Figura 4. Competitividade de oligossacarídeos e HA em ligação a CD44. (A) Reduzindo o efeito de oligossacarídeos em CD44 antigenicidade. Recombinante rato CD44 (20 ng/mL) foi misturado com ou sem FC, FOS, ou GOS para 1{{10}}} min e então analisado usando o CD44 ELISA kit. Resultados são expressos como média ± SD (n=4). *, p < 0,05 vs. o grupo controle. (B)interfere com oligossacarídeos' inibitório efeitos em CD44 e fosforilado JNK expressão. NRK-52E células foram pré-tratados com 0,1 mg/mL HA para 30 min, então administrado com 0,1 mg/mL FC, FOS, ou GOS para 30 min, e finalmente sujeitos a hipóxica cultura. Proteína expressão foi analisada por Western blot. Relativa aumentos em a proteína bandas são também apresentadas em barra gráfico forma. Resultados são expressos como média ± DP ( n=4).

A NOVO ERVA PARA CISTANCHE PARA TRABALHO?

3.5. Oligosaccharides Improve Renal Function and Reduce Inflammation in Mice with Early-Stage AKI 

Um AKI mouse modelo com IRI foi estabelecido para avaliar a influência de oligossacarídeos % c2% a0on renal função E inflamação. Renal função foi monitorado por detecção soro creatinina. O ratos foram alimentados com oligossacarídeos only at the initial stage of AKI, i.e., from 1 day before to 2 days after IRI surgery. After this period, serum creatinine significantly increased in AKI mice but not in oligosaccharide-treated AKI mice, indicating that oligosaccharides have improved renal function (Figure 5A). Serum cytokine levels were then monitored to evaluate the inflammation of AKI mice. Serum MCP-1, IL-1 , and TNF- were significantly increased in AKI mice (Figure 5B–D) but were reduced by all three oligosaccharides. IHC analysis of neutrophil marker Ly6G showed neutrophil infiltration in the kidneys of AKI mice but not in normal mice and oligosaccharide-treated AKI mice (Figure 6A). IHC analysis of macrophage marker F4/80 revealed macrophage invasion in the kidneys of AKI mice but not in normal mice and AKI mice treated with oligosaccharides (Figure 6B). Compared with those of normal mice and AKI mice fed with oligosaccharides, the renal tubules of AKI mice showed high TNF- expression (Figure 6C). These results indicate that FC, FOS, and GOS reduce renal inflammation in mice with early-stage AKI.

Figura 5. Reduzir o efeito de oligossacarídeos sobre soro creatinina e citocinas em IA camundongos em o estágio inicial. Sangue foi coletado de cada camundongo 2 dias após IRI cirurgia para medida soro creatinina (A), MCP-1 (B), IL-1 (C), e TNF- (D). Os resultados são expressos como meios ± SD (n=8). 

3.6. Oligossacarídeos Promover Rim Recuperação em Pós-LRA 

Em o sétimo dia depois IRI cirurgia% 2c o soro creatinina de AKI camundongos diminuiu mas foi ainda maior do que de normal camundongos (Figura 7A). Não diferença significativa em soro creatinina foi observada entre AKI camundongos tratados com e sem oligossacarídeos. No entanto% % 2c soro NGAL nível % 2c a AKI marcador LRA % 2c foi ainda maior em AKI camundongos do que em normal camundongos e oligossacarídeos tratados AKI camundongos (Figura 7B). Este achado indica que alimentação oligossacarídeos em o estágio inicial de IA pode reduzir lesão renal em pós-LRA. PAS coloração de camundongo rim córtex foi conduzido em 7 dias após IRI cirurgia. IA camundongos exibiu grave tubular lesão com tubular dilatação e intraluminal célula detritos por tubular necrose, e AKI camundongos tratados com oligossacarídeos mostrou apenas menor tubular lesão (Figura 7C). Portanto, a ingestão de FC, FOS, ou GOS no estágio inicial de AKI aids na recuperação de rim tecidos e renal função em AKI camundongos.

Figura 6. Inibitório efeito de oligossacarídeos em inflamação renal em camundongos com estágio inicial LRA. O rins de cada camundongo 2 dias após IRI cirurgia foram coletados para IHC coloração. (A) Ly6G IHC coloração. O branco seta indica Ly6G positivo coloração neutrófilos. (B) F4/80 IHC coloração. O branco seta indica F4/80 positivo coloração macrófagos. (C) TNF- IHC coloração

4. Discussão 

Ischemia is one of the main causes of AKI. Here, a hypoxic cell model and an AKI mouse model with IRI were established to study the influence of oligosaccharides on AKI and their underlying molecular mechanism. Hypoxia-induced the expression of MCP-1, IL-1 , and TNF- in NRK-52E cells, which were inhibited by JNK inhibitor SP600125. CD44 siRNA transfection blocked the hypoxia-induced activation of JNK. These results indicate that hypoxia upregulates CD44, which in turn activates JNK and thereby regulates cytokines in renal tubular cells. FC, FOS, and GOS reduced CD44 antigenicity and competed with the ligand HA of CD44 of renal tubular cells. This finding implies that the three oligosaccharides can directly interact with CD44 to suppress hypoxia-induced CD44 upregulation, JNK activation, and cytokine expression in renal tubular cells. Although p38 is another important MAP kinase for TNF and IL-1 during inflammatory responses [21], phosphorylated p38 was not upregulated in hypoxic NRK-52E cells. Therefore, p38 is not involved in cytokine expression in renal tubular cells and the inhibition effect of oligosaccharides on the inflammatory responses of renal tubular cells. FC and GOS upregulated p38 phosphorylation in hypoxic cells, indicating their potential influence on p38-associated inflammatory responses. In animal studies, FC, FOS, and GOS inhibited the increase in serum MCP-1 and IL-1 in mice with early-stage AKI and reduced TNF- expression in kidney tissues. Cytokines are key effectors of leukocyte recruitment. FC, FOS, and GOS reduced neutrophil infiltration and macrophage invasion in the kidneys of mice with early stage AKI. At 7 days after surgery, the reduced inflammation and neutrophil infiltration in AKI at the early stage is beneficial for recovery at the advanced stage. This study reveals for the first time that the consumption of FC, FOS, or GOS at the early stage of AKI can promote recovery by inhibiting kidney inflammation. 

To date, the influence of oligosaccharide supplementation on AKI has not been explored. Many oligosaccharides are considered prebiotics and can promote the growth of favorable microbiota [13,22]. Human milk oligosaccharides regulate the development and function of the immune system by constructing specific microbiota [23,24]. The intestinal microbiota is an important modifier of AKI outcome [20]. In theory, oligosaccharides can affect AKI severity by regulating intestinal microbiota. However, microbiota construction by oligosaccharides and immune moderation by microbiota would require more than several weeks. In the current animal study, the mice were fed with oligosaccharides from 1 day before to 2 days after IRI surgery. The results showed the positive effect of oligoscharades on AKI on the second day after IRI surgery. Therefore, the renal protection of oligosaccharides in the current system is not caused by the influence of microbiota. Many oligosaccharides, including FC, FOS, and GOS, can be absorbed by the body, enter the systemic circulation, and finally be eliminated by the kidneys [14–16,22,25]. Those oligoscharades have a chance to interact with CD44 on renal tubular cells in vivo. By influencing CD44, oligosaccharides can instantly modulate the immune response of the kidney with acute injury, thereby improving its recovery. In addition, human milk oligosaccharides directly modulate immune responses locally or systemically [22,25]. Therefore, the effect of oligosaccharides on immune and other physiological systems is attributed to their direct influences and microbiota changes.

CD44 promotes cell adhesion and acts as a signal receptor in many important physiological phenomena [26]. The interaction of CD44 and its major ligand HA, a major constituent of the extracellular matrix, can enhance cell adhesion, proliferation, migration, and immune responses [27–29]. HA, binding to CD44 initiates intracellular signaling events by modulating the downstream signaling molecules of CD44, including actin cytoskeleton [30]; ezrin, radixin, and moesin proteins [31]; ankyrin [32]; and non-receptor tyrosine kinase Src [33]. These downstream signal molecules are related to JNK activation. Reorganization of the actin cytoskeleton activates numerous signaling cascades, specifically JNK [34,35]. Ezrin binds with JNK signaling components to facilitate JNK activation in B cells [36]. Ankyrin and Src also induce JNK activation [37–39]. Therefore, the signal transduction of CD44 is associated with JNK activation. HA binding to CD44 has been proven to promote JNK activation in breast cancer cells [19]. In the present study, oligoscharades were found to interfere with the interaction between HA and CD44 to inhibit the downstream JNK signal transduction and consequently the immune response of renal tubular cells. Based on these findings, oligosaccharides show the potential to suppress other physiological responses related to CD44, especially the proliferation and migration of cancer cells promoted by CD44. 

Oligosaccharides currently available on the market mainly include FOS, GOS, FC, and isomaltooligosaccharides [40,41]. FOS, GOS, and FC can be partially absorbed by the body, enter the systemic circulation, and be ingested from vegetables, milk, and seaweed [14–16,22,25]. In this work, the influence of these three anionic oligosaccharides on AKI was explored. Although these three oligosaccharides have different monosaccharide compositions, their effects on renal tubular cells under hypoxia are highly similar, including the inhibitory effect on CD44, phosphorylated JNK, and cytokines. Moreover, the added HA interfered with the inhibitory effect of all three oligosaccharides. Therefore, the mechanism of these three oligosaccharides on renal tubular cells is the same regardless of their monosaccharide type. This mechanism may be related to the structural similarities between oligosaccharides and HA. However, the types of monosaccharides in the three oligosaccharides still cause slightly different effects in the current system. FC and FOS reduced JNK expression in hypoxic renal tubular cells, but GOS exhibited a limited effect. The influence of FOS on CD44 antigenicity was weaker than that of FC and GOS. The most effective dose of FOS (0.5 mg/mL) for inhibiting hypoxia-induced CD44 was also higher than that of FC and GOS (0.05 mg/mL). These results suggest that the inhibitory effects of FC and GOS on CD44 signaling transduction are greater than those of FOS. However, animal experiments showed similar renal protective effects from the ingestion of the three oligosaccharides at the initial stage of AKI. Comparing the renal protective effects among the three oligosaccharides in AKI mice is difficult and involves the absorption of oligosaccharides in the intestine. Most oligosaccharides are digested by intestinal bacteria, and only a small portion can be absorbed [42,43]. Digestion efficiency in the intestine varies for different oligosaccharides, which in turn affects their absorption amount. In addition, the length of oligosaccharides also affects the efficiency of their absorption in the gut. In this study, FC with the smallest average degree of polymerization would theoretically be more readily absorbed than FOS with the largest average degree of polymerization. However, animal experiments revealed that after oligosaccharides (10 mg/kg/d) are digested by the intestinal bacteria, a sufficient amount can still enter circulation to achieve a renal protective effect on AKI mice.

In summary, FC, FOS, and GOS directly interact with CD44 to inhibit hypoxia-induced CD44 upregulation, JNK activation, and cytokine expression in renal tubular cells. Ingesting FC, FOS, and GOS at early AKI stages promotes recovery by inhibiting kidney inflammation. AKI suddenly occurs in patients admitted to hospitals and intensive care units, and no treatment has been developed to avoid or ameliorate this condition. Based on these findings, the daily ingestion of oligosaccharides can reduce AKI severity and promote kidney recovery in patients with AKI. 

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