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低分子透明质酸HA35
低分子透明质酸HA35（分子量为35 kDa的透明质酸片段）是一种具有多种生物活性的生物大分子，在炎症调节、组织再生、皮肤渗透、肠道屏障功能维护以及疼痛缓解等方面展现出显著的潜力。其独特之处在于其较小的分子量赋予了它与高分子量透明质酸不同的作用机制和更强的组织渗透能力。
一、HA35的生物学特性与作用机制
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炎症调节与免疫调控
- 促炎与抗炎双重特性: 透明质酸的炎症调节作用与其分子量密切相关。高分子量透明质酸通常表现出抗炎和免疫抑制特性，而低分子量透明质酸（包括HA35）则被认为是一种有效的促炎分子。然而，研究也表明HA35在某些特定情况下具有抗炎作用。例如，HA35能够通过调节microRNA291b及其靶点Tollip来使慢性乙醇喂养大鼠肝巨噬细胞中TLR4信号正常化，从而改善乙醇诱导的炎症。在肠道炎症模型中，HA35也表现出保护作用，通过上调与杯状细胞功能和先天免疫相关的基因来降低疾病的严重程度和死亡率，这表明其可能在炎症性肠病中发挥有益作用。
- TLR4依赖性机制: HA35在缓解乙醇引起的抗微生物防御和肠道屏障完整性丧失中，是依赖于TLR4的。研究发现，HA35通过HA受体Tlr4（而非Cd44或Tlr2）发挥保护作用，防止乙醇诱导的C型凝集素抗菌肽的表达减少和屏障完整性受损。
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组织再生与伤口愈合
- 促进上皮细胞修复: HA35能够显著加速小肠类器官单层细胞的伤口愈合，这可能通过促进伤口边缘细胞增殖和向伤口区域迁移来实现。在慢性伤口治疗中，局部注射HA35能显著促进伤口愈合，表现为新生肉芽组织的形成、伤口周围皮肤状况的改善以及伤口尺寸的减小。
- 诱导紧密连接蛋白表达: HA35能够诱导紧密连接蛋白ZO-1（zonula occludens-1）的表达，这对于维持肠道屏障的完整性至关重要。这种作用对于预防肠道通透性增加、抵御病原体入侵具有重要意义。Layilin（一种透明质酸受体）在HA35诱导结肠上皮细胞中ZO-1表达过程中发挥关键作用，而TLR-4或CD44则不参与此过程。
- 影响抗菌肽表达: HA35能够诱导上皮细胞中β-防御素等抗菌肽的表达，增强宿主对病原体的先天免疫防御能力。
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皮肤渗透与美容应用
- 增强皮肤渗透与滞留: 低分子量HA（如5 kDa和8 kDa的LMW-HA）能够穿透角质层进入表皮和真皮层，而高分子量HA则滞留在角质层表面。HA35作为低分子量HA的一种，其较小的分子量使其具有更好的皮肤渗透性。LMW-HA通过与角质层中的角蛋白和脂质相互作用，并产生显著的皮肤水合作用，从而促进其穿透角质层。
- 药物递送载体: HA修饰的脂质体（HA-UP-LPs）可显著增强药物的皮肤渗透和滞留。HA的表面修饰通过直接结合皮肤细胞膜上广泛表达的CD44受体，驱动脂质体通过能量依赖性的内吞作用进入细胞。这为开发具有改进透皮渗透和滞留的新型局部制剂和护肤产品提供了机会。
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疼痛缓解
- 骨转移疼痛: 在晚期癌症患者中，骨转移引起的剧烈疼痛是一个常见问题。一项案例研究显示，皮下注射100毫克/5毫升的HA35到腹部深层脂肪组织中，能够有效缓解骨转移患者的疼痛。首次注射后20分钟内疼痛评分下降40%，40分钟后下降60%。经过50次注射后，疼痛持续控制在3分左右。HA35被认为通过阻断TRVP1钙通道来缓解疼痛。
- 慢性伤口相关疼痛: 局部注射HA35不仅促进慢性伤口愈合，还能在24小时内显著缓解与慢性伤口相关的神经性疼痛。
二、HA35在特定疾病中的应用与研究
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颞下颌关节骨关节炎（TMJ OA）
- 疗效争议: 对于TMJ OA的治疗，单独进行关节穿刺术已被证实能有效减轻疼痛并改善下颌功能。然而，在关节穿刺术后额外注射HA（无论是低分子量HA还是高分子量HA）或皮质类固醇，并不能改善最终的临床结果。这表明HA35在TMJ OA治疗中的额外益处可能有限，需要进一步的大规模、多中心研究来评估其确切疗效。
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膝骨关节炎（Knee OA）
- 高分子量HA更优: 针对膝骨关节炎的关节内注射HA治疗，现有证据表明高分子量HA在减轻疼痛方面优于低分子量HA产品。这提示HA35可能不适用于膝骨关节炎的治疗，或其疗效不如高分子量HA。
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坏死性小肠结肠炎（NEC）
- 保护作用: NEC是早产儿常见的胃肠道急症。HA35作为人乳中一种生物活性糖胺聚糖，能够加速小鼠幼崽的肠道发育，并在小鼠NEC模型中预防炎症引起的组织损伤。HA35显著降低了模型中疾病的严重程度，并有降低死亡率的趋势，同时上调了与杯状细胞功能和先天免疫相关的基因。HA35还被发现能保护小鼠免受大肠杆菌感染，并增强上皮屏障功能，特别是在健康小鼠和感染小鼠中都能诱导紧密连接蛋白ZO-1的表达。
- 人乳成分: HA35是人乳中的一种重要成分，可能通过促进肠道成熟、抗氧化和生长通路（如核因子E2相关因子介导的氧化应激反应、缺氧诱导因子-1α、雷帕霉素靶点等）以及下调细胞凋亡信号来保护新生儿肠道健康，对抗NEC。
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糖尿病相关肠道屏障功能障碍
- 对抗高血糖应激: 在高血糖环境下，肠道上皮屏障功能会受损，表现为紧密连接蛋白ZO-1和occludin的表达降低和空间分布改变。HA35能够防止这些屏障蛋白的降解或丢失，并在高血糖应激下维持其空间分布，从而发挥屏障保护作用。这一保护作用是HA受体Layilin依赖的，HA35通过调节细胞凋亡和影响黏着斑激酶（FAK）以及F-肌动蛋白的空间组织来发挥作用。
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酒精性肝病相关肠道损伤
- 缓解肠道损伤: 急性和慢性酒精暴露会损害肠道上皮完整性。口服HA35可缓解慢性乙醇暴露对小鼠肠道C型凝集素抗菌肽表达的减少、紧密连接蛋白的表达降低以及细菌移位至肠系膜淋巴结的影响。在体外研究中，HA35也能够防止乙醇诱导的肠道类器官中C型凝集素抗菌肽的表达减少和屏障完整性受损。
三、HA35的安全性与临床前景
- 安全性: 对健康人体的初步研究表明，口服HA35（140毫克/天，连续七天）是安全的，没有引起任何严重不良事件，并且不影响代谢、炎症或微生物组参数。这为HA35的进一步临床应用奠定了基础。
- 临床潜力: 鉴于HA35在促进伤口愈合、缓解疼痛、维护肠道屏障功能以及对抗多种炎症性疾病中的潜在益处，它被认为是一种有前途的治疗剂或膳食补充剂。例如，它被提议作为一种简单的膳食补充剂来增强肠道屏障防御，并在高级癌症患者的姑息治疗中提供关键支持。
- 新型HA产品: 现有技术也发展出将高分子量和低分子量HA结合的混合协同复合物（HCC），例如NAHYCO®技术生产的Profhilo® Structura，这种产品具有更高的HA浓度和独特的流变学特性，旨在恢复面部脂肪组织，但其分子量范围与HA35不同。这些新型HA产品也强调了不同分子量HA协同作用的潜力。
总结
低分子透明质酸HA35因其独特的分子量而展现出广泛的生物学功能，包括调节炎症、促进组织再生和伤口愈合、增强皮肤渗透、维护肠道屏障完整性以及缓解疼痛。尽管在某些关节疾病（如TMJ OA和膝OA）中的疗效仍需进一步证实，但在肠道疾病（如NEC、酒精性肠道损伤和糖尿病相关肠道屏障功能障碍）和慢性伤口治疗中已显示出显著的潜力。其良好的安全性和多种作用机制，使其成为再生医学、皮肤护理和临床治疗领域中一个值得关注的生物分子。
References
1Hyaluronic Acid in Inflammation and Tissue Regeneration.PubMed
Malgorzata Litwiniuk, Alicja Krejner, Marcus S Speyrer, et al.
Wounds. 2016 Mar;28(3):78-88.
Hyaluronic acid (HA), the main component of extracellular matrix, is considered one of the key players in the tissue regeneration process. It has been proven to modulate via specific HA receptors, inflammation, cellular migration, and angiogenesis, which are the main phases of wound healing. Studies have revealed that most HA properties depend on its molecular size. High molecular weight HA displays anti-inflammatory and immunosuppressive properties, whereas low molecular weight HA is a potent proinflammatory molecule. In this review, the authors summarize the role of HA polymers of different molecular weight in tissue regeneration and provide a short overview of main cellular receptors involved in HA signaling. In addition, the role of HA in 2 major steps of wound healing is examined: inflammation and the angiogenesis process. Finally, the antioxidative properties of HA are discussed and its possible clinical implication presented.
2Mechanisms of Action and Efficacy of Hyaluronic Acid, Corticosteroids and Platelet-Rich Plasma in the Treatment of Temporomandibular Joint Osteoarthritis-A Systematic Review.PubMed
Marcin Derwich, Maria Mitus-Kenig, Elzbieta Pawlowska
Int J Mol Sci. 2021 Jul 9;22(14):7405. doi: 10.3390/ijms22147405.
Temporomandibular joint osteoarthritis (TMJ OA) is a low-inflammatory disorder with multifactorial etiology. The aim of this review was to present the current state of knowledge regarding the mechanisms of action and the efficacy of hyaluronic acid (HA), corticosteroids (CS) and platelet-rich plasma (PRP) in the treatment of TMJ OA.: The PubMed database was analyzed with the keywords: "(temporomandibular joint) AND ((osteoarthritis) OR (dysfunction) OR (disorders) OR (pain)) AND ((treatment) OR (arthrocentesis) OR (arthroscopy) OR (injection)) AND ((hyaluronic acid) OR (corticosteroid) OR (platelet rich plasma))". After screening of 363 results, 16 studies were included in this review. Arthrocentesis alone effectively reduces pain and improves jaw function in patients diagnosed with TMJ OA. Additional injections of HA, either low-molecular-weight (LMW) HA or high-molecular-weight (HMW) HA, or CS at the end of the arthrocentesis do not improve the final clinical outcomes. CS present several negative effects on the articular cartilage. Results related to additional PRP injections are not consistent and are rather questionable. Further studies should be multicenter, based on a larger group of patients and should answer the question of whether other methods of TMJ OA treatment are more beneficial for the patients than simple arthrocentesis.
3Hyaluronic acid and HA-modified cationic liposomes for promoting skin penetration and retention.PubMed
Chang Ni, Zijun Zhang, Yuling Wang, et al.
J Control Release. 2023 May;357:432-443. doi: 10.1016/j.jconrel.2023.03.049. Epub 2023 Apr 18.
Hyaluronic acid (HA) has been widely used in cosmetics and topical preparations owing to its favorable moisturizing property and potential in enhancing drugs' skin permeability. Here, the influencing factors and underlying mechanism of HA on skin penetration were carefully investigated, and HA-modified Undecylenoyl-Phenylalanine (UP) liposomes (HA-UP-LPs) were designed as a proof of principle for efficacious transdermal drug delivery strategy to enhance the skin penetration and retention. An in vitro penetration test (IVPT) of HA with different molecular weights showed that low molecular weight HA (LMW-HA, 5 kDa and 8 kDa) could pass through the stratum corneum (SC) barrier and enter into the epidermis and dermis layers, whereas its high molecular counterparts (HMW-HA) were trapped on the SC surface. Mechanistic studies revealed that LMW-HA could interact with keratin and lipid in the SC meanwhile exerted a substantial skin hydration effect, which may partially contribute to the SC penetration benefit. In addition, the surface decoration of HA drove an energy-dependent caveolae/lipid raft-mediated endocytosis of the liposomes through direct binding to the CD44 receptors widely expressed on skin cell membranes. Notably, IVPT showed a 1.36-fold and 4.86-fold increase in skin retention of UP and a 1.62-fold and 5.41-fold increase in skin penetration of UP with HA-UP-LPs over UP-LPs and free UP at 24 h, respectively. As a result, the anionic HA-UP-LPs (-30.0 mV) showed enhanced drug skin penetration and retention compared with conventional cationic bared UP-LPs (+21.3 mV) on both in vitro mini-pig skin as well as in vivo mouse skin. Overall, the usage of LMW-HA might offer opportunities in developing novel topical preparations and skin care products with improved transdermal penetration and retention.
4Human skin penetration of hyaluronic acid of different molecular weights as probed by Raman spectroscopy.PubMed
M Essendoubi, C Gobinet, R Reynaud, et al.
Skin Res Technol. 2016 Feb;22(1):55-62. doi: 10.1111/srt.12228. Epub 2015 Apr 16.
BACKGROUND: Topical delivery of molecules into the human skin is one of the main issues in dermatology and cosmetology. Several techniques were developed to study molecules penetration into the human skin. Although widely accepted, the conventional methods such as Franz diffusion cells are unable to provide the accurate localization of actives in the skin layers. A different approach based on Raman spectroscopy has been proposed to follow-up the permeation of actives. It presents a high molecular specificity to distinguish exogenous molecules from skin constituents. METHODS: Raman micro-imaging was applied to monitor the skin penetration of hyaluronic acids (HA) of different molecular weights. The first step, was the spectral characterization of these HA. After, we have determined spectral features of HA by which they can be detected in the skin. In the second part, transverse skin sections were realized and spectral images were recorded. RESULTS: Our results show a difference of skin permeation of the three HA. Indeed, HA with low molecular weight (20-300 kDa) passes through the stratum corneum in contrast of the impermeability of high molecular weight HA (1000-1400 kDa). CONCLUSION: Raman spectroscopy represents an analytical, non-destructive, and dynamic method to evaluate the permeation of actives in the skin layers.
5Profhilo® Structura, Current Status and Future Perspectives: A Practical Review.PubMed
Riccardo Forte, Giovanni Salti, Antonello Tateo
Plast Aesthet Nurs (Phila). 2024;44(3):213-219. doi: 10.1097/PSN.0000000000000571. Epub 2024 Jul 3.
Hyaluronic acid (HA)-based injectables continue to grow in popularity and are used worldwide to improve facial changes associated with aging. Profhilo® Structura (IBSA Farmaceutici Italia, Lodi, Italy) is a novel HA-based product that uses nano hybrid complex of hyaluronic acid (NAHYCO)® technology to create hybrid cooperative complexes consisting of high and low molecular weight HA. Profhilo® Structura's high concentration (45 mg of total HA) and rheological properties (ie, viscosity [resistance to flow] and elasticity [stickiness]) have been specially formulated to restore facial adipose tissue. Because of their early involvement in the facial aging process, the superficial medial and lateral-temporal cheek fat compartments are good candidates for treatment. However, recommendations and guidelines are necessary to ensure that treatment is safe and effective. Three experts were invited to IBSA Farmaceutici Italia Srl to discuss clinical rationale, optimal injection techniques, and clinical outcomes for treating the superficial medial and lateral-temporal cheek fat compartments with Profhilo® Structura. These techniques were developed using patients presenting with either initial hypotrophy of fat compartments within the preauricular area (ie, sinkers) or jowl sagging due to hypotrophy within the preauricular and zygomatic areas (ie, saggers). Targeting these areas using the optimal injection site can maximize product diffusion and minimize the risk for vascular compromise. Targeting these areas also leads to lateral tightening and lipolifting effects due to adipose tissue restoration. The experts agreed that Profhilo® Structura is currently the first and only HA-based injectable treatment that can integrate and restore the adipose tissue in these affected fat compartments.
6Intradermal Injection of Hybrid Complexes of High- and Low-Molecular-Weight Hyaluronan: Where Do We Stand and Where Are We Headed in Regenerative Medicine?PubMed
Dalvi Humzah, Beatriz Molina, Giovanni Salti, et al.
Int J Mol Sci. 2024 Mar 12;25(6):3216. doi: 10.3390/ijms25063216.
Hyaluronic acid (HA) is a remarkably multifaceted biomacromolecule, playing a role in regulating myriad biological processes such as wound healing, tissue regeneration, anti-inflammation, and immunomodulation. Crosslinked high- and low-molecular-weight hyaluronic acid hydrogels achieve higher molar concentrations, display slower degradation, and allow optimal tissue product diffusion, while harnessing the synergistic contribution of different-molecular-weight hyaluronans. A recent innovation in the world of hyaluronic acid synthesis is represented by NAHYCO Hybrid Technology, a thermal process leading to hybrid cooperative hyaluronic acid complexes (HCC). This review summarizes the current literature on the in vitro studies and in vivo applications of HCC, from facial and body rejuvenation to future perspectives in skin wound healing, dermatology, and genitourinary pathologies.
7Intra-articular Hyaluronic Acid Treatments for Knee Osteoarthritis: A Systematic Review of Product Properties.PubMed
Eric Ferkel, Ajay Manjoo, Damion Martins, et al.
Cartilage. 2023 Dec;14(4):424-432. doi: 10.1177/19476035231154530. Epub 2023 Jun 14.
INTRODUCTION: There are many intra-articular hyaluronic acid (IA-HA) products on the market that have known intrinsic differences in molecular size, source, and structure. The current review summarizes existing evidence describing and assessing these differences, while also identifying whether these differences have an impact on clinical outcomes. METHODS: This systematic review summarized all literature that specifically addresses IA-HA product differences. Included studies summarized basic science and mechanism of action comparisons of IA-HA product differences, or systematic reviews that assess differences in clinical outcomes between IA-HA product differences. RESULTS: A total of 20 investigations assessed basic science differences between IA-HA products, while 20 investigations provided assessments of the clinical outcome differences between IA-HA product characteristics. The published basic science literature provided a differentiation between low molecular weight (LMW) and high molecular weight (HMW) HA with regard to changes within the synovial fluid, driven by the interactions that these molecules have with receptors in the joint space. These differences in receptor interaction manifest within clinical outcomes, as meta-analyses comparing pain relief after IA-HA suggest that pain reduction is superior in patients who receive HMW HA as opposed to LMW HA. CONCLUSION: This review highlights differences between IA-HA characteristics, and how important the molecular weight, derivation of the product, and structure are to variances in reported clinical outcomes to treat osteoarthritis (OA) of the knee. HMW IA-HAs have shown greater efficacy compared to the alternative of LMW products, while avian-derived and cross-linked products have potentially demonstrated an increase in inflammatory events over non-avian-derived, non-cross-linked HAs.
8Low molecular weight 35 kDa hyaluronan fragment HA35 in the treatment of bone metastasis pain: A case report.PubMed
Zongchun Zhang, Xiaoxiao Jia, Dylan Treger, et al.
Medicine (Baltimore). 2024 Aug 2;103(31):e39145. doi: 10.1097/MD.0000000000039145.
RATIONALE: Late-stage cancer patients often experience severe pain due to bone metastasis, caused by structural damage and cancer-induced inflammation. Hyaluronan, known to alleviate pain by blocking the TRVP1 calcium channel, faces limitations due to its high molecular weight. However, 35 kDa low molecular weight hyaluronan fragment (HA35) have shown promise in relieving various pains, including cancer-related pain. Nonetheless, evidence regarding their efficacy in bone metastasis pain remains scarce. PATIENTS CONCERNS: A 52-year-old female with a rectal malignant tumor and multiple secondary tumors in the sacrum and lungs, accompanied by bone metastasis pain. Despite undergoing radiotherapy, her pain relief was unsatisfactory. Before treatment with HA35, her numerical rating scale score was 10, severely affecting her sleep, appetite, and daily activities. DIAGNOSES: The patient was diagnosed with rectal malignant tumor with multiple metastases, presenting symptoms such as sacral metastasis pain, anal pain, lower limb pain, and anterior abdominal pain. Sacral metastasis pain and lower limb pain indicated a clear diagnosis of bone metastasis pain. INTERVENTIONS: Treatment involved subcutaneous injection into the deep fat tissue layer of the abdomen. A subcutaneous injection of 100 mg/5 mL of HA35 was administered once into the deep fat tissue of the abdomen, with subsequent injections repeated every 3 days. OUTCOMES: Following 1 injection, the patient's pain score decreased to 6 points within 20 minutes, providing 40% pain relief. After 40 minutes, the score further dropped to 4 points, with 60% pain relief. After 50 injections, pain was consistently controlled at around 3 points. LESSONS SUBSECTIONS: Subcutaneous injection of HA35 into the abdominal fat tissue effectively alleviates pain in cancer and bone metastasis patients resistant to conventional treatments. Additionally, it helps alleviate anxiety and fatigue, and improves diet and sleep, thereby offering crucial palliative care for advanced cancer patients.
9Hyaluronan 35kDa treatment protects mice from Citrobacter rodentium infection and induces epithelial tight junction protein ZO-1 in vivo.PubMed
Yeojung Kim, Sean P Kessler, Dana R Obery, et al.
Matrix Biol. 2017 Oct;62:28-39. doi: 10.1016/j.matbio.2016.11.001. Epub 2016 Nov 11.
Maintaining a healthy intestinal barrier, the primary physical barrier between intestinal microbiota and the underlying lamina propria, is critical for optimal health. Epithelial integrity is essential for the prevention of the entrance of luminal contents, such as bacteria and their products, through the large intestinal barrier. In this study, we investigated the protective functions of biosynthetic, specific sized, hyaluronan around 35kDa (HA35) on intestinal epithelium in healthy mice, as well as mice infected Citrobacter rodentium, an established model that mimics infection with a serious human pathogen, enteropathogenic E. coli (EPEC). Our results reveal that treatment with HA35 protects mice from Citrobacter infection and enhances the epithelial barrier function. In particular, we have found that HA35 induces the expression of tight junction protein zonula occludens (ZO)-1 in both healthy and Citrobacter infected mice, as demonstrated by immunoflurorescence and Western blot analyses. Furthermore, we determined that HA35 treatment enhances ZO-1 expression and reduces intestinal permeability at the early stages of dextran sulfate sodium (DSS)-induced colitis in mice. Together, our data demonstrate that the expression and functionality of tight junctions, are increased by HA35 treatment, suggesting a novel mechanism for the protection from Citrobacter infection.
10Hyaluronic Acid 35 kDa Protects against a Hyperosmotic, Formula Feeding Model of Necrotizing Enterocolitis.PubMed
Kathryn Burge, Jeffrey Eckert, Adam Wilson, et al.
Nutrients. 2022 Apr 24;14(9):1779. doi: 10.3390/nu14091779.
Necrotizing enterocolitis (NEC), an inflammatory disease of the intestine, is a common gastrointestinal emergency among preterm infants. Intestinal barrier dysfunction, hyperactivation of the premature immune system, and dysbiosis are thought to play major roles in the disease. Human milk (HM) is protective, but the mechanisms underpinning formula feeding as a risk factor in the development of NEC are incompletely understood. Hyaluronic acid 35 kDa (HA35), a bioactive glycosaminoglycan of HM, accelerates intestinal development in murine pups during homeostasis. In addition, HA35 prevents inflammation-induced tissue damage in pups subjected to murine NEC, incorporating Paneth cell dysfunction and dysbiosis. We hypothesized HA35 treatment would reduce histological injury and mortality in a secondary mouse model of NEC incorporating formula feeding. NEC-like injury was induced in 14-day mice by dithizone-induced disruption of Paneth cells and oral gavage of rodent milk substitute. Mortality and histological injury, serum and tissue cytokine levels, stool bacterial sequencing, and bulk RNA-Seq comparisons were analyzed. HA35 significantly reduced the severity of illness in this model, with a trend toward reduced mortality, while RNA-Seq analysis demonstrated HA35 upregulated genes associated with goblet cell function and innate immunity. Activation of these critical protective and reparative mechanisms of the small intestine likely play a role in the reduced pathology and enhanced survival trends of HA-treated pups subjected to intestinal inflammation in this secondary model of NEC, providing potentially interesting translational targets for the human preterm disease.
11Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing using Enteroid-derived Monolayers.PubMed
Adam Wilson, Kathryn Burge, Jeffrey Eckert, et al.
J Vis Exp. 2022 Jul 28(185). doi: 10.3791/63758.
In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. Here, we describe a protocol to generate a two-dimensional (2D) monolayer from three-dimensional (3D) non-human primate enteroids derived from intestinal crypts of the terminal ileum. These enteroid-derived monolayers were then utilized in an in vitro scratch wound assay to test the ability of hyaluronan 35 kDa (HA35), a human milk HA mimic, to promote cell migration and proliferation along the epithelial wound edge. After the monolayers were grown to confluency, they were manually scratched and treated with HA35 (50 µg/mL, 100 µg/mL, 200 µg/mL) or control (PBS). Cell migration and proliferation into the gap were imaged using a transmitted-light microscope equipped for live-cell imaging. Wound closure was quantified as percent wound healing using the Wound Healing Size Plugin in ImageJ. The scratch area and rate of cell migration and the percentage of wound closure were measured over 24 h. HA35 in vitro accelerates wound healing in small intestinal enteroid monolayers, likely through a combination of cell proliferation at the wound edge and migration to the wound area. These methods can potentially be used as a model to explore intestinal regeneration in the preterm human small intestine.
12Hyperglycemic environments directly compromise intestinal epithelial barrier function in an organoid model and hyaluronan (∼35 kDa) protects via a layilin dependent mechanism.PubMed
Samreen Jatana, Amina Abbadi, Gail A West, et al.
Matrix Biol. 2024 Nov;133:116-133. doi: 10.1016/j.matbio.2024.08.007. Epub 2024 Aug 24.
BACKGROUND: Metabolic syndrome and diabetes in obese individuals are strong risk factors for development of inflammatory bowel disease (IBD) and colorectal cancer. The pathogenic mechanisms of low-grade metabolic inflammation, including chronic hyperglycemic stress, in disrupting gut homeostasis are poorly understood. In this study, we sought to understand the impact of a hyperglycemic environment on intestinal barrier integrity and the protective effects of small molecular weight (35 kDa) hyaluronan on epithelial barrier function. METHODS: Intestinal organoids derived from mouse colon were grown in normal glucose media (5 mM) or high glucose media (25 mM) to study the impact of hyperglycemic stress on the intestinal barrier. Additionally, organoids were pretreated with 35 kDa hyaluronan (HA35) to investigate the effect of hyaluronan on epithelial barrier under high glucose stress. Immunoblotting as well as confocal imaging was used to understand changes in barrier proteins, quantitative as well as spatial distribution, respectively. Alterations in barrier function were measured using trans-epithelial electrical resistance and fluorescein isothiocyanate flux assays. Untargeted proteomics analysis was performed to elucidate mechanisms by which HA35 exerts a protective effect on the barrier. Intestinal organoids derived from receptor knockout mice specific to various HA receptors were utilized to understand the role of HA receptors in barrier protection under high glucose conditions. RESULTS: We found that high glucose stress decreased the protein expression as well as spatial distribution of two key barrier proteins, zona occludens-1 (ZO-1) and occludin. HA35 prevented the degradation or loss of ZO-1 and maintained the spatial distribution of both ZO-1 and occludin under hyperglycemic stress. Functionally, we also observed a protective effect of HA35 on the epithelial barrier under high glucose conditions. We found that HA receptor, layilin, was involved in preventing barrier protein loss (ZO-1) as well as maintaining spatial distribution of ZO-1 and occludin. Additionally, proteomics analysis showed that cell death and survival was the primary pathway upregulated in organoids treated with HA35 under high glucose stress. We found that XIAP associated factor 1 (Xaf1) was modulated by HA35 thereby regulating apoptotic cell death in the intestinal organoid system. Finally, we observed that spatial organization of both focal adhesion kinase (FAK) as well as F-actin was mediated by HA35 via layilin. CONCLUSION: Our results highlight the impact of hyperglycemic stress on the intestinal barrier function. This is of clinical relevance, as impaired barrier function has been observed in individuals with metabolic syndrome. Additionally, we demonstrate barrier protective effects of HA35 through its receptor layilin and modulation of cellular apoptosis under high glucose stress.
13Network-Based Bioinformatics Highlights Broad Importance of Human Milk Hyaluronan.PubMed
Kathryn Y Burge, Hua Zhong, Adam P Wilson, et al.
Int J Mol Sci. 2024 Nov 26;25(23):12679. doi: 10.3390/ijms252312679.
Human milk (HM) is rich in bioactive factors promoting postnatal small intestinal development and maturation of the microbiome. HM is also protective against necrotizing enterocolitis (NEC), a devastating inflammatory condition predominantly affecting preterm infants. The HM glycosaminoglycan, hyaluronan (HA), is present at high levels in colostrum and early milk. Our group has demonstrated that HA with a molecular weight of 35 kDa (HA35) promotes maturation of the murine neonatal intestine and protects against two distinct models of NEC. However, the molecular mechanisms underpinning HA35-induced changes in the developing ileum are unclear. CD-1 mouse pups were treated with HA35 or vehicle control daily, from P7 to P14, and we used network and functional analyses of bulk RNA-seq ileal transcriptomes to further characterize molecular mechanisms through which HA35 likely influences intestinal maturation. HA35-treated pups separated well by principal component analysis, and cell deconvolution revealed increases in stromal, Paneth, and mature enterocyte and progenitor cells in HA35-treated pups. Gene set enrichment and pathway analyses demonstrated upregulation in key processes related to antioxidant and growth pathways, such as nuclear factor erythroid 2-related factor-mediated oxidative stress response, hypoxia inducible factor-1 alpha, mechanistic target of rapamycin, and downregulation of apoptotic signaling. Collectively, pro-growth and differentiation signals induced by HA35 may present novel mechanisms by which this HM bioactive factor may protect against NEC.
14Layilin is critical for mediating hyaluronan 35kDa-induced intestinal epithelial tight junction protein ZO-1 in vitro and in vivo.PubMed
Yeojung Kim, Gail A West, Greeshma Ray, et al.
Matrix Biol. 2018 Mar;66:93-109. doi: 10.1016/j.matbio.2017.09.003. Epub 2017 Oct 1.
Tight junction proteins are critical in maintaining homeostatic intestinal permeability. Multiple intestinal inflammatory diseases are correlated with reduced expression of tight junction proteins. We have recently reported that oral treatment of mice with Hyaluronan 35kDa (HA35) increases colonic expression of tight junction protein zonula occludens-1 (ZO-1). Here, we investigate whether HA35 treatment enhances ZO-1 expression by direct interaction with intestinal epithelium in vitro and have identified the HA receptor responsible for HA35-mediated ZO-1 induction in colonic epithelium in vitro and in vivo. Our results reveal that HA35 treatment increases ZO-1 expression in mouse intestinal epithelial organoids, while large HA 2000kDa is not internalized into the cells. Our immunofluorescence data indicate that layilin, but neither toll-like receptor-4 (TLR-4) nor CD44, mediate the HA35-induced ZO-1 expression in colonic epithelium in vitro and in vivo. Additionally, using layilin null mice we have determined that layilin mediates HA35 induction of ZO-1 in healthy mice and during dextran sulfate sodium (DSS)-induced colitis. Furthermore, we find that while ZO-1 expression levels are reduced, layilin expression levels are equivalent in inflammatory bowel disease (IBD) patients and non-IBD controls. Together, our data suggest that layilin is an important HA receptor, that mediates the effect of oral HA35 treatment on intestinal epithelium. HA35 holds promise as a simple dietary supplement to strengthen gut barrier defense.
15Hyaluronic acid 35 normalizes TLR4 signaling in Kupffer cells from ethanol-fed rats via regulation of microRNA291b and its target Tollip.PubMed
Paramananda Saikia, Sanjoy Roychowdhury, Damien Bellos, et al.
Sci Rep. 2017 Nov 15;7(1):15671. doi: 10.1038/s41598-017-15760-4.
TLR4 signaling in hepatic macrophages is increased after chronic ethanol feeding. Treatment of hepatic macrophages after chronic ethanol feeding with small-specific sized hyaluronic acid 35 (HA35) normalizes TLR4 signaling; however, the mechanisms for HA35 action are not completely understood. Here we used Next Generation Sequencing of microRNAs to identify negative regulators of TLR4 signaling reciprocally modulated by ethanol and HA35 in hepatic macrophages. Eleven microRNAs were up-regulated by ethanol; only 4 microRNAs, including miR291b, were decreased by HA35. Bioinformatics analysis identified Tollip, a negative regulator of TLR4, as a target of miR291b. Tollip expression was decreased in hepatic macrophages from ethanol-fed rats, but treatment with HA35 or transfection with a miR291b hairpin inhibitor restored Tollip expression and normalized TLR4-stimulated TNFα expression. In peripheral blood monocytes isolated from patients with alcoholic hepatitis, expression of TNFα mRNA was robustly increased in response to challenge with lipopolysaccharide. Importantly, pre-treatment with HA35 reduced TNFα expression by more than 50%. Taken together, we have identified miR291b as a critical miRNA up-regulated by ethanol. Normalization of the miR291b → Tollip pathway by HA35 ameliorated ethanol-induced sensitization of TLR4 signaling in macrophages/monocytes, suggesting that HA35 may be a novel therapeutic agent in the treatment of ALD.
1635kDa hyaluronan ameliorates ethanol driven loss of anti-microbial defense and intestinal barrier integrity in a TLR4-dependent manner.PubMed
Semanti Ray, Emily Huang, Gail A West, et al.
Matrix Biol. 2023 Jan;115:71-80. doi: 10.1016/j.matbio.2022.11.008. Epub 2022 Nov 26.
Acute and chronic alcohol exposure compromise intestinal epithelial integrity, due to reduced expression of anti-microbial peptides (AMP) and loss of tight junction integrity. Ameliorating gut damage is beneficial in preventing associated distant organ pathologies. Orally administered purified hyaluronan (HA) polymers with an average size of 35 kDa have multiple protective effects in the gut and are well-tolerated in humans. Therefore, we tested the hypothesis that HA35 ameliorates ethanol-induced gut damage. Specifically, mechanisms that restore epithelial barrier integrity and normalize expression of the Reg3 class of C-type lectin AMPs (i.e. Reg3β and Reg3γ) were investigated. Chronic ethanol feeding to mice reduced expression of C-type lectin AMPs in the proximal small intestine (jejunum), reduced expression of tight junction proteins and increased bacterial translocation to the mesenteric lymph node. Oral consumption of HA35 during the last 6 days of ethanol exposure ameliorated the effects of chronic ethanol. Similarly, in vitro challenge of isolated intestinal organoids from murine jejunum with ethanol reduced the expression of C-type lectin AMPs and impaired barrier integrity; these ethanol-induced responses were prevented by pre-treatment with HA35. Importantly, HA receptor null jejunum-derived organoids demonstrated that the HA receptor Tlr4, but not Cd44 nor Tlr2, was required for the protective effect of HA35. Consistent with the data from organoids, HA35 did not protect Tlr4-deficient mice from chronic ethanol-induced intestinal injury. Together, these data suggest therapeutic administration of HA35 is beneficial in restoring gut epithelial integrity and defense during the early stages of ethanol-driven intestinal damage.
17Safety of Hyaluronan 35 in Healthy Human Subjects: A Pilot Study.PubMed
Annette Bellar, Sean P Kessler, Dana R Obery, et al.
Nutrients. 2019 May 22;11(5):1135. doi: 10.3390/nu11051135.
. Hyaluronan (HA) is a naturally occurring glycosaminoglycan polymer produced in all vertebrates, and usually present at the high molecular weight (>10 Da). Low molecular weight HA has signaling properties, and fragments ~35 kDa size (HA35) have biological activity in eliciting epithelial β-defensins and tight junction proteins, notably ZO1, important components of innate host defense arsenal of the gut barrier in preclinical models. Safety, tolerability, impact on metabolism, gut permeability, and microbiome composition in healthy human subjects were all evaluated prospectively. . Pharmaceutical grade HA35 (140 mg in water once daily for seven days), was administered orally to 20 healthy subjects (30.7 ± 5.6 years). Demographical, clinical, biochemical laboratory tests, metabolic function and stool microbiome composition were measured on Day 0, 8 and 28. . HA35 was tolerated well in all subjects with no serious adverse events in any subjects. No statistical differences in any of the measurements were seen among the study group over the course of the trial. In aggregate there were no changes in demographical, clinical, biochemical laboratory tests, and metabolic function or microbiome composition during the 28-day study. . Oral HA35 administration (140 mg/day) is a safe treatment in healthy individuals and does not affect metabolic, inflammatory or microbiome parameters.
18Specifically Sized Hyaluronan (35 kDa) Prevents Ethanol-Induced Disruption of Epithelial Tight Junctions Through a layilin-Dependent Mechanism in Caco-2 Cells.PubMed
Damien A Bellos, Dhara Sharma, Megan R McMullen, et al.
Alcohol Clin Exp Res. 2019 Sep;43(9):1848-1858. doi: 10.1111/acer.14140. Epub 2019 Jul 16.
BACKGROUND: Specific-sized species of the carbohydrate hyaluronan elicit a variety of cellular responses mediating tissue integrity and repair, as well as regulating inflammatory responses. Orally provided hyaluronan with an average molecular weight of 35 kDa (HA35) protects mice from short-term ethanol (EtOH)-induced liver injury. This protection was associated with maintenance of the colocalization of zonula occludens-1 (ZO-1) and occludin at tight junctions in the proximal colon. However, it is not known whether HA35 also protects other regions of the intestine or whether protection is due to a direct and/or indirect interaction of HA35 with the intestinal epithelium. METHODS: Female C57BL/6J mice were fed an EtOH containing diet or pair-fed control diet (4 days) and treated with or without HA35 via daily gavage during the last 3 days of EtOH feeding. Intestinal morphology and tight junction integrity were assessed. Differentiated Caco-2 cells were transfected or not with scrambled siRNA or siRNA targeting layilin, a hyaluronan receptor. Caco-2 cells were treated with or without HA35 prior to challenge with EtOH. Localization of tight junction proteins, fluorescein isothiocyanate (FITC)-dextran permeability, and transepithelial electrical resistance (TEER) were evaluated. RESULTS: While short-term EtOH did not result in any apparent changes in the gross morphology of the intestine, colocalization of ZO-1 and occludin at tight junctions was decreased in the proximal and distal colon. HA35 prevented these effects of EtOH. In differentiated Caco-2 cells, EtOH decreased the localization of ZO-1 and occludin at tight junctions and increased permeability of FITC-dextran. At higher concentrations, EtOH also decreased TEER. Pretreatment with HA35 prevented these changes. When the hyaluronan receptor layilin was knocked down in Caco-2 cells, HA35 no longer protected cells from EtOH-induced loss of tight junctions. CONCLUSIONS: Taken together, these data indicate that HA35 interacts with layilin on intestinal epithelial cells and maintains intestinal tight junction integrity during short-term EtOH exposure.
19A clinical study of the local injection of a freshly manufactured 35 kDa hyaluronan fragment for treating chronic wounds.PubMed
Dylan Treger, Lujia Zhang, Xiaoxiao Jia, et al.
Int Wound J. 2024 May;21(5):e14906. doi: 10.1111/iwj.14906.
This study manufactured a 35 kDa hyaluronan fragment (HA35) by enzymatically degrading high-molecular-weight HA using hyaluronidase PH20 derived from bovine testis. The research then examined the therapeutic efficacy of locally administered, tissue-permeable HA35 in alleviating chronic wounds and their associated neuropathic pain. For 20 patients with nonhealing wounds and associated pain lasting over three months, 100 mg of HA35 was injected daily into the healthy skin surrounding the chronic wound for 10 days. Self-assessments before and after treatment indicated that HA35 significantly enhanced wound healing. This was evidenced by the formation of fresh granulation tissue on the wounds (p < 0.0001); reduced darkness, redness, dryness, and damage in the skin surrounding the wounds (p < 0.0001), and a decrease in wound size (p < 0.001). Remarkably, HA35 injections alleviated pain associated with chronic wounds within 24 hours (p < 0.0001). It can be concluded that the low-molecular-weight hyaluronan fragment HA35 potentially enhances the immune response and angiogenesis during wound healing.
20Multifunctional Role of 35 Kilodalton Hyaluronan in Promoting Defense of the Intestinal Epithelium.PubMed
Sean P Kessler, Dana R Obery, Kourtney P Nickerson, et al.
J Histochem Cytochem. 2018 Apr;66(4):273-287. doi: 10.1369/0022155417746775. Epub 2018 Jan 1.
Intestinal epithelium plays a critical role in host defense against orally acquired pathogens. Dysregulation of this protective barrier is a primary driver of inflammatory bowel diseases (Crohn's and ulcerative colitis) and also infant gastrointestinal infections. Previously, our lab reported that hyaluronan (HA) isolated from human milk induces the expression of the antimicrobial peptide β-defensin in vivo and protects against Salmonella Typhimurium infection of epithelial cells in vitro. In addition, we demonstrated that commercially available 35 kDa size HA induces the expression of β-defensin, upregulates the expression of tight junction protein zonula occludens-1 (ZO-1), and attenuates murine Citrobacter rodentium infection in vivo. In this current study, we report that HA35 remains largely intact and biologically active during transit through the digestive tract where it directly induces β-defensin expression upon epithelial cell contact. We also demonstrate HA35 abrogation of murine Salmonella Typhimurium infection as well as downregulation of leaky tight junction protein claudin-2 expression. Taken together, we propose a dual role for HA in host innate immune defense at the epithelial cell surface, acting to induce antimicrobial peptide production and also block pathogen-induced leaky gut. HA35 is therefore a promising therapeutic in the defense against bacterially induced colitis in compromised adults and vulnerable newborns.