Kaempferol and Chrysin Synergies to Improve Septic Mice Survival

Previously, we reported the role of synergy between two flavonoids—namely, chrysin and kaempferol—in inhibiting the secretion of a few major proinflammatory mediators such as tumor necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2), and nitric oxide (NO) from lipopolysaccharide (LPS)-induced RAW 264.7 cells. The present study aims to evaluate the effects of this combination on a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Severe sepsis was induced in male ICR mice (n = 7) via the CLP procedure. The effects of chrysin and kaempferol combination treatment on septic mice were investigated using a 7-day survival study. The levels of key proinflammatory mediators and markers—such as aspartate aminotransferase (AST), TNF-α, and NO—in the sera samples of the septic mice were determined via ELISA and fluorescence determination at different time point intervals post-CLP challenge. Liver tissue samples from septic mice were harvested to measure myeloperoxidase (MPO) levels using a spectrophotometer. Moreover, intraperitoneal fluid (IPF) bacterial clearance and total leukocyte count were also assessed to detect any antibacterial effects exerted by chrysin and kaempferol, individually and in combination. Kaempferol treatment improved the survival rate of CLP-challenged mice by up to 16%. During this treatment, kaempferol expressed antibacterial, antiapoptotic and antioxidant activities through the attenuation of bacterial forming units, AST and NO levels, and increased polymorphonuclear leukocyte (PMN) count in the IPF. On the other hand, the chrysin treatment significantly reduced serum TNF-α levels. However, it failed to significantly improve the survival rate of the CLP-challenged mice. Subsequently, the kaempferol/chrysin combination treatment significantly improved the overall 7-day survival rate by 2-fold—up to 29%. Kaempferol and chrysin revealed some synergistic effects by acting individually upon multiple pathophysiological factors involved during sepsis. Although the kaempferol/chrysin combination did not exhibit significant antibacterial effects, it did exhibit anti-inflammatory and antioxidant activities, which translate to significant improvement in the survival rate of septic animals. These findings suggest the potential application of this combination treatment as a beneficial adjuvant supplement strategy in sepsis control.


Introduction
Sepsis is a serious life-threatening disorder that occurs as a response to invading organisms and their components. It is considered to be the leading cause of death in many intensive care units (ICUs) around the world, often with a high mortality rate, and remains without a definitive cure [1]. This complex syndrome is associated with the overproduction of inflammatory mediators [2], resulting

Effects of Chrysin and Kaempferol Individual or Combination Treatments on Endotoxin Shock
The optimal treatment doses of chrysin and kaempferol were determined separately prior to formulating the kaempferol/chrysin combination. Figure 1 shows the survival dose-response of kaempferol and chrysin, respectively, for septic mice. Taking into account the highest survival rate and first mortality occurrence, chrysin at 5 mg/kg showed the best efficacy among other tested concentrations, whereas kaempferol at 1 mg/kg significantly reduced the mortality rate to 16% (p ≤ 0.05) compared to the CLP/vehicle control group. Consequently, a fixed-ratio, serial dilution dose-response study was constructed by combining the above two optimal doses ( Figure 2). A kaempferol/chrysin combination at 3 mg/kg significantly increased survival rate up to 29% (p ≤ 0.01) compared to the CLP/vehicle control group. The sham-operated control group resulted in no mortality over the 7-day experiment, while all the mice from the CLP/vehicle-treated group died within the first 60 h.

Effects of Chrysin and Kaempferol Individual or Combination Treatments on Endotoxin Shock
The optimal treatment doses of chrysin and kaempferol were determined separately prior to formulating the kaempferol/chrysin combination. Figure 1 shows the survival dose-response of kaempferol and chrysin, respectively, for septic mice. Taking into account the highest survival rate and first mortality occurrence, chrysin at 5 mg/kg showed the best efficacy among other tested concentrations, whereas kaempferol at 1 mg/kg significantly reduced the mortality rate to 16% (p ≤ 0.05) compared to the CLP/vehicle control group. Consequently, a fixed-ratio, serial dilution dose-response study was constructed by combining the above two optimal doses ( Figure 2). A kaempferol/chrysin combination at 3 mg/kg significantly increased survival rate up to 29% (p ≤ 0.01) compared to the CLP/vehicle control group. The sham-operated control group resulted in no mortality over the 7-day experiment, while all the mice from the CLP/vehicle-treated group died within the first 60 h.

Effects of Chrysin and Kaempferol Individual or Combination Treatments on Endotoxin Shock
The optimal treatment doses of chrysin and kaempferol were determined separately prior to formulating the kaempferol/chrysin combination. Figure 1 shows the survival dose-response of kaempferol and chrysin, respectively, for septic mice. Taking into account the highest survival rate and first mortality occurrence, chrysin at 5 mg/kg showed the best efficacy among other tested concentrations, whereas kaempferol at 1 mg/kg significantly reduced the mortality rate to 16% (p ≤ 0.05) compared to the CLP/vehicle control group. Consequently, a fixed-ratio, serial dilution dose-response study was constructed by combining the above two optimal doses ( Figure 2). A kaempferol/chrysin combination at 3 mg/kg significantly increased survival rate up to 29% (p ≤ 0.01) compared to the CLP/vehicle control group. The sham-operated control group resulted in no mortality over the 7-day experiment, while all the mice from the CLP/vehicle-treated group died within the first 60 h. Kaplan-Meier plots of the survival rates (%) of cecal ligation and puncture (CLP)-challenged septic ICR mice treated with different doses of (A) kaempferol; (B) chrysin within 7 days. Animals were i.p. injected with treatments or vehicle (10 mL/kg) 1 h prior to CLP challenge, * p = 0.01.

Organ Weights
Chrysin treatment at 5 mg/kg transiently increased the mean spleen weight of the male ICR mice (p ≤ 0.05) at 6 h ( Figure 3b). This was followed by a decreasing trend in the mean spleen weights of all groups to points below the sham-operated control group. Meanwhile, the mean thymus weights decreased during the progression of sepsis from 6 to 24 h, however, none of them were significant (Figure 3a).

Organ Weights
Chrysin treatment at 5 mg/kg transiently increased the mean spleen weight of the male ICR mice (p ≤ 0.05) at 6 h ( Figure 3b). This was followed by a decreasing trend in the mean spleen weights of all groups to points below the sham-operated control group. Meanwhile, the mean thymus weights decreased during the progression of sepsis from 6 to 24 h, however, none of them were significant (Figure 3a).

Quantitative and Differential Leukocytic Count
Results from Figure 4 show an increasing trend in total leukocyte cell population from 6 to 24 h. Kaempferol at 1 mg/kg significantly increased the leukocytes numbers at 6 h (p ≤ 0.001) and maintained this high population for up to 24 h. Meanwhile, chrysin at 5 mg/kg and the combination of kaempferol/chrysin at 3 mg/kg maintained a significantly low leukocyte population at 24 h compared to the vehicle control group. Likewise, the kaempferol treatment at 1 mg/kg maintained high neutrophil, macrophage, and lymphocyte populations, as displayed in Figure 5, while the chrysin (5 mg/kg) and kaempferol/chrysin (3 mg/kg) treatments maintained significantly lower populations compared to the vehicle control group.

Quantitative and Differential Leukocytic Count
Results from Figure 4 show an increasing trend in total leukocyte cell population from 6 to 24 h. Kaempferol at 1 mg/kg significantly increased the leukocytes numbers at 6 h (p ≤ 0.001) and maintained this high population for up to 24 h. Meanwhile, chrysin at 5 mg/kg and the combination of kaempferol/chrysin at 3 mg/kg maintained a significantly low leukocyte population at 24 h compared to the vehicle control group. Likewise, the kaempferol treatment at 1 mg/kg maintained high neutrophil, macrophage, and lymphocyte populations, as displayed in Figure 5, while the chrysin (5 mg/kg) and kaempferol/chrysin (3 mg/kg) treatments maintained significantly lower populations compared to the vehicle control group.

Organ Weights
Chrysin treatment at 5 mg/kg transiently increased the mean spleen weight of the male ICR mice (p ≤ 0.05) at 6 h ( Figure 3b). This was followed by a decreasing trend in the mean spleen weights of all groups to points below the sham-operated control group. Meanwhile, the mean thymus weights decreased during the progression of sepsis from 6 to 24 h, however, none of them were significant ( Figure 3a).

Quantitative and Differential Leukocytic Count
Results from Figure 4 show an increasing trend in total leukocyte cell population from 6 to 24 h. Kaempferol at 1 mg/kg significantly increased the leukocytes numbers at 6 h (p ≤ 0.001) and maintained this high population for up to 24 h. Meanwhile, chrysin at 5 mg/kg and the combination of kaempferol/chrysin at 3 mg/kg maintained a significantly low leukocyte population at 24 h compared to the vehicle control group. Likewise, the kaempferol treatment at 1 mg/kg maintained high neutrophil, macrophage, and lymphocyte populations, as displayed in Figure 5, while the chrysin (5 mg/kg) and kaempferol/chrysin (3 mg/kg) treatments maintained significantly lower populations compared to the vehicle control group.

Bacterial Clearance
The antibacterial properties of kaempferol and chrysin individual or combination treatments were assessed by counting the colony forming units (CFUs) at 6 and 24 h post-CLP followed by a 24 h incubation period in nutrient agar. As shown in Figure 6, intraperitoneal swabs from the shamoperated group were sterile at 6 and 24 h. Even though the kaempferol (1 mg/kg) and kaempferol/chrysin (3 mg/kg) combination groups significantly decreased the CFUs at 6 h with p ≤ 0.001 and p ≤ 0.01, respectively, the chrysin (5 mg/kg) group did not significantly attenuate the CFUs at 6 h, and none of the tested groups exhibited antibacterial effects at 24 h.

Bacterial Clearance
The antibacterial properties of kaempferol and chrysin individual or combination treatments were assessed by counting the colony forming units (CFUs) at 6 and 24 h post-CLP followed by a 24 h incubation period in nutrient agar. As shown in Figure 6, intraperitoneal swabs from the sham-operated group were sterile at 6 and 24 h. Even though the kaempferol (1 mg/kg) and kaempferol/chrysin (3 mg/kg) combination groups significantly decreased the CFUs at 6 h with p ≤ 0.001 and p ≤ 0.01, respectively, the chrysin (5 mg/kg) group did not significantly attenuate the CFUs at 6 h, and none of the tested groups exhibited antibacterial effects at 24 h.

Serum AST and TNF-α Levels
The serum fibrosis marker, AST, was measured as an indicator to assess the degree of tissue damage, particularly heart and liver inflammation. Figure 7 shows an overall increase in AST levels in most of the treatment groups during the progression of sepsis from 6 to 24 h. Kaempferol at 1 mg/kg showed a significant decrease in AST levels (p ≤ 0.05) compared to the CLP/vehicle control group at 24 h, while the sham-operated group demonstrated a baseline AST level of 39 U/L. As shown in Figure 8, the serum TNF-α levels decreased during the progression of sepsis from 6 to 24 h, and all treatments showed a significant reduction in serum TNF-α levels (p ≤ 0.001) at 6 h compared to the CLP/vehicle group.

Serum AST and TNF-α Levels
The serum fibrosis marker, AST, was measured as an indicator to assess the degree of tissue damage, particularly heart and liver inflammation. Figure 7 shows an overall increase in AST levels in most of the treatment groups during the progression of sepsis from 6 to 24 h. Kaempferol at 1 mg/kg showed a significant decrease in AST levels (p ≤ 0.05) compared to the CLP/vehicle control group at 24 h, while the sham-operated group demonstrated a baseline AST level of 39 U/L. As shown in Figure 8, the serum TNF-α levels decreased during the progression of sepsis from 6 to 24 h, and all treatments showed a significant reduction in serum TNF-α levels (p ≤ 0.001) at 6 h compared to the CLP/vehicle group.

Serum AST and TNF-α Levels
The serum fibrosis marker, AST, was measured as an indicator to assess the degree of tissue damage, particularly heart and liver inflammation. Figure 7 shows an overall increase in AST levels in most of the treatment groups during the progression of sepsis from 6 to 24 h. Kaempferol at 1 mg/kg showed a significant decrease in AST levels (p ≤ 0.05) compared to the CLP/vehicle control group at 24 h, while the sham-operated group demonstrated a baseline AST level of 39 U/L. As shown in Figure 8, the serum TNF-α levels decreased during the progression of sepsis from 6 to 24 h, and all treatments showed a significant reduction in serum TNF-α levels (p ≤ 0.001) at 6 h compared to the CLP/vehicle group.

Serum AST and TNF-α Levels
The serum fibrosis marker, AST, was measured as an indicator to assess the degree of tissue damage, particularly heart and liver inflammation. Figure 7 shows an overall increase in AST levels in most of the treatment groups during the progression of sepsis from 6 to 24 h. Kaempferol at 1 mg/kg showed a significant decrease in AST levels (p ≤ 0.05) compared to the CLP/vehicle control group at 24 h, while the sham-operated group demonstrated a baseline AST level of 39 U/L. As shown in Figure 8, the serum TNF-α levels decreased during the progression of sepsis from 6 to 24 h, and all treatments showed a significant reduction in serum TNF-α levels (p ≤ 0.001) at 6 h compared to the CLP/vehicle group.   Figure 9 shows that only kaempferol at 1 mg/kg significantly reduced serum nitrate levels (p ≤ 0.05) at 6 h compared to the CLP/vehicle control group. Meanwhile, the sham-operated group maintained a baseline nitrite level.  Figure 9 shows that only kaempferol at 1 mg/kg significantly reduced serum nitrate levels (p ≤ 0.05) at 6 h compared to the CLP/vehicle control group. Meanwhile, the sham-operated group maintained a baseline nitrite level.

MPO Activity in Liver Tissue
MPO activity in liver tissue was measured as an index to assess the extent of liver injury resulting from neutrophil infiltration during the progression of sepsis. Tissue MPO activities reached a peak at 6 h after CLP operation, followed by a general decrease at 24 h. All treatment groups showed a significant reduction in MPO activities at 6 and 24 h compared to the CLP/vehicle. Out of all the groups, chrysin at 5 mg/kg and kaempferol/chrysin 3 mg/kg combination groups reduced MPO activity to nearly that of the sham-operated control group level (Table 1).

Statistical Analysis
Statistical analysis was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA, http://www.graphpad.com). Survival rates were expressed as percentages, and differences between groups were analyzed using the Kaplan-Meier log-rank and chi-square test. The data were presented as mean ± standard error of the mean (SEM) and compared using Student's t-test.

MPO Activity in Liver Tissue
MPO activity in liver tissue was measured as an index to assess the extent of liver injury resulting from neutrophil infiltration during the progression of sepsis. Tissue MPO activities reached a peak at 6 h after CLP operation, followed by a general decrease at 24 h. All treatment groups showed a significant reduction in MPO activities at 6 and 24 h compared to the CLP/vehicle. Out of all the groups, chrysin at 5 mg/kg and kaempferol/chrysin 3 mg/kg combination groups reduced MPO activity to nearly that of the sham-operated control group level (Table 1).

Statistical Analysis
Statistical analysis was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA, http://www.graphpad.com). Survival rates were expressed as percentages, and differences between groups were analyzed using the Kaplan-Meier log-rank and chi-square test. The data were presented as mean ± standard error of the mean (SEM) and compared using Student's t-test.

Animals
Male ICR mice (25-30 g) were used in this experiment. The mice were maintained in a temperature-and humidity-controlled room with a 12 h light/dark cycle and allowed free access to standard chow diet and water. The experimental protocol was approved by the Animal Care and Use Committee of the Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (Approval no. UPM/FPSK/PADS/BR-UUH/00309). Chrysin and kaempferol were purchased from Sigma-Aldrich (St. Louis, MO, USA). All compounds were dissolved in 2% dimethylsulfoxide (DMSO) and 5% Tween-20/phosphate-buffered saline (PBS).

Experimental Protocol and Combination
The mice were randomly stratified and divided into 15 groups (n = 7) including, briefly, the sham-operated group, CLP-vehicle treated group (2% DMSO and 5% Tween-20/PBS), CLP-chrysin-treated groups (with 50, 10, 5, 1, and 0.1 mg/kg), CLP-kaempferol-treated groups (with 50, 10, 5, 1, and 0.1 mg/kg), and CLP-kaempferol/chrysin combination treatment groups (with 6, 3, and 1.5 mg/kg). All mice in this study received the same volume (10 mL/kg) in a single injection 1 h prior to the CLP procedure. Combination doses were determined based on the optimum survival dose of each single compound. A fixed-ratio proportion was selected by combining the best survival dose of each single compound followed by a 2-fold serial dilution.

CLP as a Sepsis Model
Mice were acclimatized for 7 days, then starved for 12 h with free access to water to empty their bowels. Sepsis was induced in all groups of mice via the CLP procedure according to the method reported by Rittirsch et al. [29]. Briefly, mice were anesthetized with 2,2,2-tribromoethanol (St. Louis, MO, USA, 250 mg/kg i.p.) [30], and unconsciousness was tested via animal paw pinching. After shaving the lower quadrants, a 1 cm midline incision at the linea alba was made under sterile conditions, the cecum was then exposed and ligated immediately-distal to the ileocecal valve-so that the bowel continuity would be preserved. A single "through and through" perforation with an 18-gauge needle was made through the distal cecum, after which the fecal contents were extruded into the peritoneum. After the puncture, the cecum was relocated into the abdominal cavity, and the peritoneal incision was closed in layers with simple running sutures (BRILON, VIGILENZ non-absorbable, Malaysia). The sham control animals were subjected to midline laparotomy, in which their cecum was manipulated but neither ligated nor punctured. The mice were allowed to recover in their cages postoperatively with free access to rodent chow and water, and antibiotic cream was applied once at point of surgery.

Survival Rates, Harvesting Samples, and Cytokine Determination
Survival was monitored up to 7 days after the operation. In an additional set of experiments, the mice from each group were sacrificed at two time points of 6 and 24 h after operation for sample harvesting. Blood was collected via cardiac puncture, and then centrifuged and stored at −20 • C until the serum levels of AST and TNF-α were determined using commercially available ELISA kits (ID Labs Inc., London, ON, Canada, Cat No. SUP6002 and BD Pharmingen, San Diego, CA, USA, Cat No. 555268, respectively). Thymus and spleen were rapidly harvested and weighed, and liver tissues were harvested for MPO activity measurement. The peritoneal cavity was lavaged with 3 mL of sterile PBS and the obtained intraperitoneal fluid (IPF) was kept on ice for further analysis. IPF (500 µL) was fixed on slides by cytospin at 500 RCF (relative centrifugal force) for 5 min and stained with Wright's stain to determine the differential leukocyte count (1 slide/mouse, 4 mice/group, 300 cells counted/slide); 10 µL of IPF was stained with trypan blue for polymorphonuclear leukocyte quantification using a hemocytometer under a light microscope; whereas 100 µL of IPF was processed and serially diluted under sterile conditions on nutrient agar plates (Merck, Darmstadt, Germany) for bacterial-forming colony quantification. The same investigator carried out the standardized operations, sample collection, and processing of this study.

Serum Nitrite Determination
The serum nitrite concentration was determined spectrofluorometrically, as described by Fernández et al. [17]. Nitrite determination was based on its ability to react with 2,3-diaminonaphthalene (DAN) under acidic conditions to yield the fluorescent product 1-(H)-naphthotriazole. Briefly, 10 µL of freshly prepared DAN (0.05 mg/mL in 0.62 M HCl) was added to 50 µL of either the samples or nitrite standards in wells and then mixed. After 10 min incubation at room temperature in the dark, the reaction was stopped by adding 5 µL of 0.28 N NaOH per well. The formed 1-(H)-naphthotriazole was measured fluorometrically with an excitation wavelength of 350 nm, and the emission was read at 450 nm in a microplate spectrofluorometer (TECAN, Infinite M200, Männedorf, Switzerland). The concentration of nitrite in the samples was calculated from a sodium nitrite standard curve (0-100 µmol/L). The samples were performed in quadruplicates.

Measurement of MPO Activity in Liver Tissue
MPO was measured in liver tissues, as described by Hillefass et al. [31]. Briefly, 100 mg of liver tissue was homogenized and suspended in 50 mM of potassium phosphate buffer (pH 6.0) on ice. Then, it was centrifuged at 20,000× g at 4 • C for 15 min. The supernatant was discarded and pellet was resuspended in 50 mM of phosphate buffer containing 0.5% hexadecyltrimethyl-ammonium bromide (HTA-Br) (St. Louis, MO, USA). The tube contents were then homogenized for 30 s and sonicated with an ultrasonic dismembrator (Branson, Danbury, CT, USA) for 15 s at 40% power. Three cycles of freezing and thawing in liquid nitrogen were performed, followed by repeated sonication. The tube was centrifuged again at 20,000× g at 4 • C for 15 min and the supernatant aliquots (100 µL) were added to 2.9 mL of the reaction mixture, which contained 50 mM of potassium phosphate buffer, 0.167 mg/mL o-dianisidine dihydrochloride, and a 0.0005% hydrogen peroxide (H 2 O 2 ) solution. MPO activity in each sample was determined by measuring the changes in absorbance per 30 s interval over 3 min at 460 nm using a spectrophotometer (UVM 340, ASYS Hitech GmbH, Eugendorf, Austria). MPO activity was expressed as a percentage change in absorbance/100 mg liver tissue.

Discussion
Previous studies demonstrated that some flavonoids exhibited synergistic properties in vitro that were translated into an enhanced attenuation of the production of major proinflammatory mediators. Particularly, kaempferol and chrysin were reported to be highly synergistic in inhibiting the production of NO, TNF-α, and PGE 2 from LPS-induced RAW 264.7 murine macrophages by reducing the IC 50 (inhibitory concentration at 50%) of the individual compound [14]. In view of this finding, this study aims to investigate whether or not the synergistic effects previously achieved in vitro could be translated into an animal model of sepsis.
During sepsis progression, the vehicle-treated control group died rapidly within the first 3 days. A postmortem necropsy examination showed that septic mice died due to severe multiple organ dysfunction and necrosis, which were reflected by the reduction of the thymus and spleen mean weights during the progression of sepsis. On the other hand, the sham-operated group survived until the end of the experiment, and the organs of this group did not undergo any weight reduction, as they were not challenged by polymicrobial sepsis. Based on the results of our present study, kaempferol and chrysin did not synergize in inhibiting many of the proinflammatory markers, but instead exhibited different patterns of protection against multiple inflammatory factors in the septic mice. However, their combination significantly improved the outcome of sepsis as a whole, described by the increase in the survival rate of the septic animals of up to 29%, which is considered a significant improvement compared to the individual kaempferol/chrysin treatments. Therefore, this finding suggests that kaempferol/chrysin combination may possess underlying synergistic benefits.
Kaempferol or chrysin and their combination expressed an additive protective effect against the serum levels of AST, NO, and TNF-α in septic mice. Interestingly, it was noted that kaempferol or chrysin treatments and their combination significantly attenuated serum TNF-α and NO levels when these cytokines were majorly expressed in the septic animal at the early phase of sepsis, followed by a general depletion of their concentrations in all groups at a later phase, which is attributed to the short lifespan of these cytokines. These findings align with previously reported beneficial anti-inflammatory activities of kaempferol and chrysin through the inhibition of the overproduction of TNF-α and NO [32,33]. Accordingly, the abilities of kaempferol and chrysin to attenuate these cytokines as key targets in septic animals have contributed to the initial survival benefits in response to the CLP-induced lethal sepsis in this model. Sepsis is characterized by the early systemic recruitment of PMNs, which are short-lived and rapidly undergo apoptosis [34], followed by the recruitment of monocytes and lymphocytes, accordingly [35]. White blood cells (WBCs), neutrophils, monocytes, and macrophages have been reported to be highly recruited and activated following the release of inflammatory mediators such as TNF-α and ·NO in the animal system during the progression of sepsis [36]. Similarly, this study demonstrated rising trends in total WBC count and the percentages of cells also shifted from neutrophils towards lymphocytes during the progression of sepsis. Interestingly, the individual treatment of kaempferol further promoted WBC recruitment, pertaining to a significant increase in total WBC count by 2-fold at 6 h post-CLP; subsequently, a high WBC count similar to that observed in the vehicle-treated group at 24 h was maintained. On the other hand, chrysin treatment was found to attenuate WBC count to the basal levels of the sham-operated group. As expected, the kaempferol/chrysin combination treatment had an additive intermediate effect upon the WBC population when compared to that of individual treatments. This unique ability of kaempferol to increase neutrophil population could be attributed to its previously reported distinctive antimicrobial and antibacterial properties [37,38]. Interestingly, even though kaempferol treatment attenuated bacterial colonies at the early stages of sepsis, it failed to sustain a prolonged antibacterial effect. This suggests that any prolonged therapeutic effects by kaempferol is attributed to its anti-inflammatory and antioxidant activities alongside its short-term antibacterial activities. Moreover, kaempferol's ability to sustain a high neutrophil count at 24 h indicates that kaempferol may possess some antiapoptotic activities, which is supported by its ability to reduce the serum fibrosis marker (AST) levels at the same time point. Such properties of kaempferol have been previously reported during inflammation [39].
Neutrophil activation and migration to the site of infection is believed to play a complex protective role during sepsis and inflammation. Activated neutrophils release a host of mediators, cytokines, free radicals, MPO, and phagocyte bacteria before apoptosis [36]. During an impaired immune response, delayed neutrophil apoptosis has been associated with high serum levels of activated neutrophils and reported to cause tissue injury and SIRS during sepsis [40,41]. Furthermore, the neutrophil-secreted MPO enzyme has been linked to oxidative stress and tissue damage [42], and is used as an independent prognostic marker for neutrophil activation and migration [43]. Likewise, this study reported a significant increase in liver tissue MPO activities in CLP-challenged groups. However, chrysin treatment attenuated MPO activities to almost basal levels, whereas kaempferol treatment did not exhibit similar benefits. Accordingly, the kaempferol/chrysin combination expressed an intermediate additive effect upon MPO inhibition compared to that of individual treatment.
In conclusion, this study demonstrated that a combined treatment of kaempferol and chrysin significantly improved the outcome of CLP-induced septic shock. This combination may not have a superior effect on any pathophysiological factor compared to the individual treatments, for which each flavonoid acted on different factors independently. However, when combined, kaempferol and chrysin synergized and significantly increased the survival of septic animals. These findings suggest kaempferol/chrysin combinational treatment as a beneficial adjuvant therapy in sepsis control, opening further possibilities of research to investigate the potentials and mechanisms of different flavonoid combinations, synergism, and protection against sepsis.