Protective effect of Astragalus membranaceus and Astragaloside IV in sepsis-induced acute kidney injury

Chemicals and reagents

LPS (E. coli 0111: B4) was obtained from Millipore Sigma. Astragalus membranaceus was purchased from Kang Qiao Traditional Chinese Medicine Decoction Pieces Co. Ltd. Astragaloside IV (purity above 98%) was purchased from Xi’an Sobeo Pharmaceutical Technology Co., Ltd. The antibodies used for Western blotting, PI3K (#4255) and Phospho-PI3K p85 (Tyr458) (#17366) were purchased from Cell Signaling Technology. AKT (A17909) and Phospho-AKT (Ser473) (AP0637) were purchased from ABclonal.

Water extract of Astragalus membranaceus

Astragalus slices (500 g) were weighed, distilled water was added in a ration of 1:10, soaked for 2–3 h, then decocted and extracted 3 times at 90°C for 1.5 h. The decoction was filtered twice with gauze and the filtrate combined. The filtrate was concentrated to a viscous liquid using a rotary evaporator at 50°C, poured into trays, and dried in a water bath set at 65°C to form an infusion. The infusion was frozen for two days at −80°C and then lyophilized to form a powder in a freeze dryer.

Animals

Male C57BL/6J mice (18–22 g) were purchased from the Changzhou Cavens Laboratory Animal Co., China. All mice were housed in an automated 12-hour dark-light cycle at a controlled temperature of 22°C ± 2°C and relative humidity of 50–60%, with free access to standard dry feed and tap water. All animal experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011) and were approved by the Naval Medical University Committee on Animal Care (EC11-055).

Sepsis model

Sepsis was induced by CLP as previously described [14]. Isoflurane was used to completely anesthetize the mice, and a midline abdominal incision was made. The ileocecal valve was ligated on the distal 3/4 of the cecum. in a sterile environment, the cecum was perforated twice with a 21-gauge needle, and a droplet of feces was extruded from the perforations to induce polymicrobial peritonitis. Sutures were placed in two layers along the abdomen wall and 1 ml of a 0.9% sodium chloride solution was administered for fluid resuscitation. The sham group underwent laparotomy and bowel manipulation without ligation or perforation. Following recovery from anesthesia, all mice had unrestricted access to food and water. There were three groups of six animals each for Astragalus membranaceus: the sham-operated group (Sham group), the sepsis model group (CLP group), and treatment groups with Astragalus membranaceus (250 mg/kg). Astragalus membranaceus was administrated orally every day before CLP. There were four groups of six animals each in the AS-IV study, a sham group, a CLP group, and two treatment groups: AS-IV low dose (5 mg/kg) and AS-IV high dose (10 mg/kg). AS-IV was administrated orally every day before CLP.

Kidney injury study

Kidney tissues were cut into 5 μm sections and stained with H&E, PAS, and nitrotyrosine reagents, as well as KIM-1 antibody and Phospho-AKT antibody. The kidney injury score is as follows: 0, normal; 1, <10%; 2, 10–25%; 3, 25–50%; 4, 50–75%; 5, 75–100% of affected kidney areas obtained from mice exposed to sham or CLP surgery. Tubular atrophy or dilatation, loss of the brush border, vacuolization, epithelial cell shedding, and denuded tubular basement membrane were some of the criteria.

Cell culture

HK-2 cells (CRL-2190, ATCC, Rockefeller, MD, USA) were maintained in RPMI1640 (31800022, Gibco, NY, USA) supplemented with 10% v/v FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) in humidified incubators at 37°C and 5% CO₂.

Bioinformatics analysis

The TCMSP server consisted of a system-level pharmacology database with a flexible, user-friendly web interface. To gain a better understanding of the complex, network targets genes and diseases were constructed and analyzed using Cytoscape 3.0.

Western blotting

The protein samples were electrophoresed in 8–12% SDS-PAGE gels and transferred onto nitrocellulose membranes (Amersham). The membranes were blocked with 5% BSA in PBS for 1 hour at room temperature and then probed overnight at 4°C with primary antibodies (1:1000 dilution). The membranes were then incubated with secondary antibodies (1:10,000 dilution) for 50 min at room temperature. Finally, Odyssey (LI-COR, Lincoln, NE, USA) and the relative protein expression were calculated from gray-scale blots using ImageJ software.

Statistics

GraphPad Prism 9.0.1 was used to perform statistical analysis and data were expressed as mean ± SEM. Statistical significance was determined using Student’s t-test or ANOVA. P < 0.05 was considered statistically significant.

Data statement

Our data are available. Please contact the corresponding author for requirement. More detailed Materials and Methods are in the Supplementary Materials and Methods.

The effect of Astragalus membranaceus active ingredients on LPS-induced damage on HK-2 cell line

Given that sepsis’s primary target is proximal tubular epithelial cells, HK-2 cells were treated with LPS to mimic Gram (–) induced sepsis-associated tubular injury. A total of 144 Astragalus-related compounds were identified through a database search. The TCMSP database was examined for OB and DL values of each compound, and 42 compounds (28.6%) had OB values greater than 30%, 50 compounds (40%) had DL values greater than 0.18, and 22 compounds (31.4%) had optimum OB and DL values. Astragalus saponins were excluded from the study due to their failure to meet the OB and DL thresholds. However, because these components are the most often reported active ingredients in the pharmacological action of Astragalus, they were included in this study. To screen for anti-SA-AKI activity, the final 26 monomers from Astragalus were collected (Table 1). We discovered nine compounds, including isorhamnetin, kaempferol, mangosteen, and astragaloside, that have been reported in “sepsis” or “acute kidney injury” and have been verified by the official website of the China Academy of Food and Drug Control. The nine compounds were screened further using LPS-induced HK-2 cells. The MTT assay was used to examine the effects of isorhamnetin (Iso), kaempferol (Kae), formononetin (Form), astragaloside I (AS-I), astragaloside II (AS-II), astragaloside IV (AS-IV), Mairin, Jaranol, and Calycosin (Cal) on LPS-induced HK-2 cell injury in vitro (Figure 2).

The effect of LPS on HK-2 cells

LPS significantly inhibited the survival rate of HK-2 cells (P < 0.001). Astragaloside I (AS-I) and Astragaloside IV (AS-IV) demonstrated dose-dependent protective effects at 20 μM versus 50 μM concentrations (AS-I: P < 0.01, P < 0.001; Form: P < 0.001, P < 0.001; AS-IV: P < 0.001, P < 0.001). At 50 μM Iso and Cal showed significant protection (P < 0.001). In contrast, treatment of HK-2 cells with Kae, AS-II, Mairin, and Jaranol did not result in any significant changes in the LPS-induced cytotoxicity (Figure 2). Our results demonstrated that AS-I, AS-IV, Iso, and Cal all exerted protective effects against LPS-induced HK-2 cytotoxicity in renal epithelial cells at 50 μM concentration, with AS-IV exhibiting the most pronounced protective effect. Therefore, we further investigated the mechanism through which AS-IV protects against septic acute kidney injury.

AS-IV protects against renal injury in CLP-induced sepsis

The China Pharmacopeia 2015 specifies that the content of AS-IV in Astragalus should not be less than 0.040%. Using HPLC analysis, we determined the AS-IV content of Astragalus to be 0.147%, which met the quality standard requirements stipulated in the China Pharmacopeia 2015 (Supplementary Figure 1). To determine the protective effect of AS-IV on SA-AKI-induced renal damage, we subjected mice to CLP or sham surgery. The treatment group was divided into two groups, low-dose (i.p. 5 mg/kg) and high-dose (i.p. 10 mg/kg). Similarly, the AS-IV 5 mg/kg and 10 gm/kg groups had considerably fewer intracellular vacuoles (Figure 3A).

Additionally, the treatment groups showed significant loss of brush border in the renal proximal tubular epithelial cells caused by CLP (Figure 3B). After AS-IV treatment, mice showed a markedly decrease kidney injury score (Figure 3C). Immunohistochemical analysis revealed decreased staining of KIM-1 on the apical side of renal tubular epithelial cells in kidney sections from treated septic mice (Figure 3D). Next, BUN and creatinine levels were markedly decreased in mice of the treatment groups, indicating preserved renal function for AS-IV (Figure 3E and 3F). ELISA results confirmed that IL-6 and IL-1β concentrations in serum were deceased after AS-IV treatment (Figure 3G).

Astragalus membranaceus and AS-IV for SA-AKI target prediction

The therapeutic target database (TTD), Genecards, the Comparative Toxicogenomics Database (CTD), and the CTD were searched using the terms “sepsis” and “acute kidney damage (AKI). Genecards and the Comparative Toxicogenomics Database (CTD) were used to search for relevant disease targets, and the CTD database was used to identify experimentally validated disease targets designated as “marker/mechanism” or “therapeutic”. The CTD database selects targets with a high degree of confidence that has been experimentally validated with “marker/mechanism” or “therapeutic” as targets for research. Finally, 2498 sepsis-related genes and 6746 AKI-related genes were retrieved from the GeneCards database, while 48 sepsis-related genes and 120 AKI-related genes were retrieved from the CTD database. A total of 1938 targets were obtained after a comprehensive screening of all targets to eliminate duplicates, and the DAVID database validated a total of 1910 targets related to sepsis and acute kidney injury in humans. All genes were imported into the DAVID database, and the relevant genes were exported by selecting and clicking “show gene lis”. GO enrichment and KEGG pathway enrichment analyses were performed on the disease targets via the DAVID database, using P < 0.01 and False discovery rate (False discovery rate (FDR) <0.01 as the screening thresholds. A total of 686 entries for biological process (BP), 104 for cellular component (CC), and 121 for molecular function (MF) were obtained. The top 20 entries are displayed in Tables 2 and 3 below, are ordered by the magnitude of their P and FDR values. SA-AKI was predicted to be involved in biological processes such as the inflammatory response, immune response, intrinsic immune response, response to LPS, response to hypoxic factors, etc. The cellular components were involved in extracellular gaps, extracellular regions, molecular functions including protein binding, receptor binding, enzyme binding, etc. Astragalus and SA-AKI targets were intersected by Venn to obtain a total of 275 core targets (Figure 4A). AS-IV and SA-AKI targets were intersected by Venn to obtain a total of 167 core targets (Figure 4B). We further examined the relationship between the bio-active ingredients and the core targets, to construct an interaction network between the two (Supplementary Figure 2). A total of 26 active ingredients of Astragalus were identified to be critical to the core target. The high-degree nodes in the network exhibited a greater number of component-target-pathway interactions, which were likely to play a critical role in renal protective AKI. Following that, the Astragalus Target-SA-AKI Target-PPI Network input the compound targets into TCMSP and String databases. There were 275 nodes and 741 edges in the Astragalus Target-SA-AKI Target-PPI Network (Figure 4C). Similarly, the AS-IV Target-SA-AKI Target-PPI Network Input the compound targets into TCMSP and String databases. There were 167 nodes and 2413 edges in the AS-IV Target-SA-AKI Target-PPI Network (Figure 4D). We imported the 254 target genes of Astragalus anti-SA-AKI and the 167 target gene sets of AS-IV anti-SA-AKI into the DAVID version 6.7 database for Gene ontology analysis and KEGG pathway enrichment analysis, using three panels typically included in GO analysis. For Astragalus, a total of 241 MF entries, 3045 BF entries, 123 CC entries, and 169 KEGG entries were obtained. Following that, we screened at P < 0.01 and FDR <0.01 to extract significant card values and discovered 2028 entries under BP, 63 entries under CC, and 132 entries under MF. Figure 4E and Table 4 show the top 10 entries selected for visualization. As shown in Figure 4F and Table 5, 140 KEGG entries were selected for visualization. For AS-IV, 168 entries were obtained under MF, 2661 entries under BP, 64 entries under CC, and 147 entries under KEGG. We used P < 0.01 and FDR <0.01 as significant card values and obtained 1667 entries under BP, 43 entries under CC, and 89 entries under MF. The top 10 entries were selected for visualization, as shown in Figure 4G and Table 6. 114 entries under KEGG were selected for visualization, as shown in Figure 4H and Table 7.

Astragalus and AS-IV enhance the PI3K/AKT pathway in both in vivo and in vitro

Gene enrichment analysis revealed that the PI3K/AKT pathway was enriched for the most corresponding target genes, indicating that PI3K/AKT is an important intracellular pathway, and a previous study confirmed that PI3K/AKT plays an important role in SA-AKI [15, 16]. Western Blot analysis revealed that PI3K and AKT phosphorylation was inhibited in the apical kidney tissues of the mice model when compared to the sham-operated group and that PI3K and AKT phosphorylation was significantly increased in the astragalus and AS-IV administration group when compared to the CLP group in a dose-dependent manner (Figure 5A, 5B and Supplementary Figure 3A, 3B). This confirms the hypothesis in the preceding section on network pharmacology that Astragalus and AS-IV are involved in the regulation of the PI3K/AKT pathway. Additionally, immunohistochemistry was used to determine the phosphorylation levels of AKT in the kidney tissues. The results indicated that AKT phosphorylation was inhibited in the apoptotic kidney tissues of the mice model when compared to the sham-operated group, but was significantly elevated in the Astragalus and AS-IV group compared to the CLP group (Figure 5C and 5D). Furthermore, Western Blot analysis revealed that LPS suppressed PI3K and AKT phosphorylation relative to the normal group, but AS-IV significantly elevated PI3K and AKT phosphorylation compared to the LPS group in a dose-dependent manner (Figure 5E and Supplementary Figure 3C). To further establish the involvement of PI3K/AKT, we used the PI3K inhibitor IC-87114 (5 μM) in HK-2 cells followed by incubation with LPS and AS-IV (20 μM) and found that phosphorylation was inhibited (Figure 5F and Supplementary Figure 3D). Additionally, the MTT assay confirmed that inhibiting the PI3K/AKT pathway decreased the cell survival rate induced by AS-IV (Figure 5G), corroborating our network pharmacology prediction that AS-IV was involved in regulating the PI3K/AKT pathway.