INTRODUCTION:

Cancer claims the most number of lives in the world, accounting for nearly 10 million death in 2020¹ GIT mucositis is one of the excruciating conditions, the cancer patients facing during their treatment. Due to a series of reactions,and formation of reactive molecules, altered inflammatory actions and injury of enterocytes. In consequence, there can be over production of cytokines, changed cellular penetrability, entry of microbial cells and production of cellular toxins².

These processes may alter the mucosal integrity of tissues and hence the immunological activities³throughupregulation of gene expression encoding the inflammatory molecules. Study results have shown that pro-inflammatory cytokines, specifically tumor necrosis factor (TNF-alpha) and interleukin-6 (IL-6) are some of the key molecules in the development of mucositis⁴ and in the pathogenesis of chronic inflammatory bowel syndrome. Earlier animal study reports with endo-toxemia or sepsis, also supports the enhanced levels of gut pro-inflammatory cytokines⁵. Because of the above processes, normal cell vulnerability would reach to its heights during cancer therapy and may end up with delay in or termination of therapy and exacerbates patient`s health and socio-economic conditions.⁶

Oxidative tension is one of the significant contributing factors leads to mucositis during chemotherapy, which is effecting normal cells⁷. Studies have pointed out that some of the peptides with antioxidant properties derived from casein and whey proteins are accountable for the protective action on many tissues including GIT, during chemical-induced ulcerative gastric lesions. Experimental and several other studies have reported that some of such components aswell as targeted therapies are beneficial during cancer⁸therapy. A diet comprising mainly of whey protein showed negative impact on the development of malignant tumors in mice induced by dimethyl hydrazine⁹and glutathione in liver and heart in aging mice and increased their life span¹⁰. Some of the studies reported that immunological modulation is of significant in post chemotherapy period. Mice fed with a diet with sufficient quantities of lactalbumin and shown to increase the immunological actions and mitogenic reactions¹¹. A concentrate of whey protein also shown to inhibit the formation of gastric ulcerative lesions in animal studies. Study on oral mucositis reports that use of some moth washes¹², honey¹³ as well as whey protein concentrate^14,15helped to reduce the severity and duration of mucositis Components of whey–lactalbumin, lactoglobulins, lactoferrin, lactoperoxidase, glycol-macropeptide etc. exert varied effects on different organs and immune system¹⁵. Studies reported that dietary supplementation of antioxidants and management of immunological factors are effective during chemotherapy^7,16. Since there is a need of such agent- that can protect the normal tissues from these toxic events of chemotherapeutic drug and Whey preparation, derived from bovine milk, available easily and having antioxidant properties could be one of such adjuvant. Hence, this work was designed to evaluate the influence of Whey preparation on intestinal mucositis in a rat model.

MATERIALS AND METHODS:

Experimental design:

Adult albino rats (10±1 week old) of Wistarstrain, were used for the study. Animals weighing between 220-250 g were obtained from central animal house, Kasturba Medical College, Mangalore, India, was used. The animal studies carried out with institutional animal ethical committee approval (Ref No. IAEC/KMC/ 06/11/2012). Animals were subjected to experimental procedures after two-weeks of acclimatization. They received standard pellet and water ad libitum. Whey Preparation (WP) was administered daily via orogastric gavage. At 0h, all rats were intraperitoneally injected with a single dose of either saline (control group) or etoposide (60mg of per kg). Freshly prepared whey preparation was used for the study. 25ml of milk was boiled and was curdled when it was hot by adding few drops of lime juice, kept for some time the supernatant was used for the study.

Grouping of animals:

Rats were coded in groups of two per cage and were randomly allocated to one of the following groups (n=6):

Group 1: Normal control.

Group 2: Etoposide control (i.p, 60mg per kg body weight). (ET)

Group 3: Etoposide (i.p) followed by whey preparation 100mg per kg body weight/day, at 0hr, 24 and 48 hrs.(WP+ET-100)

Group 4: Etoposide (i.p) followed by whey preparation, 200mg per kg body weight/day, once in a day at 0hr, 24 and 48hrs. (WP+ET -200)

Group 5: Etoposide (i.p) at zero hour, whey preparation in a dose of 100mg per kg body weight/day once in a day at from -120hrs to 48hrs. (WP+ET+WP-100)

Group 6: Etoposide (i.p) at zero hour, whey preparation in a dose of 200mg per kg body weight/day once in a day at from -120hrs to 48 hrs (WP+ET+WP-200)

Rats were sacrificed by cervical dislocation 72 hrs after etoposide administration. The small intestine was dissected and a duodenum section (first part) was immediately removed for the assessment of the biochemical parameters. These samples were homogenized immediately with the respective buffers (sucrose buffer for Na-K-ATPase and PBS) and stored at -20ºC until use.

Small intestinal sucrase activity:

Small intestinal sucrase activity was estimated by using 50μl of suitably diluted homogenates with 50μl of 0.2M sucrose, incubate at 37°C for 30 minutes. Sucrose cleaved to its constituents, glucose and fructose by sucrase in the homogenate. Amount of Glucose released was measured using spectrophotometer.¹⁷

Estimation of Na+/K+ATPase:

By the method of Adam et al,¹⁸. Na+/K+ ATPase activity was measured by incubating 50μl of enzyme preparation with 1M NaCl, 1M KCl, 0.1M MgCl2, 0.2M EDTA, 0.5M Tris-HCl, pH-7.4 for 5min. 10mM ouabain was added. Reaction was initiated by the addition of 50 μl of ATP solution and the reaction was terminated by the addition of 10% TCA after 10min. In a similar way enzyme blank was run and enzyme was added after TCA. After 10 mins. the tubes were centrifuged for 5min, to remove the precipitate. The supernatant was used for phosphate estimation by Fiske-SubbaRow method¹⁶

Estimation of Reduced Glutathione:

GSH concentration was estimated by the method of Ellman¹⁹. One milliliter of supernatant was treated with 1ml of metaphosphoric acid and cold digested at 4ºC for 1h. The samples were centrifuged at 1,200g for 15min at 4ºC. To 1ml of this supernatant, 2.7ml of phosphate buffer and 0.2ml of 5, 5’ dithio-bis-2-nitrobenzoic acid (DTNB) was added. The yellow color that developed was read immediately at 412nm using a Systronic-117 UV-Visible spectrophotometer. The values were expressed as mg/g of tissue.

Nitric Oxide (NO) levels:

Duodenal tissue nitric oxide level was determined using method of Griess reagent²⁰. One ml of the suitably diluted tissue homogenate was incubated with Griess reagent for 30minutes at 37⁰C and OD was measured at 540nm. Level of NO was expressed as mg/g tissue.

Myeloperoxidase (MPO):

In duodenum was determined using kineticmethod described by Pulli B et al.²¹. Samples were centrifuged at 10000rpm for 10min, the supernatant was discarded and the tissue homogenate was re-suspended in 0·5% hexadecyltrimethyl ammonium bromide, vortexed for 2 min and centrifuged at 5000rmp for 2min. After the addition of 25mM of 4- amino antipyridine and 1.7mM of hydrogen peroxide, the change in absorbance was measured at 450nm. One unit of MPO activity is defined as that required to convert 1µmole of hydrogen peroxide to water per min/g tissue.

Estimation of Cyclooxygenase (COX):

Enzymatic activities of COX was measured according to the method by Copeland ^22. The assay mixture contained Tris-HCl buffer (100mM, pH 8.0), Hematin (15µM), EDTA (3µM) and 0.75ml of sample. The mixture was preincubated at 25ºC for 15 min and then the reaction was initiated by the addition of H₂O₂and TMPD in a total volume of 3ml. The enzyme activity was measured by estimating the velocity of TMPD oxidation at intervals of 1min for 5min at 603nm. One unit of enzyme activity is defined as the amount of enzyme required to cause a change in TMPD absorbance at 603 nm per min in gram tissue.

Estimation of TNF-α and IL-6 levels-by ELISA:

The levels of intestinal tissue tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were determined using specific enzyme-linked immunosorbent assay kits (Raybio, inc Rat TNF-α ELISA and Raybio, inc Rat IL-6 ELISA).

Kit from TaKaRa Bio Inc. was used to perform TUNEL assay to analyze the status of apoptosis using In situ apoptosis Detection Kit.

Statistical analysis:

All data were expressed as mean±SEM and analyzed using GraphPad Prism (GraphPad Software, Inc. version 5.00). Statistical analysis for multiple comparisons was performed by one-way analysis of variance (ANOVA) with Bonferroni’s corrections. Pvalue <0.05 was considered as significant.

RESULTS:

Sucrase:

Fig.1: Effects of etoposide and whey preparation on the sucrase (µM/min/g tissue) activity in the rat duodenum.

ANOVA significance (Bonferroni’s test, each bar represents mean±SEM, n=06 per group) P<0.0001 and F=18.42 group-1 Vs group-2,* p<0.05,group-2 Vs group-3, group-4, group-5 and group-6, ### p<0.001, One unit= One µmole of glucose produced/min/g tissue

Nitric Oxide:

ET group showed a significant rise in NO levels. Both doses of WP, 100mg and 200mg/Kg body wt and showed a good recovery towards normal levels when compared with ET controls.

Fig. 2: Duodenal nitric oxide level (mg/g tissue) in rats.

ANOVA significance (Bonferroni’s test, each bar represents mean±SEM, n=06) P<0.0053, F=3.543 , group-1 Vs group-2, *p<0.05, group-2 Vs group-3, group-5 & group-6, ## p<0.05 #p<0.05

GSH: A significant decrease in reduced glutathione levels was observed in etoposide treated group compared with normal controls. There was significant recovery in the same in the animals treated with etoposide followed by whey preparation. A similar trend was observed in the sucrase activity in the study groups.

Fig. 3: Duodenal reduced glutathione level (mg/g tissue) in rats. Non-parametric test (kruskullwallies test) and Mann- whitney test P<0.018, group-1 Vs group-2, ** p<0.01, group-2 Vs group-3, group-4, group-5 & group-6, ## p<0.05

Na⁺-K⁺-ATPase:

The tissue Na⁺, k⁺ -ATPase activity was decreased significantly (p=0.001) in all study groups compared to normal control group (group-1). However, pre-treatment (group-5) with whey preparation showed a preventive effect on the loss of Sodium potassium ATPase activity.

Fig.4: Duodenal Na⁺, K⁺ -ATPase activity in rats.

Non-parametric test (Kruskull Wallies test) and Mann- whitney test p<0.0005. One Unit= One µmloe of Pi produced/min/g tissue group-1 Vs group-2, ** p<o.o1, group-1 Vs group-2,## p<0.01, #p<0.05

MPO: Animals exposed to etoposide followed by whey preparation (100 and 200mg/kg b.w) showed a significant decrease in MPO level in the duodenum compared to etoposide treated rats. Animals received whey preparation (100 and 200mg/kg b.w) before and after etoposide showed a significant decrease in MPO level in the duodenum compared to etoposide controls.

Fig.5:Estimation of MPO activity in rats.

ANOVA significance (Bonferroni’s test, each bar represents mean±SEM, n=06 per group) P=0.0001 and F=70506 group-1 Vs group-2, ***p<0.001, group-2 Vs group-3, group-4, group-5 and group-6, ## p<0.01

Cyclooxygenase:

A highly significant elevation in the COX activity was seen in ET group in comparison with normal controls. In all the treated groups, COX activity showed a significant decrease when compared with etoposide group and remained in the normal control levels.

Fig.6: Duodenal COX activity in rats.

ANOVA significance (Bonferroni’s test, each bar represents mean±SEM, n=06 per group) P=0.0001 and F=13.17 One unit= amount of enzyme required to cause a change in TMPD per min group-1 Vs group-2, *** p<0.001, group-2 Vs group-3, group-4, group-5 and group-6, ### p<0.001

TNF-α:There was significant increase in TNF-α concentration of the intestinal tissue among the rats that received chemotherapy compared to normal control rats. TNF-α concentrations decreased significantly in the all the different groups treated with whey preparation, showed a tendency to revert back towards normal.

Fig.7: Duodenal tumor necrosis factor-alpha level (ng/g tissue) in rats.

Non-parametric test (Kruskalwallis test) and Mann- Whitney test P<0.01 group-2 Vs group-3, group-4, group-5 and group-6, ## p<0.05

IL-6: The intestinal tissue IL-6 levels were higher in the etoposide group than in the control (P<0.001). There were significant differences between the etoposide and group 3 and group 4 (P<0.05), whereas the decreased levels of IL-6 in the ET+WP (200) compared to etoposide groups were statistically significant (P<0.01)

Fig.8: Duodenal Interleukin-6 levels (pg/g tissue) in rats.

Non-parametric test (Kruskullwallies test) and Mann- Whitney test P<0.0015 group-1 Vs group-2, * p<0.05

BrDU TUNEL Assay For apoptotic study:

Table -1: % of apoptosis

Groups	Apoptotic cells (%)
Control	28.5
Etoposide (ET)	76.3
ET+whey preparation (WP) (100 mg /kg)	29.5
ET+WP (200 MG/KG)	28.3
WP+ ET+ WP (100 MG/KG)	26.9
WP+ ET+ WP (200 MG/KG)	24.4
WP alone (100 mg/kg)	26.7
WP alone (200 mg/kg)	26.7

DISCUSSION:

Mucositis is a significant and common clinical challenge in many cancer patients during therapy. Cancer Chemotherapy generally results in loss of epithelial cells, which alters the structural and functional integrity of the intestinal mucosa. Recent studies have indicated that inflammation is one of the influencing factor which regulates the activity of gut stem cells during acute epithelial damageThe inflammatory flow that develops on exposure to etoposide could be an important pathway in the pathogenesis of intestinal mucositis. Study on mucositis models reported an increase in the inflammatory mediators of the gastrointestinal tract and are parallel to the amount of the cellular damage along with loss of gut barrier function²³. Chemotherapy also tempts DNA strand break leads to damage or death of the basal epithelial cells through ROS generation directly or by eliciting secondary signals.

Exposure to etoposide resulted in severe stress, which wasreflected by lethargic appearance, decreased intake of food and water in rats. Oxidative stress appears to be a critical mediator of chemotherapy induced intestinal injury. It is of notable prominence because of the role of oxidants in inflammation²⁴, which may lead to variation in mucosal barrier permeability and initiation or agitation of mucosal inflammation and injury²⁵with unusual production of free radicals. These reactive species damage the cells of gastrointestinal tract, which ultimately leads to severe mucositis.In the present study with etoposide induced rat mucositis model, we have observed an increase in the levels of COX and MPO, reflected the development of inflammation during mucositis. Elevated levels of IL-6 and TNF- α reinforces the involvement of inflammatory and immunological processes accompanying mucositis. These changes in inflammatory cytokines in combination with increased levels of free radicals result in mucosal barrier injury or severe mucositis. As stated in other studies, it is evident that oxidative stress is one of the main contributing factors for mucositis, evident by reduced GSH concentration and altered NO levels. Etoposide elicits the formation of free radicals during its metabolism in the body, along with its action on DNA. Free radicals formed in large quantity during the development of mucositis also results in DNA damage and strand breaks. Present study results also showed an increased rate of intestinal cell apoptosis due to chemotherapy again strengthens the engrossment of free radicals. Further, study results also supports the preventive/protective action of the WP during the process of mucositis. Increased free radicals and the developed inflammatory responses could be managed by modifying the action of COX²⁶. That could one of the explanation for the observed normal COX levels after WP administration. Compared to normal control, single dose of etoposide injection resulted in considerable decrease in tissue GSH levels, which is in line with reports of previous studies. GSH, an important contributing influence of redox signaling, as well as one of the essentials during the detoxification of foreign bodies. Adding to these it also influences the rate of cell proliferation, apoptosis and immune function²⁷. Being a rich source of -SH groups, WP fed rats showed normal levels of GSH.Rats fed with a diet with casein hydrolysates, shown positive influence on nitrogen balance and upregulation of mucin genes²⁸. Recent studies have been reported the protective effects exerted by cheese whey protein could be due to the presence of lactalbumin and lactoglobuins along with its ferroxidase activity, which have the capacity to restrict oxidative stress, immunological changes and-inflammatory activities and are closely interconnected during their protective effects^28,29. Sucrose can serve as marker for small intestine permeability. Animal studies have reported that the intestinal permeability to sugars is an effective way to assess the cellular permeability status of Chemotherapy Induced Mucositis^30,31. Compared to controls, etoposide administration significantly reduced the intestinal sucrase levels, which can be a marker of intestinal epithelium damage and indicates the defective absorption process due to change in intestinal morphology and permeability. WP treatment considerably increased the enzyme levels, suggestive of the restoration of mucosal barrier function. WP improved the sucrose levels as compared to ET control. Na⁺, K⁺-ATPase is one of the key enzymes that regulates the transport of ion processes across epithelial cells. Decreased level of this key enzyme in post etoposide exposure mainly reflect the disturbed membrane potential due to altered flux of sodium and potassium ions and thus altered membrane integrity³². Earlier studies have been reported that PGE2-, one of the product of COX shows an inhibitory effect on Na⁺, K⁺-ATPase on various organs^33,34. WP administration significantly restored the above membrane pump. Which also could be due to established intact mucosal membrane. All these properties of whey preparation could be due to their antioxidant and anti-inflammatory properties, which may fine tune the expression of some genes. TNF α has been considered a precarious cytokine in the development of inflammation through modulation of immunological processes³⁵. Hindrance of mucositis could result by endogenous mechanism that antagonizes such inflammatory processes. In the present study, occurrence of inflammatory process is evident with increased MPO and COX levels in the animals exposed to etoposide. Administration of whey preparation indicated that the components of the whey preparation could preserve and restore the normal environment of the intestine to large extent and is at par with earlier studies^14,15. Thus, pre-treatment with anti-inflammatory agents may help to prevent the mucosal barrier loss. Several of the previous studies on animals, and in patients with diabetes as well as with stroke support the vital role of whey proteins as antioxidant enhancers and anti-inflammatory agents –by regulating the expressions of interleukins and TNF-alpha. Thus, it may be a major role in limiting the oxidative stress. It is said that several of the genes related to inflammatory process are regulated through NF-KB- by whey protein combination ³⁶.This could be a mechanism underlying the healing /preventive action of the whey preparation against mucositis observed in the present study. Study showed that 200mg/kg body wt. of WP is better than that of 100mg/kg body wt. Which may need a deeper study to understand the detailed signal mechanisms.

CONCLUSION:

The results of this study showed that the protective effects of orally administered whey protein extract against the etoposideinduced mucositis were mediated through the anti-inflammatory, antioxidant and cytoprotective actions.

CONFLICT OF INTEREST:

The authors declare that they do not have any conflicts or competing interests

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Received on 28.11.2022 Modified on 11.03.2023

Research J. Pharm. and Tech 2023; 16(12):5860-5866.

DOI: 10.52711/0974-360X.2023.00949