Pharmaceutical composition for improving acute kidney injury caused by sepsis as well as preparation method and application of pharmaceutical composition

Abstract

The invention belongs to the technical field of traditional Chinese medicine and biological medicine, and provides a pharmaceutical composition SGD for improving sepsis acute kidney injury and a preparation method and application thereof, the pharmaceutical composition SGD is prepared from the following raw materials by weight: 25-35 parts of raw white peony root, 8-13 parts of honey-fried licorice root, 10-20 parts of winged euonymus twig, 10-20 parts of caulis sinomenii, 25-35 parts of lalang grass rhizome, 8-13 parts of patrinia and 8-13 parts of sargentgloryvine stem. The SGD is used for treating S-AKI for the first time, on one hand, the SGD reduces oxidative stress and systemic inflammatory response by inhibiting a PI3K-AKT signal channel, on the other hand, the SGD can improve the renal function of S-AKI and the survival rate of mice, and participates in regulating the disease mechanism of S-AKI. The potential protection effect of the SGD on S-AKI, systemic inflammation and oxidative stress is discussed, and the protection mechanism of the SGD is further discussed by paying attention to a PI3K-AKT signaling pathway. And further evidence can be provided for SGD treatment of S-AKI.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine and biomedicine, and specifically relates to a pharmaceutical composition for improving acute kidney injury caused by sepsis, a preparation method and an application thereof. The pharmaceutical composition is Shengbaishao Gancao Decoction (SGD). Background Art

[0002] Sepsis is a potentially life-threatening complication caused by an imbalanced host response to bacterial infection. The kidney is one of the organs most susceptible to sepsis. Acute kidney injury (AKI) occurs in 50% of patients with sepsis, with a mortality rate of up to 60%. Sepsis-induced acute kidney injury (S-AKI) is one of the most common and severe complications of sepsis, leading to prolonged hospital stay and poor prognosis. Sepsis can lead to immune dysfunction and oxidative stress. Risk factors in the inflammatory response, such as nitric oxide (NO) and reactive oxygen species (ROS), can trigger mitochondrial oxidative stress and apoptosis. In the case of sepsis, microorganisms release pathogen-associated molecular patterns (PAMPs), triggering the activation of immune cells and the massive release of inflammatory mediators. These inflammatory mediators can directly damage renal tubular and endothelial cells, leading to renal insufficiency. ROS can directly damage lipids, proteins, and DNA of renal cells, leading to cell dysfunction and death. In addition, oxidative stress can promote the release of inflammatory mediators by activating inflammatory signaling pathways, thereby aggravating the inflammatory response. At the same time, the inflammatory response can further induce the production of ROS, forming a vicious cycle, thereby aggravating renal damage. Due to the limited availability of effective drug treatments for inflammatory responses and oxidative stress, the challenge of reducing the mortality rate of severe S-AKI remains. The current standard management of S-AKI in clinical practice includes fluid resuscitation, the application of vasopressors, and the early use of antibiotics. However, these strategies for treating S-AKI are supportive and non-specific. Therefore, the discovery of new effective drugs for S-AKI is urgent and of great clinical significance.

[0003] Shengbaishaogancao decoction (SGD) is a traditional Chinese medicine compound that has multiple beneficial effects on diseases. Previous studies have reported that SGD has multiple pharmacological effects, such as regulating inflammation, regulating iron deficiency, and protecting renal function. However, it is still unclear whether SGD has a therapeutic effect on S-AKI. Summary of the invention

[0004] In view of the above problems, the present invention provides a pharmaceutical composition SGD for improving acute kidney injury caused by sepsis, and a preparation method and application thereof, explores the potential protective effect of SGD on S-AKI, systemic inflammation and oxidative stress, and further explores its protective mechanism by focusing on the PI3K-AKT signaling pathway, which may provide further evidence for the treatment of S-AKI with SGD.

[0005] The present invention is achieved by the following technical scheme: a pharmaceutical composition for improving acute kidney injury caused by sepsis, which is prepared from the following raw materials in parts by weight: 25-35 parts of raw white peony root, 8-13 parts of roasted licorice root, 10-20 parts of Psoralea corylifolia, 10-20 parts of Caulis Sinomenii, 25-35 parts of Imperatae cylindrica, 8-13 parts of Patrinia scabra, and 8-13 parts of Caulis Sargentodoxae.

[0006] Furthermore, it is prepared from the following raw materials in parts by weight: 30 parts of raw white peony root, 10 parts of roasted licorice root, 15 parts of Psoralea corylifolia, 15 parts of Caulis Sinomenii, 30 parts of Imperata rhizome, 15 parts of Patrinia scabra, and 15 parts of Caulis Scutellariae.

[0007] The method for preparing the pharmaceutical composition for improving acute kidney injury caused by sepsis comprises mixing raw white peony root, roasted licorice root, Chinese prickly ash, caulis cylindrica, cyperus rotundus, patrinia ulmoides, and scutellaria baicalensis in proportion, and then boiling twice with distilled water, the first time for 30 minutes and the second time for 20 minutes; taking the solution boiled twice, mixing and concentrating, and drying to a drug concentration of 1.1 g / ml; the main active ingredients are catechins, quercetin, gallic acid, ellagic acid, naringenin, kaempferol, chlorogenic acid methyl ester, and paeoniflorin.

[0008] The present invention also provides the use of the pharmaceutical composition for improving sepsis-acute kidney injury in the preparation of a drug for treating sepsis-acute kidney injury (S-AKI).

[0009] Furthermore, the pharmaceutical composition is used in the preparation of a drug for improving the survival rate of S-AKI mice.

[0010] Furthermore, the pharmaceutical composition is used in the preparation of a drug for inhibiting inflammatory factors IL-1β, IL-6 or TNF-α.

[0011] Furthermore, the pharmaceutical composition is used in the preparation of drugs for inhibiting renal tubular epithelial cell shedding and inhibiting cell apoptosis.

[0012] The pharmaceutical composition is used in preparing a drug for inhibiting acute kidney injury markers, such as blood creatinine, urea nitrogen or kidney injury molecules.

[0013] The pharmaceutical composition is used in the preparation of antioxidant drugs that promote S-AKI.

[0014] The pharmaceutical composition is used in preparing a drug for inhibiting the PI3K-AKT signaling pathway to reduce oxidative stress and systemic inflammatory response.

[0015] The present invention is the first to use SGD to treat S-AKI. On the one hand, SGD reduces oxidative stress and systemic inflammatory response by inhibiting the PI3K-AKT signaling pathway. On the other hand, it can improve the renal function and survival rate of mice in S-AKI, and participate in regulating the disease mechanism of S-AKI. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a diagram of the preparation and drug administration pattern of the mouse model; Figure 2 This is the survival curve of mice in each group within 7 days; Figure 3 The inflammatory expression of mice in each group; Figure 4 This is the improvement of renal injury in each group of mice; Figure 5 HE staining of renal tissue and renal cell apoptosis of mice in each group; Figure 6 The oxidative stress in the kidneys of mice in each group; Figure 7 The expression of the antioxidant function of SGD on S-AKI; Figure 8 is the SGD principal component identification result; Fig. 9 For network pharmacology results; Fig.10 is the molecular docking diagram; Fig.11 is the cluster heat map; Fig.12 For volcano pictures; Fig.13 This is the GO analysis graph; Fig.14 This is the KEGG pathway enrichment analysis diagram; Fig.15 Results of proteomics KEGG targeting PI3K-AKT signaling pathway; Fig.16 The expression and quantification results of PI3K and AKT in kidney cells of each group of mice. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0019] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0020] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0021] 1. Preparation of Chinese medicine preparation: Soak 30g of white peony root, 10g of roasted licorice root, 15g of Radix Pseudostellariae, 15g of Caulis Cibotii, 30g of Imperatae Rhizome, 15g of Patriniae Herba and 15g of Caulis Sargentodoxae in cold water for 30 minutes, with the amount of water exceeding the medicine by 5 cm, decoct for 30 minutes, filter, and obtain decoction A and dregs A. Add cold water exceeding the medicine by 3 cm to the obtained dregs A, decoct for 20 minutes, filter, and obtain decoction B and dregs B. Combine the obtained decoction A and decoction B, and concentrate to a liquid with a concentration of 1.1 g / ml.

[0022] 2. Preparation and administration of mouse models Experimental animals: SPF male C57BL / 6 mice were purchased from Beijing Huafukang Biotechnology Co., Ltd., 6-8 weeks old, 20-22g. All experimental animals were humanely treated in accordance with the "Guidelines for the Care and Use of Laboratory Animals". The experimental protocol was approved by the Experimental Animal Ethics Committee of Shanxi Medical University. Before the start of the experiment, all mice were free to forage for food, drink water, and natural light.

[0023] Cecal ligation and puncture (CLP) was used to induce a sepsis model in mice: 1.5 ml / kg of sodium pentobarbital was injected intraperitoneally for anesthesia according to the weight of the mice. The mice were fixed and depilated. After disinfection with iodine, the skin and muscular layer were cut along the right lower abdomen of the mice. The cecum was carefully turned out, and the feces in the intestine were squeezed to the blind end. After ligating one-third of the cecum, a 10 mL syringe was used to puncture the blind end and squeeze out a small amount of feces. Finally, the cecum was returned and the skin and muscular layer were sutured. After 1 ml of normal saline was given, the mice were kept warm and resuscitated on a 37 ° C warming blanket. Compared with the model group, the sham operation group had the same anesthesia method and surgical laparotomy steps as the model group. After turning out the cecum, the intestinal tract was directly returned to the abdomen and the skin and muscular layer were sutured.

[0024] According to the experimental requirements, the mice were randomly divided into 4 groups, namely, sham operation group, CLP model group, SGD low concentration group and SGD high concentration group. The four groups of mice were given ordinary feed and free to eat. After 3 days of adaptive feeding, they were pretreated with drugs for 7 days. The specific treatment methods were as follows: the sham operation group and the model group were given intragastric administration of normal saline, and the low-concentration and high-concentration treatment groups were given low-concentration SGD (22 g / kg) and high-concentration SGD (44 g / kg), respectively. One hour after the end of the seventh day of medication, the mice underwent cecal ligation and puncture surgery to induce a moderate sepsis acute kidney injury model. The model diagram is as follows Figure 1 shown.

[0025] The survival status of mice in each group was observed within 7 days, and the survival curve of mice in each group was drawn within 7 days. Figure 2 As shown in the data, SGD can reduce the 7-day mortality of mice with acute kidney injury caused by sepsis within a 7-day observation period. 3. Application of the Chinese herbal compound SGD in improving acute kidney injury The animal experimental model and grouping were the same as above. After the modeling was completed, half of the mice were fed for 24 hours, and the remaining half of the mice were fed for 72 hours. After that, the relevant indicators were tested. The specific treatment methods were as follows: (1) Serum was collected from each group of mice 24 hours after surgery, and the expression of inflammation-related genes was detected by ELISA. The mRNA expression levels of IL-1β, IL-6, and TNF-α in the kidney, liver, and lung tissues of each group of mice were also detected according to the instructions of the ELISA kit provided by Shanghai Jianglai Biotechnology Co., Ltd., China. The results are as follows: Figure 3 shown. The experimental results showed that compared with the sham group, the levels of serum IL-1β, IL-6, and TNF-α in the CLP group were significantly increased, and the SGD group showed a significant decrease in the levels of these inflammatory cytokines. In addition, CLP surgery increased the mRNA expression of proinflammatory factors, while SGD inhibited the expression of these cytokines in the kidney, liver, and lung. It can be seen that SGD can alleviate CLP-induced sepsis by reducing the overexpression of proinflammatory cytokines.

[0026] Renal tissues of mice in each group were obtained 24 hours after surgery to detect the mRNA expression of KIM-1 in renal tissues; serum of mice in each group was obtained 72 hours after surgery to detect the expression of blood creatinine and urea nitrogen in renal tissues. The serum creatinine determination kit was provided by Nanjing Jiancheng Biotechnology Co., Ltd., China, and the blood urea nitrogen (BUN) level detection kit was provided by ElabscienceBiotech Co, Ltd (Wuhan, China).

[0027] The results are as follows Figure 4The experimental results showed that compared with the sham operation group, the levels of KIM-1 mRNA, serum creatinine and urea nitrogen in the CLP group mice were increased, and SGD pretreatment of CLP-induced mice reduced the expression levels of KIM-1 mRNA, serum creatinine and urea nitrogen.

[0028] The kidney tissues of mice in each group were obtained 72 hours after surgery. The degree of renal injury in mice was observed by HE staining and TUNEL staining. The kidney tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced. The slices were dewaxed with xylene, and the xylene was removed by gradient alcohol, then washed with water and PBS. Cell apoptosis was detected by Meilun One Step TUNEL Cell Apoptosis Detection Kit (FITC). Proteinase K was incubated at 37°C for 20 minutes and washed with PBS; terminal deoxynucleotidyl transferase (TdT) enzyme and fluorescein-12 deoxytriuracil phosphate (FITC-12dUTP) were incubated in the dark at 37°C for 1 hour and washed with PBS three times. Finally, the slices were covered with anti-fading medium containing DAPI and imaged under a fluorescence microscope. TUNEL-positive cells were recorded, and the relative proportion of apoptotic cells was calculated according to (number of apoptotic cells / total number of cell nuclei) × 100%.

[0029] The results are as follows Figure 5 As shown. The results of HE analysis of renal paraffin sections showed that the number of vacuoles in the renal tubules of mice in the CLP group increased compared with the mice in the sham operation group and the SGD pretreatment group. There were epithelial cells and cell fragments in the lumen of the renal tubules to varying degrees (black triangles), and the intercellular spaces of the renal tubular epithelial cells were enlarged (red triangles). SGD pretreatment can improve pathological renal structural damage. The above results indicate that the degree of renal pathological damage is reduced. Further, TUNEL staining was used to detect the apoptosis of renal tissue. The incidence of TUNEL-positive cells in the sham operation group was low, and the number of TUNEL-positive cells in the CLP group was significantly higher than that in the sham operation group. However, SGD pretreatment resulted in a significant decrease in the number of apoptotic TUNEL-positive cells.

[0030] 24 hours after surgery, kidney tissues of mice in each group were collected and made into paraffin sections. TOMM20 immunofluorescence staining and DHE staining were used to observe renal oxidative stress. Figure 6 As shown. The results of TOMM20 immunofluorescence of paraffin sections of renal tissue showed that CLP surgery reduced the fluorescence intensity of the mitochondrial outer membrane, indicating a decrease in mitochondrial biogenesis. However, SGD pretreatment increased the fluorescence intensity of mitochondria. In addition, DHE staining was used to detect the production of reactive oxygen species (ROS) in renal tissue cells. Compared with the sham operation group, the ROS production expression in the CLP-induced group was upregulated. The SGD pretreatment group alleviated the abnormal ROS production.

[0031] The kidney tissues of mice in each group were taken 24 hours after surgery to detect the expression levels of GSH and SOD. Figure 7 To quantify the antioxidant function of SGD against S-AKI, the levels of GSH and SOD in all groups were evaluated. The data showed that the antioxidant function of the CLP group was significantly decreased, while that of the SGD group was significantly increased compared with the sham group.

[0032] Network pharmacology: The targets of SGD were identified using the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), Traditional Chinese Medicine Integrated Database (TCMID), and Comparative Toxicogenomics Database (CTD). Disease targets were obtained using CTD. Common genes obtained from cross-combination related targets and disease-related targets were imported into the STRING11.5 database to create a protein-protein interaction network (PPI), and common genes were imported into the GO database to analyze the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.

[0033] SGD principal component analysis, network pharmacology and molecular docking were used to predict potential therapeutic targets. Figure 8 , Fig. 9 , Fig.10 shown.

[0034] The composition of SGD was identified by UPLC-HRMS analysis, and the data showed that there were 1844 compounds in SGD, including alkaloids, amino acids and peptides, carbohydrates, fatty acids, polyketides, oxalic acid, phenylpropanoids and terpenes. The comprehensive research results are shown in Figure 8 , Figure 8 A and B are the chromatograms of the positive and negative ion base peaks of SGD, respectively.

[0035] After identifying the components of SGD, the potential mechanism of action of SGD on S-AKI was further discovered using network pharmacology and molecular docking techniques. Based on the drug and disease screening conditions, 107 active compounds and 4283 targets were obtained from the seven SGD herbs. In addition, the disease database provided 16966 and 21497 disease targets for sepsis and AKI, respectively. The Venn diagram showed that there were 3987 common targets in SGD, sepsis, and AKI. The results are shown in Fig. 9 A. To screen the key targets closely related to the effects of SGD on S-AKI, we selected the top 100 targets for PPI analysis. Fig. 9 B The results showed that AKT1, IL-6 and MAPK1 may be its important targets. In addition, GO and KEGG enrichment analysis showed that SGD pretreatment was mainly related to inflammatory response-related signaling pathways (such as toll-like receptors, MAPK pathways, T cell receptors, chemokine pathways) and cell apoptosis. The results are shown in Fig. 9 C, D.

[0036] To further explore the molecular mechanism of SGD in the treatment of S-AKI, we used molecular docking technology. Based on the PPI network, GO, KEGG and compound-target interaction network, we selected the top three core targets (AKT1, HMOX1 and BCL2) and eight active ingredients (catechins, quercetin, quercetin, gallic acid, ellagitannin, naringenin, kaempferol, chlorochloride and paeoniflorin). The results are shown in Fig. 9 E. Molecular docking see Fig.10 Molecular docking revealed that three core targets could penetrate and attach to most of the active ingredients.

[0037] Renal tissues of mice in each group were obtained 24 hours after surgery for proteomic analysis to further explore the molecular mechanism of SGD on S-AKI. Fig.11 , Fig.12 , Fig.13 , Fig.14 , Fig.15 shown.

[0038] First, differentially expressed proteins were selected, with a threshold of p<0.05, and then the fold change was selected as >1.3. In order to analyze the protein expression pattern of all samples, a hierarchical clustering algorithm was used to compare the differentially expressed proteins of the two groups. The clustering heat map results are shown in Fig.11 The results showed that there were significant differences in the levels of differentially expressed proteins within each group. Fig.12 The results showed that compared with the CLP group, 87 proteins were up-regulated and 106 proteins were down-regulated in the SGD pretreatment group. GO analysis was used to analyze the biological functions of the differentially expressed proteins, such as Fig.13 As shown in Figure 3, its antioxidant activity and immune system processes are closely related to the beneficial effects of SGD on S-AKI. Next, KEGG enrichment analysis was performed to identify signaling pathways that may be associated with inflammation and oxidative stress. Fig.14 The results showed that the SGD pretreatment group had more than 100 KEGG enriched pathways compared with the CLP group. We selected the top 20 most significant pathways to draw a pathway map. Fig.15 The PI3K-AKT signaling pathway was significantly enriched.

[0039] Renal tissues of mice in each group were obtained 24 hours after surgery, and the expression of PI3K, p-PI3K, AKT and p-AKT proteins was detected by immunofluorescence staining and Western blot to further verify its molecular mechanism. Total protein was separated from mouse kidney tissue using cell lysis buffer, and protein concentration was determined using a BCA kit. The protein extract was separated by SDS-PAGE on a Bio-Rad gel electrophoresis system, and the protein was electrophoresed onto a 0.45um PVDF membrane, and then 5% skim milk was added to TBST to block nonspecific binding. Then, the primary antibody was incubated at 4°C for 4h, washed, and incubated with the secondary antibody at room temperature for 30min. The protein bands were visualized and photographed using a chemiluminescent solution, and the blots were quantified using Image J software. The results are shown in Fig.16 shown.

[0040] To determine whether SGD has a protective effect against S-AKI in vivo through the PI3K-AKT pathway, Western blot was used to detect the protein levels of PI3K, p-PI3K, AKT, and p-AKT in renal tissues. Fig.16 AC showed that the protein levels of p-PI3K / PI3K were increased in the CLP group. In contrast, SGD pretreatment significantly reduced the protein levels of p-PI3K and p-AKT. At the same time, immunofluorescence staining was used to detect the protein levels of p-PI3K and p-AKT in each group. Fig.16 DG showed that the protein levels of p-PI3K and p-AKT in the CLP group were significantly higher than those in the sham group. However, the levels of these phosphorylated proteins were significantly lower in the SGD group than in the CLP group. The above findings suggest that the PI3K-AKT signaling pathway is activated in S-AKI mice, and SGD pretreatment can inhibit this pathway to protect the systemic inflammation and oxidative stress of S-AKI.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pharmaceutical composition for improving acute kidney injury caused by sepsis, characterized in that: The invention is prepared from the following raw materials in parts by weight: 25-35 parts of raw white peony root, 8-13 parts of roasted liquorice root, 10-20 parts of Psoralea corylifolia, 10-20 parts of Caulis Sinomenii, 25-35 parts of Imperatae cylindrica, 8-13 parts of Patrinia scabra and 8-13 parts of Caulis Sargentodoxae.

2. A pharmaceutical composition for improving acute kidney injury caused by sepsis according to claim 1, characterized in that: The invention is prepared from the following raw materials in parts by weight: 30 parts of raw white peony root, 10 parts of roasted liquorice root, 15 parts of Psoralea corylifolia, 15 parts of Caulis Sinomenii, 30 parts of Imperatae cylindrica, 15 parts of Patrinia scabra and 15 parts of Caulis Sargentodoxae.

3. A method for preparing the pharmaceutical composition for improving acute kidney injury caused by sepsis according to claim 1, characterized in that: Raw white peony root, roasted liquorice, Euphorbia obesa, Caulis Cibotii, Imperatae cylindrica, Patrinia scabra and Caulis Scutellariae were mixed in proportion and boiled twice with distilled water, the first time for 30 minutes and the second time for 20 minutes; the solutions boiled twice were mixed and concentrated, and dried to a drug concentration of 1.1 g / ml; its main active ingredients are: catechins, quercetin, gallic acid, ellagic acid, naringenin, kaempferol, chlorogenic acid methyl ester and paeoniflorin.

4. Use of the pharmaceutical composition for improving sepsis-induced acute kidney injury according to claim 1 or 2 in the preparation of a medicament for treating sepsis-induced acute kidney injury (S-AKI).

5. The use according to claim 4, characterized in that: The pharmaceutical composition is used in preparing a drug for improving the survival rate of S-AKI mice.

6. The use according to claim 4, characterized in that: The pharmaceutical composition is used in preparing a drug for inhibiting inflammatory factors IL-1β, IL-6 or TNF-α.

7. The use according to claim 4, characterized in that: The pharmaceutical composition is used in preparing drugs for inhibiting renal tubular epithelial cell shedding and cell apoptosis.

8. The use according to claim 4, characterized in that: The pharmaceutical composition is used in preparing a drug for inhibiting acute kidney injury markers, such as blood creatinine, urea nitrogen or kidney injury molecules.

9. The use according to claim 4, characterized in that: The pharmaceutical composition is used in the preparation of antioxidant drugs that promote S-AKI.

10. The use according to claim 4, characterized in that: The pharmaceutical composition is used in preparing a drug for inhibiting the PI3K-AKT signaling pathway to reduce oxidative stress and systemic inflammatory response.

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