著降低，治疗后血浆中9种氨基酸的水平显著降低，包括精氨酸、天冬氨酸、谷氨酸、瓜氨酸、丝氨酸、甘氨酸、组氨酸、赖氨酸、苯丙氨酸。

3、SIL纠正了INR异常的NAD+补救合成途径

HIV感染IR组和INR组血浆NAM均显著高于HC组（P < 0.001），但NAD+均低于NC组（P < 0.05），且INR组NAD+水平更低；IR组NAMPT活性显著高于HC组和INR组（P < 0.0001），提示HIV感染导致NAD+合成障碍，即使IR者NAMPT代偿性激活仍不能将NAD+恢复至正常水平，而INR者NAMPT活性不足，更加无法有效的将过量NAM转化为NAD+。然而，INR者经SIL治疗后血浆NAMPT活性显著增加（P < 0.0001），NMN水平升高（P < 0.001），NAD+和NADH水平显著升高（P < 0.001），提示水飞蓟宾可通过提高NAMPT活性，促进NAM转化为NAD+。

4、水飞蓟宾治疗后富集的有益菌具有表达NAMPT的潜力

水飞蓟宾治疗后显著富集的菌（Blautia和Eubacterium hallii）携带高度同源性的NAMPT功能基因（PF04095结构域，E-value：2.9E-7至9.1E-6），提示富集的这些有益菌可能通过表达NAMPT，介导NAM向NAD+的转化。Blautia wexlerae菌在有/无NAMPT抑制剂FK866条件下与NAM体外共培养，结果显示FK866处理后B. wexlerae生长显著减缓，进一步证明B. wexlerae能够通过表达NAMPT，介导NAM向NAD+转化，加速生长。NAM灌胃的SPF小鼠肠道组织中NAMPT水平高于GF小鼠，提示肠道细菌可以编码并表达NAMPT，促进NAM转化为NAD+。

5、SIL和NAD+处理可在体外细胞水平调节T细胞功能

SIL以剂量依赖性方式抑制T细胞活化和耗竭，外源性NAD+同样能够以剂量依赖性方式抑制T细胞的活化。外源性NAD+提升Jurkat细胞线粒体膜电位，ATP浓度呈现NAD+剂量依赖性上升趋势。

结论：本研究首次揭示了水飞蓟宾通过调节"肠道菌群-NAD+代谢轴"促进HIV感染INR免疫重建的全新机制。创新性地证实SIL通过重构肠道微生物组，特异性富集携带NAMPT功能基因的有益菌群，这种微生态重构显著增强NAM向NAD+的转化。同时研究也揭示INR普遍存在NAM异常积累和NAMPT代偿功能不足，形成“NAM堆积-NAD+耗竭”的恶性循环，导致T细胞能量代谢障碍。

Objective: This study aims to investigate the clinical efficacy and potential mechanisms of silibinin in treating HIV-infected immunological non-responders (INR). Specifically, it examines whether silibinin (SIL) can alleviate chronic inflammation and abnormal immune activation in HIV-infected INRs by modulating the gut microbiota, thereby increasing CD4+ T cell counts. Additionally, this study seeks to elucidate the molecular mechanisms underlying SIL-induced immune reconstitution, providing a theoretical and practical foundation for clinical treatment strategies targeting INRs.

Methods:

Participant Recruitment and Drug Intervention: This study enrolled 53 HIV-infected INRs undergoing antiviral therapy at the AIDS Care Clinic of the First Affiliated Hospital of China Medical University, all of whom exhibited liver function impairment. INRs were defined as individuals who had received antiviral treatment for over two years, with a viral load below 50 copies/mL and CD4+ T cell counts less than 500 cells/μL. All participants were men who have sex with men (MSM). A 12-week silibinin intervention was conducted, followed by a 48-week follow-up period.

Clinical Indicator Collection and Detection: Biochemical indicators such as liver function parameters (ALT, AST, ALP, GGT, direct bilirubin, indirect bilirubin, etc.) were collected via the hospital's LIS system to evaluate SIL's hepatoprotective effects. T cell counts and peripheral blood immune cell phenotypes were assessed using flow cytometry before and after SIL treatment. Changes in 92 inflammation-related proteins (IRPs) were analyzed using Olink proteomics technology to determine SIL's clinical efficacy in addressing HIV immune non-response. KEGG pathway analysis was performed on differentially expressed IRPs to explore potential pathways and targets of SIL in treating HIV immune non-response.

3. Gut Microbiota and Metabolite Analysis: Fecal bacterial composition and functional differences were analyzed using 16S rDNA sequencing before and after SIL treatment. Fecal metabolite composition and functional differences were evaluated using liquid chromatography-tandem mass spectrometry to identify potential pathways through which SIL regulates HIV immune non-response via the gut microbiota. Amino acids and water-soluble vitamins in feces and plasma were detected using liquid chromatography-tandem mass spectrometry. Network pharmacology was employed to analyze common gene targets among differentially expressed bacteria, metabolites, and HIV infection-related immune exhaustion. PPI protein interaction networks were constructed using STRING, and highly interconnected networks were identified using Cytoscape's MCODE plugin. KEGG and GO analyses were performed using the DAVID database.

4. Detection of NAD+ Salvage Synthesis Pathway: The levels and/or activities of key molecules in the NAD+ salvage synthesis pathway were measured using ELISA in healthy controls (HC), immunological responders (IR), and INRs to clarify abnormalities in the NAD+ salvage synthesis pathway in INRs.The levels and/or activities of key molecules in the NAD+ salvage synthesis pathway were also measured before and after SIL treatment to demonstrate SIL's potential role in improving immune disorders by correcting the NAD+ salvage synthesis pathway.

5. Correlation Analysis of Beneficial Bacteria and NAD+ Salvage Synthesis Pathway: Whole genome sequences of Blautia and Eubacterium hallii bacteria, which were significantly enriched post-SIL treatment, were retrieved from the NCBI Genome database. These genomes were analyzed to determine whether they contained the characteristic domain of nicotinamide phosphoribosyltransferase (NAMPT). Blautia wexlerae bacteria were co-cultured with nicotinamide (NAM) and the NAMPT-specific inhibitor FK866 to confirm whether Blautia bacteria encode and express NAMPT.Molecular docking analysis using Autodock Vina was conducted to examine the interaction mode between SIL and the NAMPT-NAM complex. GF mice and SPF mice were gavaged with NAM, and intestinal tissues were collected for NAMPT immunohistochemical staining and serum NAM level detection to prove the metabolic effect of the intestinal flora on NAM.

6. In Vitro Verification of Silybin and NAD+ Effects on T Cell Function: CCK-8 assays were used to assess the cytotoxicity of various SIL concentrations on cells. Primary peripheral blood mononuclear cells from HIV-infected INRs were treated with different SIL or NAD+ concentrations, and cell activation and exhaustion were evaluated using flow cytometry. JC-1 fluorescence probe technology was employed to evaluate the effect of NAD+ on mitochondrial membrane potential, and ATP content was measured.

Results:

SIL improved the cellular immune status of INRs, reduced inflammation levels, and demonstrated clinical safety.

After 12 weeks of SIL treatment, CD4+ T cell and CD8+ T cell counts significantly increased (P < 0.0001), indicating SIL's ability to promote immune reconstitution in INRs. Post-SIL treatment, the activation levels of CD4+ T cells and CD8+ T cells significantly decreased (P < 0.001), while regulatory T cells (Treg), Th17 cells, memory T cells, and T cell senescence did not show significant changes, suggesting that SIL may exert therapeutic effects by selectively inhibiting immune activation. Olink multiplex inflammatory protein analysis revealed that SIL significantly altered the inflammatory protein profile of INRs: upregulated proteins were primarily associated with immune regulation and tissue repair, while downregulated proteins were mainly enriched in virus-cytokine interactions, IL-17 signaling pathways, and NF-κB signaling pathways. Post-SIL treatment, ALT and AST levels in INRs significantly decreased, indicating SIL's hepatoprotective effects. SIL treatment did not affect lipid or ion metabolism, confirming its good clinical safety.

2. SIL treatment significantly altered the composition of the intestinal flora and related metabolites.

α-diversity analysis showed that fecal bacterial richness significantly decreased post-SIL treatment (P < 0.0001), and β-diversity analysis indicated systematic reconstruction of the flora, as evidenced by significant partitioning in principal coordinate analysis (PCoA) and non-metric multidimensional scaling analysis (NMDS) (P < 0.001). At the genus level, SIL treatment significantly increased the abundance of beneficial bacterial genera, such as Bifidobacterium, Blautia, and Eubacterium hallii group. Correlation analysis identified 54 bacterial genera significantly associated with immune indicators (P < 0.05), presenting two distinct regulatory patterns: promoting microbiota (e.g., Blautia and Eubacterium hallii group) positively correlated with T cell counts and negatively correlated with T cell activation; inhibitory microbiota (e.g., Ruminococcus gnavus group) negatively correlated with T cell counts and positively correlated with T cell activation. Functional prediction suggested that bacteria highly enriched post-SIL treatment were involved in amino acid, carbohydrate, and vitamin metabolism, particularly the vitamin B3 (niacin and nicotinamide) metabolic pathway. Targeted metabolomics revealed that fecal and plasma NAM levels significantly decreased post-treatment, and nine plasma amino acids (arginine, aspartic acid, glutamic acid, citrulline, serine, glycine, histidine, lysine, and phenylalanine) were significantly reduced.

3. SIL corrected the abnormal NAD+ salvage synthesis pathway in INRs.

Plasma NAM levels in HIV-infected IR and INR groups were significantly higher than those in the HC group (P < 0.001), but NAD+ levels were lower than those in the HC group (P < 0.05), with even lower levels in the INR group. NAMPT activity in the IR group was significantly higher than in the HC and INR groups (P < 0.0001), suggesting that HIV infection disrupted NAD+ synthesis. Despite compensatory NAMPT activation in IR individuals, NAD+ levels could not be restored to normal, while INR individuals exhibited insufficient NAMPT activity, making it difficult to effectively convert excessive NAM into NAD+. Post-SIL treatment, plasma NAMPT activity in INRs significantly increased (P < 0.0001), NMN levels increased (P < 0.001), and NAD+ and NADH levels significantly increased (P < 0.001), indicating SIL's ability to promote NAM conversion to NAD+ by enhancing NAMPT activity.

4. Beneficial bacteria enriched post-SIL treatment potentially express NAMPT.

Bacteria significantly enriched post-SIL treatment (Blautia and Eubacterium hallii) carried highly homologous NAMPT functional genes (PF04095 domain, E-value: 2.9E-7 to 9.1E-6), suggesting these enriched beneficial bacteria might mediate NAM-to-NAD+ conversion by expressing NAMPT. Blautia wexlerae co-cultured with NAM in the presence or absence of the NAMPT inhibitor FK866 showed significantly slowed growth post-FK866 treatment, further confirming B. wexlerae's ability to mediate NAM-to-NAD+ conversion by expressing NAMPT and accelerate growth. Intestinal tissues of NAM-gavaged SPF mice exhibited higher NAMPT levels than GF mice, suggesting intestinal bacteria encode and express NAMPT to promote NAM-to-NAD+ conversion.

5. SIL and NAD+ treatment regulated T cell function at the in vitro cellular level.

SIL inhibited T cell activation and exhaustion in a dose-dependent manner, and exogenous NAD+ similarly inhibited T cell activation in a dose-dependent manner. Exogenous NAD+ increased the mitochondrial membrane potential of Jurkat cells, and ATP concentration exhibited a dose-dependent increase trend with NAD+.

Conclusion: This study first revealed a novel mechanism by which silibinin promotes immune reconstitution in HIV-infected INRs through regulating the "gut microbiota-NAD+ metabolism axis." It confirmed that SIL reconstructs the intestinal microbiota and specifically enriches beneficial bacteria carrying the NAMPT functional gene, significantly enhancing NAM-to-NAD+ conversion. Furthermore, the study revealed that INRs generally exhibit abnormal NAM accumulation and insufficient compensatory NAMPT function, forming a vicious cycle of "NAM accumulation - NAD+ depletion," leading to energy metabolism disorders in T cells.