背景

奥沙利铂是临床常用的化疗药物，以奥沙利铂为基础的化疗方案已成为治疗结肠癌的一线方案，主要通过对癌细胞的直接抑制和细胞毒性作用阻止肿瘤进一步发展。但由于它主要针对高有丝分裂率的细胞，缺乏靶向性，在清除癌细胞的同时也会对迅速增殖的肠上皮细胞、骨髓细胞等产生致死作用，可引起许多不良反应，如骨髓抑制、胃肠道毒性等，不仅会导致化疗中断或提前终止从而影响治疗效果，还可能增加患者的医疗负担、降低患者的生活质量甚至危及患者的生命健康。已有研究表明，炎症反应在奥沙利铂引起的化疗损伤中发挥关键作用。因此，针对奥沙利铂导致化疗损伤的机制，研发、筛选能够缓解/救治奥沙利铂化疗引起的机体损伤的新产品、新方法或新策略，将更大限度地促进奥沙利铂的临床应用，提高患者预后的生活质量，具有重要的临床应用价值和社会意义。

静注免疫球蛋白是从数千名健康献浆者的混合血浆中制备的免疫球蛋白混合制剂，包含约10⁷种抗病毒、抗细菌等外源性抗体以及针对自身抗原的抗体，具有广谱性和特异性，可发挥广泛的抗炎和免疫调节作用。IVIG临床应用广泛，可用于治疗免疫缺陷病、自身免疫性疾病等适应证，在各种自身免疫性疾病和炎性疾病等超适应证（如血液病、皮肤病、神经性肌肉疾病等）中也具有良好的临床疗效。IVIG具有广泛的抗炎和免疫调节作用，而炎症反应又在奥沙利铂诱导的化疗损伤中起重要作用，因此从理论上推测IVIG可能缓解/救治奥沙利铂诱导的化疗损伤。

目的

探究IVIG能否改善奥沙利铂诱导的化疗损伤

方法

1.以BALB/c小鼠为模型动物，将其随机分为3组：空白对照组和化疗组和干预组。（1）化疗损伤小鼠的构建方式（化疗组）为：3 mg/kg奥沙利铂腹腔注射连续给药4天，Day 5~8正常饲养，Day 9进行解剖；（2）IVIG对化疗损伤小鼠的干预方式（干预组）为：800 mg/kg IVIG在奥沙利铂给药30 min后进行

腹腔注射，连续给药7天，Day 8正常饲养，Day 9进行解剖；（3）空白对照组小鼠：腹腔注射生理盐水。

2.通过外周血细胞计数、解剖学、HE（苏木精-伊红）染色、PAS（过碘酸雪夫）染色及Luminex等方法分别检测各组小鼠的外周血组成、脾指数、脾脏和肠道的病理学特征、结肠中炎性因子水平等，分析IVIG对化疗损伤小鼠的救治作用。

3.通过非标蛋白质组定量技术检测化疗组和空白对照组、干预组和化疗组小鼠的结肠、脾脏差异表达的蛋白质，并对其进行分析，筛选出IVIG发挥救治作用的关键蛋白质或信号通路。利用PRM对蛋白质组学结果进行验证。

结果

1.与空白对照组相比，化疗组小鼠外周血中的白细胞（10⁹/L, 2.45±0.51 vs 0.75±0.27, P<0.000 1）、红细胞数量（10¹²/L, 8.37±0.34 vs 7.37±0.35, P<0.000 1）和血小板（10⁹/L, 1249.00±100.04 vs 347.55±110.33, P<0.000 1）显著减少，淋巴细胞比例（75.37±7.30% vs 62.57±12.09%，P<0.001）、降钙素原（ng/mL, 0.69±0.08 vs 0.22±0.07, P<0.000 1）、血红蛋白浓度（g/L, 126.50±5.50 vs 113.30±5.27, P<0.000 1）和红细胞压积（37.63±1.22% vs 33.38±1.10%, P<0.000 1）明显下降，脾指数显著降低（P<0.000 1），结肠中的杯状细胞减少（P<0.05）、促炎因子TNF-α和IFN-γ（P<0.01）升高、抗炎因子IL-10和IL-13（P<0.05）降低。与化疗组相比，IVIG干预则明显增加了小鼠外周血中的白细胞数量（10⁹/L, 0.75±0.27 vs 1.11±0.30, P<0.05）和血小板数量（10⁹/L, 347.55±110.33 vs 698.49±182.86, P<0.000 1），提高了淋巴细胞比例（62.57±12.09% vs 71.40±9.08%, P<0.05）和降钙素原水平（ng/mL, 0.22±0.07 vs 0.44±0.11, P<0.000 1），脾指数也明显升高（P<0.000 1），增加了小鼠结肠中杯状细胞的数量（P<0.01），降低促炎因子TNF-α（P<0.05）、升高抗炎因子IL-10和IL-13（P<0.05）水平；但没有改变低水平的红细胞数量(10¹²/L, 7.37±0.35 vs 7.20±0.40, P>0.05)、血红蛋白浓度 (g/L, 113.30±5.27 vs 110.00±4.78, P>0.05)和红细胞压积 (33.38±1.10% vs 32.55±1.54%, P>0.05)。

2.通过非标蛋白质组定量技术，相对于空白对照组，在奥沙利铂处理的小鼠结肠和脾脏中分别鉴定出290（137个蛋白质水平上调，153个蛋白质水平下调）和832个差异表达的蛋白质（396个蛋白质水平上调，436个蛋白质水平下调）；与化疗组相比，在IVIG干预的化疗损伤小鼠结肠和脾脏中则分别鉴定出91（61个蛋白质水平上调，30个蛋白质水平下调）和736（409个蛋白质水平上调，327个蛋白质水平下调）个差异表达蛋白质。

3.通过GO、KEGG和蛋白质互作分析发现，奥沙利铂可能通过降低DNA复制和DNA损伤修复相关蛋白质（脾脏）、干扰素反应和突触膜组装过程相关蛋白质（结肠）的表达水平对小鼠造成化疗损伤（相较于空白对照组）；IVIG则可能通过促进DNA复制、DNA损伤修复和氧化磷酸化相关蛋白质（脾脏）的表达及补体反应、急性期反应和突触膜组装过程相关蛋白的表达（结肠）从而改善小鼠的化疗损伤。

4.对结肠组织进行PRM验证发现，Itagm（CD11b）可能是奥沙利铂和IVIG发挥作用的关键蛋白。

结论

IVIG干预能显著改善奥沙利铂引起的化疗损伤。

Background

Oxaliplatin is a commonly used chemotherapy drug in clinical treatment, which has directly inhibition and cytotoxicity on tumor cell to limit the evolution of cancer. Currently, the oxaliplatin-based chemotherapy regimens become a first-line treatment for colon cancer. Oxaliplatin not only eliminates cancer cells, but also has a lethal effect on rapidly proliferating intestinal epithelial cells and bone marrow cells, because it acts on the cell with mitotic rate and lacks targeting.Therefore, oxaliplatin oxaliplatin lead to severe adverse reactions, including gastrointestinal toxicity and bone marrow suppression, which affect treatment effectiveness by chemotherapy interruption or early termination, increase the patients’ medical burden, decrease quality of life, and even endanger patients health and safety. Previous studies have shown that the inflammatory response plays a crucial role in chemotherapy damage caused by oxaliplatin. Therefore,It is necessary to develop and figure out new products, methods, or strategies that reduce chemotherapy injury in patients, which promote clinical application of oxaliplatin in wider space. It is beneficial for improving the patient’s prognosis and quality of life, and has important clinical application value and social significance.

Intravenous immunoglobulin is a mixture of immunoglobulin, including  approximately 10⁷ types of exogenous antibodies (antiviral and antibacterial antibodies) and autoantibodies againsting autoantigens, which is prepared from the mixed plasma of thousands of healthy plasma donors. IVIG is broad-spectrum and specific, producing a wide anti-inflammatory and immunomodulatory effects. IVIG has a wide range of clinical applications. IVIG is used to treat immune deficiency diseases and autoimmune diseases, and has good clinical efficacy in various autoimmune and inflammatory diseases, including hematological diseases, skin diseases, neuromuscular diseases.

Objective

The aim is to investigate whether IVIG improve oxaliplatin-induced chemotherapy injury.

Methods

1.The BALB/c mice were randomly divided into three groups as model animals:(1) the chemotherapy injury group: the mice were intraperitoneally injected with oxaliplatin(3 mg/kg) for 4 days and fed normally on Day 5-8. The mice dissection on Day 9; (2) the treatment group：the mice were intraperitoneally injected with IVIG(800 mg/kg) after 30 min of the oxaliplatin (3 mg/kg) administration for 7 days and fed normally on Day 8. The mice dissection on Day 9; (3) the control group：the mice were intraperitoneally injected with normal saline.

2.The effect of IVIG on oxaliplatin-induced chemotherapy injury was evaluated by comparing the differences in the cell composition of peripheral blood, splenic index, pathological features of spleen and colon and colon inflammatory cytokines, which were detected by peripheral blood cell counts, anatomy, hematoxylin-eosin staining, periodic acid-schiff staining and luminex, respectively.

3.The differential proteins in the colon and spleen of the mice in the chemotherapy group and the blank control group, the intervention group and the chemotherapy group were identified by label-free proteomic quantitative technology and were analyzed to screen out the key proteins or signaling pathways. PRM was used to validate the proteomic results.

Results

1.Compared with the control group, the counts of white blood cell ( 10⁹/L, 2.45±0.51 vs 0.75±0.27, P<0.000 1), red blood cell (10¹²/L，8.37±0.34 vs 7.37±0.35，P<0.000 1) and platelet (10⁹/L, 1249.00±100.04 vs 347.55±110.33, P<0.000 1), the proportion of lymphocytes (75.37±7.30% vs 62.57±12.09%, P<0.001), procalcitonin (ng/mL, 0.69±0.08 vs 0.22±0.07, P<0.000 1), Hemoglobin (g/L, 126.50±5.50 vs 113.30±5.27, P<0.000 1) and hematocrit (37.63±1.22% vs 33.38±1.10%, P<0.000 1) in peripheral blood descend significantly in chemotherapy injury group. The splenic index remarkably declined (P<0.000 1) and the number of goblet cells was also decreased (P<0.05) in chemotherapy-injured mice. Furthermore, the proinflammatory factors TNF-α and IFN-γ increased (P<0.01) and the anti-inflammatory factors IL-10 

and IL-13 decreased (P<0.05) in chemotherapy-injured mice. Compared with the chemotherapy injury group, IVIG treatment greatly increased the numbers of white blood cell (10⁹/L, 0.75±0.27 vs 1.11±0.30, P<0.05) and platelet (10⁹/L, 347.55±110.33 vs 698.49±182.86, P<0.000 1) in peripheral blood of mice , and raised the proportion of lymphocytes (62.57±12.09% vs 71.40±9.08%, P<0.05) and procalcitonin (ng/mL, 0.22±0.07 vs 0.44±0.11, P<0.000 1). In addition, IVIG not only increased the spleen index of mice, the number of goblet cells and the levels of IL-10 and IL-13 in the colon of mice, but also decreased the colonic proinflammatory factor TNF-α levels. However, IVIG did not change the low level of red blood cells (10¹²/L, 7.37±0.35 vs 7.20±0.40, P>0.05), hemoglobin (g/L, 113.30±5.27 vs 110.00±4.78, P>0.05) and hematocrit (33.38±1.10% vs 32.55±1.54%, P>0.05).

2.Compared with the blank control group, a total of 290 (137 up-regulated proteins and 153 down-regulated proteins) and 832 (396 up-regulated proteins and 436 down-regulated proteins) differentially expressed in the colon and spleen of oxaliplatin-treated mice were identified by label-free proteomic quantitative technology, respectively. Compared with the chemotherapy group, 91 (61 up-regulated and 30 down-regulated proteins) and 736 (409 up-regulated and 327 down-regulated proteins) differentially expressed proteins were identified in the colon and spleen of IVIG treated chemotherapy-injured mice, respectively.

3.GO, KEGG and protein interaction analysis showed that oxaliplatin caused chemotherapy damage in mice by reducing the expression levels of proteins related to interferon response and synaptic membrane assembly process (in the colon tissues), and proteins involved in DNA replication and DNA damage repair (in the spleen tissues). IVIG may improve the chemotherapeutic damage in mice by promoting the expression of proteins related to complement response, acute phase response and synaptic membrane assembly process (in the colon tissues), and proteins involved in DNA replication, DNA damage repair and oxidative phosphorylation (in the spleen tissues). 

4.PRM results showed that Itagm (CD11b) may be a key protein in the action of oxaliplatin and IVIG.

Conclusion 

IVIG significantly improve oxaliplatin-induced chemotherapy injury.