Abstract Background: Acute kidney injury (AKI) induced by renal ischemia-reperfusion injury (RIRI) is associated with a high incidence of mortality. Existing therapies are mainly supportive, with no available nephroprotective agent. The purpose of this study is to examine the potential protective effect of Agathis robusta Bark Extract (ARBE) in RIRI. Methods: The chemical composition of ARBE was examined by LC-ESI-MS/MS. Network pharmacology was utilized to identify the RIRI molecular targets that could be aimed at by the identified major components of ARBE. Experimentally validated protein–protein interactions (PPIs) and compound-target networks were constructed using the STRING database and Cytoscape software. Molecular docking studies were employed to assess the interaction of the most relevant ARBE compounds with the hub RIRI-related targets. Furthermore, ARBE was tested in a rat model of RIRI. Results: The phytochemical analysis identified 95 components in ARBE, 37 of which were majors. Network analysis identified 312 molecular targets of RIRI that were associated with ARBE major compounds. Of these 312, the top targets in the experimentally validated PPI network were HSP90, EGFR, and P53. The most relevant compounds based on their peak area and network degree value included narcissoside, isorhamnetin-3-O-glucoside, and syringetin-3-O-glucoside, among others. Docking studies of the most relevant compounds revealed significant interactions with the top RIRI-related targets. In the in vivo RIRI experiments, pretreatment of ARBE improved kidney function and structural changes. ARBE reduced the renal expression of p-NfkB and cleaved caspase-3 by downregulating HSP90 and P53 in rats exposed to RIRI. Conclusion: Taken together, this study revealed the chemical composition of ARBE, depicted the interrelationship of the bioactive ingredients of ARBE with the RIRI-related molecular targets, and validated a nephroprotective effect of ARBE in RIRI. Keywords: Agathis robusta , renal ischemia-reperfusion, acute kidney injury, network pharmacology 1. Introduction Acute kidney injury (AKI) characterized by abrupt decline in renal function, is associated with several short and long-term complications and a high risk for mortality [[32]1]. Renal ischemia-reperfusion injury (RIRI) is a common cause of AKI that occurs in clinical settings, e.g., kidney transplantation, heart surgery, and shock [[33]2]. Management of this condition is mainly supportive, and survivors have markedly reduced health-related quality of life (HRQL) and continue to develop long-term adverse outcomes, including chronic kidney disease (CKD) [[34]3]. Therefore, continuous search for novel nephroprotective agents, including natural products, in the setting of RIRI-induced AKI is essential. Conifers constitute the main and most distinct set of living gymnosperms and include more than 600 species and 60–65 genera distributed in seven families, the Pinaceae, Cupressaceae, Podocarpaceae, Taxaceae, Cephalotaxaceae, Taxodiaceae, and Araucariaceae [[35]4]. The latter is categorized under the order Pinales (Coniferales). Taxonomically, the family is classified into four genera, Araucaria Juss., Agathis Salisb., Columbea Salisb., and Wollemia W.G. Jones, K.D. Hill, and J.M. Allen [[36]5]. Agathis is an interesting genus comprising about 18 species, widely distributed in the Philippines, New Zealand, Australia, New Guinea, Melanesia, and Malaysia. Various phytochemical classes have been reported in Agathis species, including essential oils, fatty acids, flavonoids (in particular, the bioflavonoids), tannins, phenolics, and diterpenes. Moreover, the extracts and essential oils of Agathis species revealed several bioactivities, such as antimicrobial, anti-inflammatory, and cytotoxic properties [[37]5]. One of the most ancient Agathis trees, Agathis robusta (C. Moore ex F. Muell.) F. M. Bailey, is primarily famed for the common names of Kauri pine, smooth-barked Kauri or Queensland Kauri, and it is native to New Guinea, the Bismarck Archipelago, and Queensland. It is a long-lived monoecious species characterized by a straight trunk with smooth brownish bark, which grows up to 50 m in height and 3 m in diameter [[38]6,[39]7]. Few phytochemical studies have been conducted on A. robusta. These studies have mainly focused on the investigation of the essential oil composition and diterpenoid content [[40]7,[41]8,[42]9,[43]10,[44]11,[45]12]. Besides, only one phytochemical analysis was conducted for the ethanolic leaf extract of A. robusta, resulting in the isolation and identification of six compounds; agathisflavone, 7″-O-methyl-agathisflavone, cupressuflavone, rutin, shikimic acid, and (2S)-1,2-Di-O-[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl]-3-O-β-d-galactop yranosylglycerol [[46]13]. Moreover, few studies explored the biological activities of this species, revealing that the leaf ethanolic extract possesses a considerable anti-inflammatory activity, while the essential oil has an antibacterial effect [[47]7,[48]14]. Recently, the bark essential oil of A. robusta has been shown to have antiviral activity against SARS-CoV2 [[49]12]. However, no previous works about the total phytochemical composition of A. robusta bark extract nor its possible biological activities have been performed. Therefore, the aim of the present work was to profile the chemical composition of A. robusta ethanolic bark extract (ARBE) and to examine its potential protective effect in RIRI using in silico and in vivo validation studies. 2. Results This study included a rational experimental design ([50]Figure 1), involving the identification of the chemical composition profile of ARBE, using Liquid Chromatography combined with Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS). The identified compounds were subjected to in silico strategies, including network pharmacology as a modern method to investigate the interactions of active components with a disease’s molecular targets, followed by molecular docking analysis. The in vivo validation comprised a rat model of RIRI followed by functional and immunohistochemical analyses. Figure 1. [51]Figure 1 [52]Open in a new tab Flowchart of the workflow showing the experimental design of this study, including phytochemical, network pharmacological, and experimental studies for the investigation of the effect of ARBE in RIRI. 2.1. LC-ESI-MS/MS Profile In the present work, the phytochemical profiling of A. robusta bark by LC-ESI-MS/MS (negative and positive mode ESI) revealed the presence of 95 secondary metabolites, including mainly diterpenoid acids, biflavonoids, procyanidins, phenolic acids, and their derivatives, in addition to other classes, such as carboxylic acids, nucleobases, amino acids, stilbene and their derivatives, coumarins, flavonoid aglycones, flavonoid glycosides, iridoids, and norlignan. The major compounds (37 compounds) were determined on the basis of their peak area values ([53]Table S1). [54]Table 1 represents the data of LC-ESI-MS/MS analysis of A. robusta bark metabolites and [55]Figure 2 displays the total ion chromatogram (TIC) of the extract in positive and negative mode ESI. The mass spectra of the identified compounds are depicted in [56]Figure S1. Table 1. Metabolites identified in ARBE using LC-ESI-MS/MS in negative and positive ionization modes. No. Rt. [M+H]^+ [M−H]^− MS^2 Fragments (m/z) Tentative Identification Class Ref. 1. 1.065 191.056 173, 85 Quinic acid Cyclohexanecarboxylic acid [[57]15] 2. 1.117 169.014 125 Trihydroxy benzoic acid (gallic acid) * Phenolic acid [[58]16] 3. 1.143 117.019 73, 99 Succinic acid Omega-dicarboxylic acid [[59]17] 4. 1.144 153.019 109 Dihydroxybenzoic acid (protocatechuic acid) Phenolic acid [[60]16] 5. 1.145 181.013 137, 113, 109 Dihydrocaffeic acid [3-(3,4-Dihydroxyphenyl)propionic acid] Phenolic acid [[61]16] 6. 1.145 197.0106 179, 153,109 β,3,4-Trihydroxy benzenepropanoic acid * Phenolic acid [[62]18] 7. 1.147 104.107 60, 58 N-methyl alanine Amino acid [[63]19] 8. 1.169 109.028 81 1,2-Benzenediol Phenol dvs. [[64]20] 9. 1.173 104.105 71, 60, 58 Choline Cholines [[65]21] 10. 1.182 137.025 93 Salicylic acid* Phenolic acid [[66]16] 11. 1.185 261.045 97 Sorbitol 6-phosphate Sugar [[67]22] 12. 1.186 110.060 93, 80, 69 4- Aminophenol Phenol derivative [[68]23] 13. 1.211 136.061 119, 109, 92, 65 Adenine Nucleobase (a purine derivative) [[69]24] 14. 1.237 315.071 153 Protocatechuic acid 4-glucoside Phenolic acid hexoside [[70]16] 15. 1.246 173.081 129, 36 Shikimic acid Cyclohexanecarboxylic acid [[71]25] 16. 1.349 167.035 152 Methoxy hydroxy benzoic acid (Vanillic acid) Phenolic acid [[72]16] 17. 1.351 229.151 142, 114, 96, 70 N,N,N-trimethyl-l-alanyl-l-proline betaine Stilbene [[73]26] 18. 1.380 179 161, 135 Caffeic acid * Phenolic acid [[74]27] 19. 1.401 193.073 147 Ferulic acid Phenolic acid [[75]16] 20 1.501 225.044 181, 151 Dihydrosinapic acid * Phenolic acid [[76]28] 21. 1.658 161.047 117, 73 6-Hydroxycoumarin Coumarin [[77]29] 22. 2.212 118.086 59, 58 Glycine- betaine Alpha amino acid [[78]30] 23. 2.232 609.127 483, 441, 423, 305, 303, 177 [(epi)gallocatechin-(epi)gallocatechin] * Procyanidin [[79]31] 24. 3.618 593.208 425, 407,289 [(epi)catechin-(epi)gallocatechin] Procyanidin [[80]31] 25. 3.643 593.205 425, 407,289, 177 [(epi)catechin-(epi)gallocatechin] Procyanidin [[81]31] 26. 3.684 593.208 425, 407 [(epi)catechin-(epi)gallocatechin] Procyanidin [[82]31] 27. 3.815 609.240 483, 441, 423, 305, 303, 177 [(epi)gallocatechin-(epi)gallocatechin] * procyanidin [[83]31] 28. 4.036 305.114 261, 221, 219, 179, 165 (epi)gallocatechin Procyanidin [[84]32] 29. 4.495 609.131 483, 441, 423, 305, 303, 177 [(epi)gallocatechin-(epi)gallocatechin]* Procyanidin [[85]31] 30. 4.515 122.079 Cysteine Amino acid [[86]33] 31. 5.150 195.065 151, 150 Hydroxycaffeic acid* Phenolic acid [[87]34] 32. 5.608 477.177 315, 300, 269 Isorhamnetin-O-glucoside Flavone glucoside [[88]35] 33. 5.629 137.131 119, 110, 64 Hypoxanthine Nucleobase (a purine derivative) [[89]36] 34. 5.854 405.191 243 3,4,3′,5′-Tetrahydroxystilbene 3′-glucoside Stilbene glucoside [[90]37] 35. 5.903 177.019 149, 133, 109 6,7-dihydroxycoumarin (esculetin) Coumarin [[91]38] 36. 6.424 611.166 449,465, 303 Quercetin-rhamnose-hexose Flavonol glycoside [[92]39] 37. 6.625 267.088 252, 131 Formononetin Isoflavone [[93]40] 38. 6.697 197.0091 179, 153, 135 Danshensu (α,3,4-trihydroxy benzenepropanoic acid) Phenolic acid [[94]18] 39. 6.816 593.156 285, 284 Kaempferol-7-neohesperidoside Flavonol glycoside [[95]41] 40. 6.847 465.235 303 Quercetin hexoside Flavonol glycoside [[96]42] 41. 6.852 449.113 287 Okanin-4′-O-glucoside [2′,3′,4′,3,4-pentahydroxychalcone glucoside]* Chalcone glucoside [[97]42] 42. 6.960 623.166 315, 314 Isorhamnetin-3-O-rutinoside (narcissoside)* Flavonol glycoside [[98]41] 43. 7.011 493.200 [M-H], 539.267[M+HCOO] 493,331,313, 145 6-O-p-coumaroyl ajugol Coumaroyl iridoid glycoside [[99]43] 44. 7.098 285.115 225 Agatharesinol Norlignan [[100]44] 45. 7.293 477.106 315, 314 Isorhamnetin-3-O-glucoside* Flavonol glycoside [[101]41] 46. 7.353 289.145 245 (epi)catechin* Procyanidin [[102]32] 47. 7.412 449.108 287 Kaempferol hexoside Flavonol glycoside [[103]45] 48. 7.451 301.107 153, 229, 257 3, 5, 7-trihydroxy-4′-methoxyflavone (Diosmetin) Flavonols [[104]27] 49. 7.584 433.127 271, 85 Apigenin-O-hexoside Flavonone -O-glycosides [[105]46] 50. 7.913 435.130 273, 167, 123 Phlorizin* Dihydrochalcone glucoside [[106]47] 51. 7.942 509.099 347, 332 Syringetin-3-O-glucoside Flavonol glycoside [[107]48] 52. 8.024 (331.191) M+ 313, 287, 255 7-Hydroxy-8-methoxydedihydrorutaecarpine (7-Hydroxy-8-methoxy,7,8-Dehydrorutaecarpine) Alkaloid [[108]49] 53. 8.092 317.029 179, 151, 137 Myricetin* Flavone [[109]50] 54. 8.192 163.076 131, 103 Umbelliferone Coumarin [[110]51] 55. 8.274 177.091 162, 149 Hymecromone (4-methylumbelliferone) Coumarin [[111]52] 56. 8.296 431.099 285,284, 255 luteolin 7-O-rhamnoside Flavone glycoside [[112]53] 57. 8.375 319.045 301, 273, 217 Steviol Ent-kaurane diterpenoid [[113]54] 58. 8.397 347.187 303, 301, 285 16-Hydroxy-8(17),13-labdadien-15,16-olid-19-oic acid Diterpenoid acid [[114]42] 59. 9.094 377.165 359 Angelol A Coumarins [[115]51] 60. 9.668 541.239[M+HCOO], 495[M-H] 349,333, 163 6-O-p-coumaroyl dihydroajugol Coumaroyl iridoid glycoside [[116]43] 61. 9.704 303.123 259, 285 Copalic acid* Diterpenoid acid [[117]55] 62. 9.771 303.050 257, 201 Abietic acid Diterpenoid acid [[118]56] 63. 10.399 301.216 283, 255 Retinoic acid Retinoids [[119]57] 64. 10.934 301.217 255, 173, 147,133, 109 Dehydroabietic acid Diterpenoid acid [[120]56] 65. 11.015 305.174 287, 259, 159 Taxifolin (dihydroquercetin) Flavanonols [[121]58] 66. 11.106 287.055 269, 153 Luteolin Flavones [[122]27] 67. 11.401 347.223 303, 301 15-Hydroxypinusolidic acid Diterpenoid acid [[123]42] 68. 11.814 377.143 359 Isoangelol Coumarins [[124]51] 69. 12.278 897.071 693, 289 (epi)gallocatechin-(epi)gallocatechin-(epi)catechin Procyanidin [[125]31] 70. 12.419 577.117 289 (epi)catechin-(epi)catechin Procyanidin [[126]32] 71. 12.57 305.212 261, 287 3-hydroxy-(13S)-l6-nor-pimar-7-en-l5-oic acid * Diterpenoid acid [[127]44] 72. 12.658 331.189 203 15-hydroxy-7-oxo-dehydroabietic acid Diterpenoid acid [[128]56] 73. 12.711 329.177 285,311 Carnosol Phenolic diterpene [[129]55] 74. 12.788 301.0349 255, 179, 151 Quercetin* Flavonol [[130]32] 75. 13.014 565.113 389 Isoginkgetin Biflavonoids [[131]59] 76. 13.058 315.161 199, 187 7-oxo-dehydroabietic acid Diterpenoid acid [[132]56] 77. 13.36 283.061 268 Chrysin-6-methyl-ether Flavonone [[133]60] 78. 13.449 283.061 268,239 Physcion dihydroxyanthraquinone [[134]61] 79. 14.413 331.229 287 Pinusolidic acid * Diterpenoid acid [[135]42] 80. 14.567 271.228 253,227, 225 Hydroxypalmitic Acid Fatty acid [[136]55] 81. 14.76 299.166 284, 255 4′-Hydroxywogonin Trihydroxy-methoxyflavone [[137]29] 82. 15.382 539.168 539, 387 2″,3″-Dihydro-3′,3‴-biapigenin Biflavonoids [[138]59] 83. 15.602 536.883 537, 385 2′,8″-Biapigenin Biflavonoids [[139]59] 84. 15.618 565.114 445, 388, 403, 456, 471 Robustaflavone 7,4′-dimethyl ether Biflavonoids [[140]59] 85. 15.911 537.305 537, 193, 192 Delicaflavone Biflavonoids [[141]59] 86. 15.929 303.229 285, 257, 239 Abietic acid isomer Diterpenoid acid [[142]56] 87. 16.014 301.216 283, 255, 133, 109 Dehydroabietic acid isomer Diterpenoid acid [[143]56] 88. 17.120 313.179 211, 197, 185 Dehydrated 15-hydroxy-7-oxo-dehydroabietic acid Diterpenoid acid [[144]56] 89. 17.17 363.212 [M+HCOO] 317, 159 Agarotetrol formate adduct 2-(2-phenylethyl) chromones [[145]62] 90 17.679 315.195 199, 187 7-oxo-dehydroabietic acid isomer Diterpenoid acid [[146]56] 91. 19.553 303.199 285, 257, 109 Abietic acid isomer Diterpenoid acid [[147]56] 92. 20.795 301.217 255, 173, 147,133, 109 Dehydroabietic acid isomer Diterpenoid acid [[148]56] 93. 20.857 301.216 255, 173,133, 109 Dehydroabietic acid isomer Diterpenoid acid [[149]56] 94. 20.883 301.217 255, 173, 147,133, 109 Dehydroabietic acid isomer Diterpenoid acid [[150]56] 95. 22.529 315.196 297,199, 187 7-oxo-dehydroabietic acid isomer Diterpenoid acid [[151]56] [152]Open in a new tab * Identified also in positive mode. Bold compounds refer to the major ones. Figure 2. [153]Figure 2 [154]Open in a new tab LC-MS total ion chromatograms of ARBE. (a) The TIC in negative ion mode. (b) The TIC in positive ion mode. (c) The structure of some LC-MS identified major components of ARBE. Numbers in red (in (a–c)) are related to [155]Table 1. 2.2. The Network Pahrmacology Analysis 2.2.1. Pharmacokinetics of ARBE Major Compounds To identify potential bioactive components, the 37 major compounds of ARBE were screened based on their pharmacokinetics and drug-likeness properties ([156]Table S2). Indeed, most of these compounds showed higher bioavailability scores (OB > 0.55) and complied with Lipinski’s rule of five, a rule of thumb to evaluate drug likeness. However, a few compounds did not meet the screening criteria perfectly, including the procyanidins, which are known to have multiple biological activities [[157]63]. Therefore, we included all the major ARBE compounds in our study for a comprehensive analysis. 2.2.2. Molecular Targets of ARBE Major Compounds To identify the molecular targets associated with the major constituents of ARBE, the Swiss Target Prediction database was utilized. This analysis resulted in 741 targets after the removal of duplicates ([158]Table S3). 2.2.3. Molecular Targets of ARBE Associated with RIRI To retrieve the molecular targets associated with RIRI, three disease-related databases, i.e., DisGeNeT, GeneCards, and OMIM, were accessed. The resulting targets were 1745 and were cut down to 1646 after removal of duplicates ([159]Table S4). Among these targets, 312 ([160]Table S5) overlapped with the 741 targets associated with ARBE major compounds ([161]Figure 3). Figure 3. [162]Figure 3 [163]Open in a new tab Overlapping molecular targets between RIRI and major compounds of ARBE. ARBE, Agathis Robusta bark extract; RIRI, renal ischemia reperfusion injury. 2.2.4. Protein–Protein Interaction (PPI) Network of the 312 Disease-Compounds Targets To reveal the potential molecular mechanisms of ARBE to protect from RIRI, a network of the experimentally validated PPIs of the 312 disease-compounds common targets was constructed in STRING database ([164]Figure 4a, PPI). This network contained 312 nodes connected by 598 edges. Then, we ranked the key essential targets in the network based on their degree value, i.e., the number of connecting edges. Indeed, a higher degree value could pinpoint a more relevant target in the network. The top three targets included HSP90AA1 (degree = 42), EGFR (degree = 39), and TP53 (degree = 33). The top 20 targets with their degree value are shortlisted in [165]Table S6 and shown in [166]Figure 4b. Figure 4. [167]Figure 4 [168]Open in a new tab Protein–protein interaction (PPI) network of ARBE molecular targets associated with RIRI. (a) PPI network. (b) Top 20 targets in the PPI network based on degree value. 2.2.5. Top ARBE Compounds Associated with RIRI Targets To determine the most significant compounds of ARBE associated with the 312 RIRI targets, we constructed a compound-target network in Cytoscape ([169]Figure 5). Then, we ranked the compounds based on degree value ([170]Table 2). Figure 5. [171]Figure 5 [172]Open in a new tab Agathis Robusta bark extract (ARBE) compounds—RIRI targets network. Table 2. Major compounds of ARBE ranked by the Degree method. Rank Target Name Score 1 Narcissoside 60 2 Isorhamnetin-3-O-glucoside 58 2 Syringetin-3-O-glucoside 58 4 6-O-p-coumaroyl ajugol 56 5 Luteolin 7-rhamnoside 55 6 Robustaflavone 7,4′-dimethyl ether 53 6 Ferulic acid 53 6 2″,3″-Dihydro-3′,3‴-biapigenin 53 9 3-hydroxy-(13S)-16-nor-pimar-7-en-15-oic acid 52 9 Caffeic acid 52 11 Agatharesinol 51 12 Delicaflavone 50 12 Dehydrated 15-hydroxy-7-oxo-dehydroabietic acid 50 12 Diosmetin 50 15 Esculetin 49 15 Hydroxycaffeic acid 49 17 Hymecromone 48 17 Myricetin 48 17 Isoginkgetin 48 17 16-Hydroxy-8(17)13-labdadien-1516-olid-19-oic acid 48 21 Quercetin 47 21 [(epi)gallocatechin-(epi)gallocatechin] 47 21 Luteolin 47 21 Umbelliferone 47 25 15-hydroxy-7-oxo-dehydroabietic acid 46 25 7-oxo-dehydroabietic acid 46 27 Phlorizin 45 27 Protocatechuic acid 4-glucoside 45 29 6-Hydroxycoumarin¬† 44 29 3,4-Trihydroxy benzenepropanoic acid 44 29 Dehydroabietic acid 44 32 [(epi)catechin-(epi)gallocatechin] 43 32 Gallic acid 43 34 Pinusolidic acid 42 35 Hydroxypalmitic Acid 41 35 15-Hydroxypinusolidic acid 41 37 Carnosol 40 [173]Open in a new tab 2.2.6. Gene-Ontology (GO) and KEGG Pathway Enrichment Analysis of 312 Common Targets To verify the relevant biological and functional characteristics of the 312 disease-compound common targets, GO enrichment analysis was performed in biological processes (BP), molecular functions (MF), and cellular components (CC) based the number of targets that were enriched in those categories ([174]Figure 6a–c). Top BP included signal transduction, inflammatory response and apoptotic process; Top MF included protein binding, enzyme binding, and protein kinase activity; Top CC included plasma membrane, cytoplasm, and nucleus. Detailed information of GO analyses is shown in [175]Supplementary Tables S7–S9. Figure 6. [176]Figure 6 [177]Open in a new tab Gene Ontology (GO) analyses. (a) Biological process. (b) Molecular function. (c) Cellular component. To recognize the possible pathways involved in the protective effects of ARBE in RIRI, KEGG pathway enrichment analysis of the 312 disease-compounds common targets was performed (p < 0.05). Top enriched pathways included lipid and atherosclerosis, HIF-1 signaling pathway, and PI3K-Akt signaling pathway ([178]Figure 7). KEGG pathway results are presented in detail in [179]Table S10. Figure 7. [180]Figure 7 [181]Open in a new tab Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. 2.3. Molecular Docking Study To examine the interaction of ARBE compounds with key RIRI molecular targets, we performed a molecular docking analysis. Ten compounds ([182]Table 3), selected according to their peak area values and score based on degree value ([183]Table 2), and the three top targets of RIRI (HSP90, EGFR, and p53) were included in the analysis. Table 3. Docking details of the selected components of ARBE on HSP90A. Component S Score Kcal/mol H-Bond Interactions Pi-H Interactions 7-oxo-dehydroabietic acid −6.5439 THR184 ASN51 Caffeic acid −5.1359 SER52 - Narcissoside (Isorhamnetin-3-O-rutinoside) −8.4051 ASP102 ASN106 Isorhamnetin-3-O-glucoside −7.6758 ASP93 - Syringetin-3-O-glucoside −8.0354 ASP93 ASN106 15-hydroxy-7-oxo-dehydroabietic acid −5.5907 MET98, PHE 138 - 6-O-p-coumaroyl ajugol −7.5511 GLU47, ASN 51 - Luteolin 7-rhamnoside −7.1269 GLY97 ASN106 Robustaflavone 7,4’-dimethyl ether −7.0831 MET98 - Ferulic acid −5.0937 ASN51 - T5M −6.4559 THR184 ASN51 [184]Open in a new tab Heat shock protein HSP 90-Alpha in complex with T5M (PDB code: 2XHX) [[185]64], epidermal growth factor receptor (EGFR) tyrosine kinase in complex with erlotinib (PDB code: 1M17) [[186]65], and cellular tumor antigen P53 (PDB code: 3Q01) [[187]66] were implemented to provide insight on binding affinity of ARBE with the active pockets of the targeted proteins. 2.3.1. Docking with Heat shock Protein HSP 90-Alpha The adenine-binding site of HSP90-A (PDB code: 2XHX) is a hydrophobic pocket. Re-docking the co-crystallized ligand, 2-tert-butyl-4-(1,3-dihydro-2H-isoindol-2-ylcarbonyl)phenol (T5M) revealed the reliable active site as the RMSD is 1.2702 Å and the energy score (S) is −6.4559 kcal/mol. Docking simulations of the selected components of ARBE inside the HSP90-A active pocket exhibited that 7-oxo-dehydroabietic acid, caffeic acid, narcissoside (isorhamnetin-3-O-rutinoside), isorhamnetin-3-O-glucoside, syringetin-3-O-glucoside, 15-hydroxy-7-oxo-dehydroabietic acid, 6-O-p-coumaroyl ajugol, luteolin 7-rhamnoside, robustaflavone 7,4’-dimethyl ether, and ferulic acid oriented within the active pocket with reasonable docking energy score, ranging from −8.4051 to −5.0937 kcal/mol ([188]Table 3). The docked components showed not only H-bond interactions but also arene-H bond interactions with the crucial amino acid residues described in detail in [189]Table 3. Narcissoside (isorhamnetin-3-O-rutinoside), isorhamnetin-3-O-glucoside, and syringetin-3-O-glucoside have lower S scores than the co-crystallized ligand (T5M), indicating better binding affinity within the pocket ([190]Figure 8). Figure 8. [191]Figure 8 [192]Figure 8 [193]Open in a new tab 3D and 2D docking plots of narcissoside (isorhamnetin-3-O-rutinoside), isorhamnetin-3-O-glucoside, and syringetin-3-O-glucoside, respectively, on HSP90A (PDB code: 2XHX). 2.3.2. Docking with Epidermal Growth Factor Receptor (EGFR) Docking studies reveal that 7-oxo-dehydroabietic acid, caffeic acid, narcissoside (Isorhamnetin-3-O-rutinoside), isorhamnetin-3-O-glucoside, syringetin-3-O-glucoside, 15-hydroxy-7-oxo-dehydroabietic acid, 6-O-p-coumaroyl ajugol, luteolin 7-rhamnoside, robustaflavone 7,4’-dimethyl ether, and ferulic acid on epidermal growth factor receptor (EGFR) tyrosine kinase (PDB code: 1M17) reached the binding site of the enzyme. In comparison to the co-crystallized ligand (AQ4: erlotinib), the docked components showed a good binding affinity, where the docking energy score ranged from −9.0112 to −5.1249 kcal/mol. The components confirmation within the active pocket stabilized by the H-bond interaction with the crucial amino acid residues. [194]Table 4 shows the detailed amino acid residues involved in the interactions with the docked components. One of hydroxyl group of narcissoside (isorhamnetin-3-O-rutinoside), isorhamnetin-3-O-glucoside, and syringetin-3-O-glucoside acts as an anchor, forming an H-bond interaction with acidic ASP831. This is allowed the rest of the component structure to fill the active site properly, forming more H-bond interactions with MET742, GLN767, and MET769 in addition to a pi-H bind interaction with LEU694 ([195]Figure 9). Table 4. Docking details of the selected components of ARBE on EGFR. Component S Score Kcal/mol H-Bond Interactions Pi-H Interactions 7-oxo-dehydroabietic acid −5.7797 MET769, THR830 GLY772 Caffeic acid −5.2224 MET742, GLN767, ASP831 - Narcissoside (Isorhamnetin-3-O-rutinoside) −9.0112 MET742, MET769, ASP 831 LEU694 Isorhamnetin-3-O-glucoside −8.42596 MET742, ASP 831 - Syringetin-3-O-glucoside −8.5239 MET742, ARG817, ASP 831 LEU694 15-hydroxy-7-oxo-dehydroabietic acid −5.9105 LYS721 VAL702 6-O-p-coumaroyl ajugol −7.4587 MET742, GLU780 - Luteolin 7-rhamnoside −7.2894 THR766, ASP 831 LEU694, GLY772 Robustaflavone 7,4′-dimethyl ether −8.4544 LYS721 LEU694, VAL 702 Ferulic acid −5.1249 MET769 - erlotinib −7.76417 VAL702, MET769, - [196]Open in a new tab Figure 9. [197]Figure 9 [198]Open in a new tab 3D and 2D docking plots of narcissoside (isorhamnetin-3-O-rutinoside), isorhamnetin-3-O-glucoside, and syringetin-3-O-glucoside, respectively, on EGFR (PDB code: 1M17). 2.3.3. Docking with Cellular Tumor Antigen P53 The P53 active pocket is lined with the following amino acid residues: PRO98, SER99, ARG158, ALA159, MET160, LEU206, ASP208, ARG213, SER215, ILE254, ILE255, THR256, GLU258, GLY262, LEU264, and ARG267. 7-Oxo-dehydroabietic acid, caffeic acid, narcissoside (isorhamnetin-3-O-rutinoside), isorhamnetin-3-O-glucoside, syringetin-3-O-glucoside, 15-hydroxy-7-oxo-dehydroabietic acid, 6-O-p-coumaroyl ajugol, luteolin 7-rhamnoside, robustaflavone 7,4′-dimethyl ether, and ferulic acid were docked into P53 (PDB code: 3Q01) with a good binding affinity to the active site, with the docking energy score ranging from −7.1544 to −4.5554 kcal/mol. The components confirmation within the active pocket stabilized by H-bond interaction with the amino acid residues. [199]Table 5 shows the detailed amino acid residues involved in the H-bond and pi-H bond interactions with the docked components. Narcissoside (isorhamnetin-3-O-rutinoside), isorhamnetin-3-O-glucoside, and syringetin-3-O-glucoside have good binding interactions with the amino acid residues of the active pocket, represented on [200]Figure 10. Table 5. Docking details of the selected components of ARBE on P53. Component S Score Kcal/mol H-Bond Interactions Pi–H Interactions 7-oxo-dehydroabietic acid −4.9993 GLY262 - Caffeic acid −4.5554 SER99 THR256 Narcissoside (Isorhamnetin-3-O-rutinoside) −7.1544 SER96 PRO 98 Isorhamnetin-3-O-glucoside −7.1453 ASP208 THR256, LEU 264 Syringetin-3-O-glucoside −6.7796 SER96, ASP 208, GLY262 PRO98 15-hydroxy-7-oxo-dehydroabietic acid −5.0645 ASP208 - 6-O-p-coumaroyl ajugol −6.7463 GLU 204, ILE 254 - Luteolin 7-rhamnoside −5.9100 SER260, ARG 267 - Robustaflavone 7,4′-dimethyl ether −6.6223 SER99, ARG 158, ASP 208 THR256 Ferulic acid −4.5974 SER99, GLY 262 THR256 [201]Open in a new tab Figure 10. [202]Figure 10 [203]Figure 10 [204]Open in a new tab 3D and 2D docking plots of narcissoside (Isorhamnetin-3-O-rutinoside), isorhamnetin-3-O-glucoside, and syringetin-3-O-glucoside, respectively, on P53 (PDB code: 3Q01). 2.4. In Vivo Validation To validate our in silico findings, we next tested whether ARBE is nephroprotective in an RIRI model in rats. RIRI in rats resulted in a decline in kidney function as indicated by increased serum creatinine and blood urea nitrogen, while pretreatment with ARBE improved kidney function ([205]Figure 11a,b). Histopathological studies ([206]Figure 11c) of the kidneys showed structural and pathological changes after RIRI, which were mitigated in the ARBE-pretreated animals. In [207]Figure 11c, HE staining, sham kidneys demonstrated normal organized histological features of renal parenchyma with abundant records of apparent intact renal corpuscles (star), renal tubular segments with almost intact tubular epithelium (arrow), as well as intact vasculatures without abnormal morphological changes records; RIRI kidneys showed sever diffuse tubular epithelial loss and necrotic changes of different nephron segments (red arrow) alternated with abundant figures of degenerated pyknotic tubular epithelium with marked tubular dilatation. Significant congested glomerular tuft capillaries with significant dilatation of Bowman’s spaces (star) with focal interstitial extravasation of blood. Marked records of intraluminal eosinophilic casts (yellow arrow) with moderate interstitial mononuclear inflammatory cells infiltrates (arrow head); kidneys of ARBE-pretreated rats showed significant protective efficacy on renal tubular epithelium with persistent moderate records of tubular epithelial degenerative changes (red arrow), alternated with relative higher records of apparent intact tubular segments (black arrow). Mild persistent records of tubular dilatations were shown as well as persistent dilatation of bowman’s spaces (star). Moreover, there were minimal records of interstitial inflammatory cells infiltrate (arrow head) and intraluminal casts with intact vasculatures. In the immunohistochemical analysis, in comparison to the vehicle, ARBE pre-treatment in rats with RIRI significantly dampened renal inflammation and apoptosis as indicated by reducing p-NfKB and cleaved caspase-3 expression, respectively ([208]Figure 11c,d). Figure 11. [209]Figure 11 [210]Open in a new tab In vivo validation of the protective effect of ARBE against RIRI. (a) Serum creatinine. (b) Blood urea nitrogen (BUN). (c) Microscopic examination and immunohistochemical studies (Scale bar, 50 μm). (d) Quantification of immunohistochemical (IHC) expression. * p < 0.05 vs. sham, # p < 0.05 vs. RIRI. Next, we examined whether these nephroprotective activities of ARBE in RIRI are due to modulating the top RIRI molecular targets which were identified by network analysis and confirmed by docking. Therefore, we assessed the renal expression of two of these targets, i.e., HSP90 and P53, which have been shown to contribute to kidney dysfunction, inflammation, and cell death in previous studies [[211]67,[212]68]. Indeed, RIRI led to increased expression of both proteins in rat kidney, yet, pretreatment with ARBE attenuated this effect ([213]Figure 11c,d). 3. Discussion Based on phytochemical characterization followed by network pharmacology, docking, and preclinical validation, we here report a potential protective effect of ARBE against RIRI. This study revealed the most biologically significant components of ARBE and their possible molecular targets and mechanisms of actions in attenuation of RIRI-induced AKI. In the phytochemical analysis, LC-ESI-MS/MS chromatographic technique was employed to characterize the phytometabolites of ARBE. In total, 95 compounds were tentatively identified according to their MS and MS^2 fragmentation data and retention times. The major identified chemical classes include diterpenoid acids, such as 7-oxo-dehydroabietic acid, abietic acid, and pinusolidic acid; biflavonoids, such as robustaflavone 7,4′-dimethyl ether and delicaflavone; procyanidins, such as as [(epi)gallocatechin-(epi)gallocatechin] and [(epi)catechin-(epi)gallocatechin]; and phenolic acids, such as caffeic and gallic acids. The presence of such diverse chemical components in herbs makes it complex to assess the potential therapeutic action by the single component-single target paradigm [[214]69]. Therefore, network pharmacology-based strategy could be a potential successful tool in this context. This approach utilizes a computational action plan to uncover the possible component-target-disease associations, and has been recognized as an efficient method in multiple previous studies [[215]69,[216]70,[217]71,[218]72]. Applying network pharmacology in this study led to the identification of 312 RIRI-related molecular targets that can be potentially aimed at by major components of ARBE. Of those targets, the most relevant hubs, based on experimentally validated PPIs, included molecular chaperones, i.e., HSPs, which regulate protein folding, intracellular transport, and repair or degradation [[219]73]. Proteins involved in signaling pathways, cell survival, and cell division, e.g., EGFR and P53, were also included as significant targets [[220]74,[221]75]. Docking is a useful tool to predict the chemical reactivity of compounds towards molecular targets [[222]76]. In this study, ten ARBE compounds with high peak area and degree value scores showed substantial binding with the top three RIRI targets in the experimentally validated PPI network, i.e., HSP90A, EGRF, and P53. The molecular chaperone HSP90A is a homodimeric protein [[223]77]. The HSP90A structure consists of three domains: N-terminal domain (N-domain), middle domain (M-domain), and C-terminal domain (C-domain). The N-domain, the catalytic domain, binds with ATP. M-domain associates both N- and C-domains, while the N-domain connects with its partner domain in the other subunit to form the dimer. The HSP90 chaperone cycle includes the turnover of ATP to ADP through the ATPase action in the N-terminal domain. The ATP binding site has been described by NMR and/or X-ray diffraction by the binding modes of a number of reported HSP90A inhibitors [[224]78]. It was reported that resorcinol-bearing compounds are considered as lead compounds for discovering inhibitors or modulators of HSP90 [[225]64,[226]79]. The docked components have hydroxyl/phenolic groups exhibited interactions with the amino acids present in the adenine-binding site of HSP90A, proposing that ARBE may be effective in prevention or treatment of a diseases mediated by HSP90A. The EGFR kinase domain (EGFR) has a characteristic bilobate-fold. The N-terminal domain is formed from mostly β-strands and one α-helix, whereas the larger C-terminal domain is formed from mostly α -helices. The two domains are separated by a cleft similar to those in which ATP, ATP analogues, and ATP inhibitors have been found to bind [[227]65]. Docked components ARBE showed promising interactions through fitting into EGFR adenine pocket. Cellular tumor antigen P53 (PDB code: 3Q01) was crystallized as a homodimer [[228]66]. The resolved P53 protein structure has no co-crystallized ligand, so both Computed Atlas for Surface Topography of Proteins (CASTp) [[229]80] and Site Finder module in MOE 2019.0102 were used to find potential 3D pockets for P53 protein. Molecular docking of the components within P53 active pocket revealed that they exhibited promising binding energies with potential activity on P53. Computational analysis without experimental validation could be non-sufficient and meaningless. Thus, a preclinical model of RIRI was used in this study to examine the potential activity and mechanism of ARBE. This experiment revealed a nephroprotective effect of ARBE in RIRI by diminishing renal inflammation and apoptosis. These effects were due to downregulation of both HSP90 and P53 by ARBE. Indeed, previous studies showed that both HSP90 and P53 are upregulated after RIRI resulting in inflammation, oxidative stress, cell death, and structural changes. Both genetic and pharmacological inhibition of these proteins in RIRI are associated with better outcomes [[230]67,[231]68,[232]81,[233]82]. For instance, AT13387, an HSP90 inhibitor, ameliorated RIRI by abolishing Toll-like receptor 4 (TLR4)-mediated NF-κB activation [[234]67]. Furthermore, intravenous injection of synthetic siRNA to p53 after ischemic injury protected both proximal tubule cells and kidney function in rats [[235]83]. In a clinical study (ClinicalTrials.gov identifier: [236]NCT00802347), a single systemic administration of QPI-1002, a siRNA-based p53 inhibitor, reduced the incidence of delayed graft function in deceased donor allograft recipients by downregulating p53 following reperfusion [[237]84]. 4. Materials and Methods 4.1. Plant Material and Extraction A. robusta fresh bark (50 years old cultivated tree) was collected in July 2021 from El-Orman Botanical Garden, Giza, Egypt. The plant identification was confirmed by Dr. Therese Labib Youssef, ExManager and Taxonomist at Orman Botanical Garden, Giza, Egypt. A voucher sample [Reg. No. (ZU-Ph-Cog-0200)] was deposited within the Herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Zagazig University. For extraction, the dried powdered bark (280 g) was macerated with 70% ethanol (3 × 1 L). The extract was evaporated at reduced pressure to afford 80 g of viscous residue. 4.2. LC-ESI-MS/MS Analysis The bark extract was reconditioned in (Water: Methanol: Acetonitrile, 50:25:25 v/v), and analyzed by LC-ESI-MS/MS using ExionLC (High flow LC, Sciex^®, Framingham, MA, USA), coupled with TripleTOF 5600+Time-of-Flight (IDA Acquisition, Sciex^®) and Analyst TF 1.7.1 (LC-Triple TOF control, Sciex^®). The injection concentration and volume were 2.5 µg/µL and 10 µL, respectively. The pre-column used consisted of In-Line filter disks (0.5 µm × 3.0 mm, Phenomenex^®[,] Torrance, CA, USA), while the used column was X select HSS T3 (2.5 µm, 2.1 × 150 mm, Waters^® [,] Milford, MA, USA) and the column temperature was set at 40 °C. The flow rate was 0.3 mL/min, and the elution was carried out using a buffer system of 1% methanol in 5 mM ammonium formate at pH 3 as solvent A for positive mode, at pH 8 as solvent B for negative mode and 100% of acetonitrile as solvent C. Gradient elution was carried out as follows: 90% solvent A or B and 10% of solvent C were injected for 20 min, then turned to 10% of solvent A or B to 90% of solvent C for the next 5 min, and finally, by the starting elution mixture was applied for the last 3 min. PeakView was employed for peaks extraction from the total ion chromatogram (TIC), on the basis of that the peaks should possess a signal-to-noise ratio greater than 5 (non-targeted analysis); in addition, the peak intensities of the sample-to-blank should be greater than 3. The interpretation of data was achieved using a ReifycsAbf (Analysis Base File) Converter for Wiff file conversion (Reifycs^®, Tokyo, Japan) and MS-DIAL 4.6 (RIKEN^® Tokyo, Japan). The compounds were tentatively identified according to their retention time, MS, and MS^2 fragmentation using PeakView^TM software version 2.1, and the peak area values were calculated using the XIC Manager in this software. For each identified compound, extracted ion chromatograms (XICs) were automatically produced and compared to a user-defined threshold [[238]85]. 4.3. Network Pharmacology 4.3.1. Acquisition of Pharmacokinetics and Associated Targets of ARBE Major Compounds SMILES of the major compounds of ARBE were obtained from PubChem ([239]https://pubchem.ncbi.nlm.nih.gov/, accessed on 25 June 2022) database or using ChemDraw v20.0.0.41 (PerkinElmer Informatics, Inc., UK). The pharmacokinetics of these compounds were acquired by using the SwissADME web tool ([240]http://www.swissadme.ch/, accessed on 25 June 2022) [[241]86]. The molecular targets linked to the identified major compounds of AR were predicted using the SwissTargetPrediction ([242]http://www.swisstargetprediction.ch/, accessed on 1 July 2022) database [[243]87]. 4.3.2. Identification of RIRI-Associated Targets The molecular targets associated with RIRI were obtained from DisGeNeT ([244]https://www.disgenet.org/search, accessed on 24 June 2022) [[245]88], GeneCards ([246]https://www.genecards.org/, accessed on 24 June 2022) [[247]89,[248]90], and Online Mendelian Inheritance in Man (OMIM, [249]https://www.omim.org/, accessed on 24 June 2022) databases [[250]91]. 4.3.3. Construction of PPI and Compound-Target Networks The common overlapping targets between ARBE major compounds and RIRI targets were identified in Microsoft Excel and represented as a Venn diagram. Protein–protein interactions (PPI) network of the intersected targets between the AR compounds and RIRI-related targets was constructed using the STRING database Version 11.5 ([251]https://string-db.org/, accessed on 2 July 2022) [[252]92]. A confidence level of >0.4 in protein interactions was applied, and only experimentally validated PPI were included. A compound-target network was also constructed between the major compounds of AR and the intersected targets. The Cytoscape 3.9.1 software program (NIGMS, USA) [[253]93] was utilized to visualize the networks. The key essential targets and top compounds were ranked based on degree by applying the CytoHubba plugin contained in Cytoscape [[254]94]. 4.3.4. The GO Analysis and KEGG Pathway Enrichment A database for Annotation, Visualization and Integrated Discovery (David database, [255]https://david.ncifcrf.gov/tools.jsp, accessed on 3 July 2022) [[256]95] was employed to perform GO analysis and KEGG pathway enrichment. A p-value < 0.05 was used as a cutoff and was corrected using a false discovery rate (FDR) error control technique. 4.4. Molecular Modelling 4.4.1. Molecular Docking Study Molecular docking studies of the following components: 7-oxo-dehydroabietic acid, caffeic acid, narcissoside (Isorhamnetin-3-O-rutinoside), isorhamnetin-3-O-glucoside, Syringetin-3-O-glucoside, 15-hydroxy-7-oxo-dehydroabietic acid, 6-O-p-coumaroyl ajugol, luteolin 7-rhamnoside, robustaflavone 7,4′-dimethyl ether, and ferulic acid were performed to evaluate their binding affinity with the targeted active sites of HSP90A, EGFR, and P53 proteins. Molecular Operating Environment MOE version 2019.0102 software (Chemical Computing Group, Montreal, CA) [[257]96] was used for the docking studies. The exploited docking placement methodology is triangle matcher. Each ligand was allowed to be flexible, while the protein structure was kept rigid. The scores of the docking energy for the best-fitted poses of the components with the protein active pocket were recorded. 4.4.2. Protein Preparation The crystal structures of HSP90A (PDB code: 2XHX/2.80 Å), EGFR (PDB code: 1M17/2.60 Å), and P53 (PDB code: 3Q01/2.10 Å) were obtained from the Protein Data Bank ([258]http://www.rcsb.org) [[259]97] ([260]Table S11). The crystallized water molecules and repeated chains were deleted from the protein complexes. The structures of HSP90A, EGFR, and P53 were prepared individually, using the MOE Quick Preparation protocol using the Amber10: EHT forcefield. 4.4.3. Ligand Preparation 7-Oxo-dehydroabietic acid, caffeic acid, narcissoside (isorhamnetin-3-O-rutinoside), isorhamnetin-3-O-glucoside, syringetin-3-O-glucoside, 15-hydroxy-7-oxo-dehydroabietic acid, 6-O-p-coumaroyl ajugol, luteolin 7-rhamnoside, robustaflavone 7,4′-dimethyl ether, and ferulic acid were drawn through the Chemdraw^® (PerkinElmer Informatics, Inc., UK), then transferred to the MOE using smiles strings. The energy of the components was minimized with root mean square (RMS) gradient 0.1 kcal/mol/Â^2 and finally preparing a database file. The co-crystallized ligands for both HSP90A, EGFR were re-docked for the validation process of the active site. P53 has no crystallized ligand; therefore, the Computed Atlas for Surface Topography of Proteins (CASTp; [261]http://sts.bioe.uic.edu/castp/index.html, accessed on 18 July 2022 ) server was used for the active pocket prediction [[262]80]. The score of docking energy (S; kcal/mol) and visual inspection of both two-dimensional and three-dimensional planes of the component-targeted protein interactions were exploited for the results analysis. 4.5. In Vivo Experiments 4.5.1. Animals, Ethical Statement, and Experimental Design All experiments were approved by the Institutional Animal Care and Use Committee of Zagazig University (ZU-IACUC/3/F/111/2022). The procedures were carried out according to the National Institute of Health guidelines. A total of 24 Wistar rats (weighing 150–180 g) were used in this study. Prior to the experiment, all rats were allowed free access to standard diet and water and were subjected to a circadian rhythm with a 12 h day and 12 h night at an ambient temperature of 24~26 °C with 50~60% humidity for 1 week. The rats were randomly divided into three groups including a sham group (n = 8), a RIRI group (n = 8), and a RIRI group treated with ARBE (n = 8). 4.5.2. Induction of Renal Ischemia-Reperfusion (RIRI) Renal ischemia-reperfusion injury (RIRI) was performed as previously described [[263]98]. Briefly, ARBE (10 mL/kg, p.o., corresponding to 400 mg/kg) or vehicle was administered twice: 180 min and 15 min prior to ischemia. Dose was chosen based on a pilot study. Rats were anesthetized with thiopental sodium (Sigma Tec, Giza, Egypt) (25 mg/kg) administered i.p. before surgery. The left kidney was accessed through abdominal incision, and a non-traumatic vascular clamp was applied to the left renal artery for 60 min. Then, the clamp was removed to induce renal reperfusion. Finally, the abdominal cavity was closed. After the 24 h reperfusion period, the animals were decapitated for sample collection. The animals in the sham group underwent abdominal incision without clamping of the left renal arteries. Blood samples were collected via venipuncture and then were centrifuged, and the serum was stored at −80 °C for further analysis. Parts of the kidneys were fixed in 10% buffered formalin for histological and immunohistochemical studies. The rest of kidneys were snap frozen in liquid nitrogen and stored at −80 °C. 4.5.3. Kidney Function Assessment Serum samples were assayed for blood urea nitrogen (BUN) and serum creatinine by using a Urea Nitrogen Kit (BioDiagnostic, Giza, Egypt) and the Creatinine Kit (BioDiagnostic, Giza, Egypt), respectively, according to the manufacturer’s protocol. 4.5.4. Histopathology Kidney tissue samples were fixed in 10% neutral buffered formalin for 72 h. Samples were processed in serial grades of ethanol, cleared in Xylene, then infiltrated and embedded into Paraplast tissue embedding media. Next, 5 μm thick serial sections were cut by rotatory microtome for demonstration of renal parenchyma in different samples and mounted on glass slides. Tissue sections were stained by Hematoxylin and Eosin as a standard staining method for blinded light microscopic examination by an experienced histologist. 4.5.5. Immunohistochemistry Five µm thick paraffin embedded tissue sections were prepared. Deparaffinized retrieved tissue sections were treated by 0.3% H[2]O[2] for 20 Mins. Then, they were incubated with anti-Cleaved Caspase-3 (GB11532, service bio. (Wuhan, China)—1: 300), Anti-HSP 90α Antibody (sc-515081, Santa cruz Biotechnology, Inc., CA, USA—1:100), anti p-NFκB p65 antibody (GTX54672, GeneTex Inc. (Hsinchu, Taiwan)—1:100), and anti P53 (Abcam (Cambridge, UK)-ab131442—1:100) overnight at 4 °C. Tissue sections were washed out by PBS followed by incubation with secondary antibody HRP Envision kit (DAKO) for 20 min, and then washed out and incubated with diaminobenzidine (DAB) for 15 min. Next, they were washed by PBS and counter stained with hematoxylin, dehydrated and cleared in xylene, then cover slipped for microscopic examination. Histological analysis was performed according to a previous study [[264]99]. At least six non-overlapping fields were randomly selected and scanned from kidney tissue sections of each sample for the determination of relative area percentage of immunohistochemical expression levels of Cleaved Caspase 3, p-NFkB, HSP90, and P53 in immunohistochemically stained sections. All light microscopic examinations and data were obtained by using a Leica Application module for histological analysis, attached to a Full HD microscopic imaging system (Leica Microsystems GmbH, Wetzlar, Germany). 4.6. Statistical Analysis GraphPad Prism version 9.4.1 (CA, USA) was used for the statistical analyses, and the results are presented as the means ± standard error of the mean (SEM). Comparisons between the groups were performed using the one-way ANOVA and Tukey’s multiple comparison test; significance was accepted at p < 0.05. 5. Conclusions Overall, our findings, using phytochemical, in silico network, and docking approaches, as well as further in vivo preclinical validation, show that ARBE could protect from RIRI by combating inflammation and apoptosis. Mechanistically, this might be due to the downregulation of HSP90 and P53. Yet, the effect of ARBE on other molecular targets cannot be excluded. Further drug discovery and preclinical and clinical studies are warranted on certain major components of ARBE, e.g., syringetin-3-O-glucoside and narcissoside, given their predicted interaction with multiple RIRI-related targets, in particular the hubs. These compounds may represent a base for the generation of new molecules that could be utilized as nephroprotective agents against RIRI. Acknowledgments