Combined ameliorative effect of N-Acetylcysteine and Vitamin C on Cyclophosphamide-Induced Immunosuppression in Balb/C mice via Immunomodulatory, anti-oxidant and downregulation of pro-inflammatory markers

doi:10.21203/rs.3.rs-4350705/v1

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Combined ameliorative effect of N-Acetylcysteine and Vitamin C on Cyclophosphamide-Induced Immunosuppression in Balb/C mice via Immunomodulatory, anti-oxidant and downregulation of pro-inflammatory markers

https://doi.org/10.21203/rs.3.rs-4350705/v1

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Background:

The present study investigated immunomodulatory effects of N -Acetylcysteine and Vitamin C on Cyclophosphamide induced Immunosuppression in Balb/C mouse models.

Methods:

Thirty two Balb/c mice randomly divided into 4 groups. One group of healthy mice acted as normal control. 80 mg/kg/day cyclophosphamide (i.p.) was administered on day 1-3 and 9 for all other groups. From day 4 to 21, the cyclophosphamide treated mice were administered as: saline in positive control, N- Acetylcysteine (200mg/kg), N-Acetylcysteine (200mg/kg) + Vitamin C (200mg/kg). On 22^nd day, thymus and spleen tissues isolated, processed for histopathological examination. Blood samples were analyzed for hematological, biochemical, immunological and parameters.

Results: NAC and Vit C prevented the Cyclophosphamide induced spleen and thymus injury compared with normal control, prevented the decrease in numbers of peripheral RBC, WBC, Hb and platelets, providing supportive evidence for their anti-leukopenia activities; reversed decreased activities of catalase (CAT), glutathione (GSH), GR and rise in MDA. NAC and Vit C also enhanced humoral immune function through increasing level of IgG; significantly decreasing the proinflammatory cytokines-TNF-α and IL-2 levels in serum; produced an increase in circulating serum immunoglobulins in haemagglutinating antibody titre test. Decrease in foot pad edema in delayed-type hypersensitivity reaction test; showed an increase in the neutrophil adhesion to the nylon fibres.

Conclusion: Taken together, our results provide scientific evidence for synergic effect between NAC and Vit C as a co-treatment for enhancing humoral and cellular immune functions, augmenting antioxidant, anti-inflammatory and immunomodulatory effects.

Immunomodulatory effect

N- Acetylcysteine

Vitamin C

hematological

Cellular and humoral response

cytokines

antioxidant activity

By producing an immune response in response to external stimuli like viruses or diseases, the immune system defends the host [1]. Through innate immunity and adaptive immunity, the immune response that takes place in the immune system defends the host from invading pathogens [2]. The enhancement of immune function is crucial for maintaining health [3]. The simplest strategy to increase immunity in the body is to adopt healthier eating habits. There are now much different functional food on the market [4].

In cancer chemotherapy, cyclophosphamide, an alkylating drug, disrupts RNA transcription and DNA replication [5]. Hematopoietic stem cells and dendritic cells directly trigger immunological responses in response to cyclophosphamide [6]. It has a negative impact on lymphocyte proliferation and is frequently used to treat various tumors, malignancies, and autoimmune diseases. Cyclophosphamide treatment that is dose-dependent also improves immunological responses. Despite having chemotherapeutic effects, cyclophosphamide treatment has been linked to a number of negative side effects, including pulmonary fibrosis, cardiac toxicity, genotoxicity in human lymphocytes, and hemorrhagic cystitis in bone marrow transplantation [7–9]. Nevertheless, due to its ability to suppress the immune system, cyclophosphamide is frequently utilized as an anticancer drug [10].

By interacting with and neutralizing oxidizing free radicals, antioxidants stop cell damage. Consequently, they are useful in chemotherapy. Recent data shows that 13 to 87% of cancer patients take antioxidant supplements. Antioxidant N-acetyl cysteine (NAC) works by directly scavenging reactive oxygen species to shield cells from oxidative damage and by promoting glutathione production [11]. NAC also controls liver damage, inflammation of the small intestine, and oxidative stress [12]. In test animals like mice and rats, the NAC reduces the genotoxicity and gonadotoxicity brought on by cyclophosphamide [13, 14].

For humans, vitamin C is a necessary micronutrient having pleiotropic activities associated with its capacity to transfer electrons. It functions as a cofactor for a variety of biosynthetic and gene regulatory enzymes and is a strong antioxidant. By assisting numerous cellular processes of the innate and adaptive immune systems, vitamin C supports immunological defense. Due to its antibacterial and immunomodulatory characteristics, particularly at high doses, vitamin C lowers the risk of infections [15]. Nuclear factor kappa-B (NF-B), a major Proinflammatoy transcription factor important for the regulation of cytokines, chemokines, inflammatory mediators, adhesion molecules, and apoptosis inhibitors, can be inhibited by vitamin C [16]. The benefits of vitamin C can also be seen in the inhibition of TNF- and IL-6 production.

Finding the substances that can lessen Cyclophosphamide's negative effects on various organs is crucial. Through hydrogenating free radical molecules, enzymatic antioxidants like vitamins E and C have the capacity to stop the cascade events brought on by free radicals [17]. Antioxidants, however, use many ways to prevent oxidative damage to organs and to have anticancer effects [18]. Therefore, the purpose of this study was to assess how well Vitamin C and NAC modulated the immunosuppressive effects of Cyclophosphamide in Balb/c mice.

Positive immunosuppressant medications include NAC (acetyl cysteine 600 mg; SEDICO, Cairo, Egypt), cyclophosphamide (CAS No.-6055-19-2), and Endoxan-N by Baxter Oncology. All additional chemicals were purchased from Hi-media in India and Merck, SRL. Dextrose, sodium citrate, sodium chloride, and distilled water were dissolved to create Alsevier's solution. The pH of the solution was then adjusted to 6 using either 0.1 N HCl or NaOH. For the immunization and challenge, sheep red blood cells (SRBCs) were collected in Alsevier's solution (1:1), rinsed in equal amounts of sterile normal saline solution three times, and adjusted to a concentration of 1108 cells/0.1 ml.

Experimental animals

32 Male Balb/c mice were used for the experiment. They were housed in polypropylene box type cages, bedded with rice husk and kept in air-conditioned room, at 23° (± 2°) and RH 50 ± 5%, were fed with a pelleted diet (Amruth Feeds, India) and water ad libitum. A 12:12, light: dark cycle was followed. Racks were positioned in a room so as to optimize air exchange. Six to eight week old animals with average body weight of 18–30 g were used for the experiments. Eight animals were used for each treatment and control group. Care and experimental procedures were conducted as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals, India. All groups of animals were kept under an absolute hygienic condition as per the recommended procedures by fulfilling the necessary ethical standards. In vivo animal studies were conducted after obtaining the prior approval from Institutional Animal Ethics Committee.

Experimental Design

After being adapt to environment for one week, these mice were randomly divided into 4 groups consisting of 8 mice each. One group of healthy mice were used as a normal control group (NC) treated once daily with the physiological saline solution by oral route for 21 days. From days 1 to 3 and 9, the other groups were given 80 mg/kg/day cyclophosphamide (i.p.) [19]. From day 4 to 21, the Cy-treated mice were administered as the following: the physiological saline solution in positive control group, Animals of third group was treated with N- Acetyl cysteine (200mg/kg) administered orally. Animals of fourth group were treated with N-Acetyl cysteine (200mg/kg) along with Vitamin C (200mg/kg) administered orally. On 22nd day, the thymus and spleen tissues were isolated and stored in buffered formalin and further processed for histopathological examination. The blood samples are collected into heparinized tubes and were analyzed for hematological, biochemical and immunological parameters. The initial & final body weight of the animals of each group were measured and recorded before, during & after the treatment.

Measurement of physiological parameters

Prior to the experiment, on the 7th and 14th days, and on the 21st day, each mouse's weight was noted. At the moment of sacrifice, the weight of the organs like the spleen and thymus was also noted.

Effect on Mouse Spleen and Thymus Indices

The mice were weighed, decapitated, and the thymus and spleen of each mouse were taken and weighed right away to determine the immune organ indices using the formula below. This was done 24 hours after the previous dose.

Index (mg/g) = weight of spleen or thymus/ body weight x 100%

HE Staining of Spleen and Thymus Tissues

Spleen and thymus tissues were removed from mice, fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4-mm sections. Following deparaffinization, hematoxylin and eosin staining was performed on these sections. The slices were mounted for imaging after being dehydrated.

Measurement of hematological parameters

Using a haematology analyzer (Unitron Bio-Medicals, India), haematological indices such as total RBC, WBC, platelet, and Hb count were revealed to be essential components of the immune system. Wright's staining procedure was used to determine the differential WBC count.

Determination of Immunoglobulin and Cytokines in Serum by ELISA

Whole blood was centrifuged at 3500 rpm for 10 minutes at 4 C to prepare serum samples. According to the directions on the ELISA kit, the levels of immunoglobulin (IgG) and cytokines for TNF- and IL-2 were evaluated in the serum.

Measurements of Antioxidant Enzyme Activities and the MDA Level

Using a Teflon homogenizer and phosphate buffer saline (50 mM, pH 7.4), spleen tissues (100 mg) were produced. After centrifuging the homogenate at 12,000 rpm for 10 min at 4°C, the supernatants were utilised to assay the activity of GR, CAT, GSH, and MDA. Using assay kits from the Nanjing Jiancheng Bioengineering Institute in Nanjing, China, all biochemical parameters were determined in accordance with the manufacturer's instructions.

Measurement of immunological parameters

Haemagluttination antibody titre Assay

The usual procedure for the hemoglutination test was used [20]. Serum samples were serially diluted twice in 96 well U-bottomed haemagglutination microtitre plates with 100 µl of phosphate-buffered saline. Then, 50 l of 1% SRBC suspension was poured into each well. After a gentle shake and two-hour incubation at room temperature, the plates were checked visually for agglutination. The antibody titre was defined as the serum concentration at which there was a visible haemagglutination [21].

Determination of Delayed-Type Hypersensitivity Response

In order to test the mice on day 14 of treatment, 20µl of 5×10⁹ sheep red blood cells were subcutaneously injected into the hind right foot pad. Vernier calipers were used to measure the left foot's foot thickness both before and 24 hours after the challenge. The differences between the thicknesses of the mice's right and left footpads before and after the challenge were utilized to determine the responsiveness of the antigen to delayed type hypersensitivity reactions, and they were reported as a mean percent increase in thickness [22].

Neutrophil Adhesion Test [23]

The retro-orbital plexus was punctured on the twenty-first day of medication therapy to obtain blood samples, which were then analyzed for total leucocyte counts (TLC) and differential leucocyte counts (DLC). Blood samples were treated with nylon fibers for 15 minutes at 37°C after the initial counts. The total leukocyte count and differential leucocyte count were once more determined for the incubated blood samples. The product of Total Leucocyte Count and percentage Neutrophil gives Neutrophil index (NI) of blood sample. Percentage of neutrophil adhesion was calculated as shown below

Neutrophil Adhesion (%) = {(Niu- Nit)/Niu} × 100

Where,

Niu = Neutrophil index of untreated blood sample and

Nit = Neutrophil index of treated blood sample.

NI = TLC x %NEUTROPHIL

Statistical analysis

Data are showed as mean ± SEM. The statistical significance of the differences between various groups were determined by either a Student t-test or an ANOVA analysis for multiple comparisons by Prism version 5.0 (Graph Pad Software, Inc.). Values of p < 0.05 were considered statistically significant.

Body weights and Immune organ Index

There was a remarkable decrease in body weights in the positive control group after challenging. While administrated with NAC, NAC + Vit C, the weights of the mice recovered signiﬁcantly (Fig 1). As shown in Fig 2, compared with the normal group, the spleen and thymus indices signiﬁcantly decreased (p<0.01) in the Cyclophosphamide treated mice. After administrated with NAC, NAC + Vit C, there was a signiﬁcant recovery of spleen and thymus indices compared with the positive control group (p < 0.05 or p < 0.01). The degree of recovery in the spleen was more pronounced than that in the thymus.

Hematological parameters

We looked at how NAC and NAC + Vit C affected the RBC, WBC, and platelet counts in the mice treated with cyclophosphamide in order to assess the hemopoietic function. Cyclophosphamide significantly reduced the counts of RBC, WBC, Hb, and platelets from peripheral blood, as seen in Tables 1 and 2, as compared to the normal group (p 0.01). The most severe decrease was in leukocyte count. NAC and NAC + Vit C markedly increased the lowered RBC, WBC, Hb, and platelet levels. WBC and RBC counts were practically restored to normal levels after treatment with co-administration of NAC + Vit C (p 0.01).

Effect on Immunoglobulin Levels in Serum

As shown in Fig 3, we found that the production of IgG (p < 0.01) in the sera was noticeably suppressed in the positive control compared with the normal control. NAC (p < 0.05) and NAC + Vit C (p < 0.01) were found to signiﬁcantly prevent the decline in the levels of IgG.

Changes in Proinflammatoy biomarkers

We found that highest concentrations of TNFα and IL-2 were in the cyclophosphamide treated group (p < 0.01) and the lowest were in the control group. In the NAC (p < 0.05), NAC+ Vit C group (p < 0.01), there is a signiﬁcant decrease in proinﬂammatory cytokines (Fig 4).

Delayed-Type Hypersensitivity Response

Large influxes of nonspecific inflammatory cells, including the macrophage, are what define a DTH reaction. When antigen activates sensitised T cells, a type IV hypersensitivity reaction occurs. Several cytokines, including as interleukin-2, interferon, macrophage migration inhibition factor, and tumour necrosis factor, are secreted when antigen is delivered by the right antigen-presenting cells to activate T cells [21]. As a result of the T-cell-mediated TH response to sheep RBC, test groups' paw volumes were less than those of the control group. The greatest reduction in paw volume was seen in the NAC + Vit C treated group (p 0.01) (Fig. 5).

Haemagglutinating Antibody Titre

Both primary and secondary HA titre value was significantly increased in animals that received vaccination along with the NAC treatment (p < 0.05) as compared to animals that received vaccination alone (P < 0.01). Co-administration of NAC + Vit C the antibody titre improved significantly (p < 0.01) (Fig 6).

Neutrophil adhesion test

Incubation of blood with nylon fibres (NF) produced a decrease in the neutrophil counts due to adhesion of neutrophils to the fibres in positive control group (p < 0.01). Treatments of NAC (p < 0.05) and NAC + Vit C (p < 0.01) showed significant increase in the neutrophil adhesion when compared to control. The co-administration of NAC + Vit C was found to be more effective than NAC alone. There was also rise in neutrophil count in untreated blood of all treatment groups (Fig 7).

Effect on Redox Imbalance in Cy-Treated Mice

We measured the activity of antioxidant enzymes like GR, GSH, and CAT as well as the level of MDA in the mouse spleens of all the groups tested in order to further study the protective mechanisms of NAC and NAC + Vit C in the Cyclophosphamide treated mice. The positive control group's GR, CAT, and GSH activities were significantly lower than those of the normal group (Fig. 8 A, B, C, and D), however treatment with NAC (p 0.05) and NAC + Vit C (p 0.01) completely reversed all of them. Additionally, varied degrees of NAC + Vit C therapy successfully reduced the rise of MDA brought on by cyclophosphamide.

Histological Observations of Spleen and Thymus

In the current investigation, an optical microscope was used to analyse the histomorphology of the spleen and thymus. The spleens and thymuses of healthy control mice showed large, densely packed, highly pigmented splenocytes and thymocytes with a clear nucleus (Figs. 9.1 and 10.1). Splenocyte and thymocyte counts in the positive control group were lower (Figs. 9.2 and 10.2). In addition, the HE stained histopathology images showed necrotic regions devoid of cell structures. The spleen and thymus cells were compactly packed in good order, with distinct nuclei and less intercellular space, which were similar to those in the normal group, in the NAC (Fig 9.3 and 10.3) and NAC + Vit C treated groups. The results indicate that treatment with NAC + Vit C (Fig 9.4 and 10.4) signiﬁcantly prevented the damage to the spleen and thymus in the mice induced by Cyclophosphamide.

This interventional study examined the effects of vitamin C and N-acetyl cysteine in minimizing the negative effects of cyclophosphamide because of the side effects associated with this drug, particularly in the immune system. In the present investigation, we discovered that the combination of vitamin C and NAC prevented animal body weight loss, which may have been a result of a protective effect against cyclophosphamide toxicity.

Due to immune system injury, immunosuppression is a condition in which the immune system is temporarily or permanently dysfunctional [24]. Finding immunomodulatory drugs with roots in medicinal plants to treat immunosuppressive illnesses is a field of considerable attention.

This interventional study assessed the effects of vitamin C and N-acetyl cysteine in minimising the negative effects of this substance due to the side effects documented for cyclophosphamide, notably in the immune system.

In the current investigation, we discovered that using vitamin C and NAC together prevented the weight loss of the animals, which may have been a result of a protective effect against cyclophosphamide toxicity. Due to immune system impairment, immunosuppression, which can be either temporary or permanent, can increase an organism's susceptibility to infections [24]. There is a lot of interest in finding immunomodulatory drugs with roots in medicinal plants to treat immunosuppressive illnesses.

Cyclophosphamide is a well-known cytotoxic medication that is frequently used to treat tumours [25]. Its cytotoxic effects are caused by chemically reactive metabolites that cross-link proteins and DNAs by alkylating them [26]. According to reports, cyclophosphamide can impair immune function by causing immunological organ atrophy, weight loss, and an imbalance of different leukocytes in mice's peripheral blood [27, 28]. Therefore, immune organ indices, blood cell counts, and spleen and thymus morphology were investigated in this study. Our results show that the corresponding therapies stopped Cyclophosphamide-induced declines in the thymus and spleen indexes. Important immunological organs that play a vital role in preserving immune homeostasis are the thymus and spleen [29].

Figures 9 and 10 from the HE staining of the spleen and thymus demonstrate that NAC, NAC, and vitamin C were able to prevent Cyclophosphamide's damaging effects on the organs' ability to function as an immune system suppressor. NAC, NAC + Vit C supplementation also resulted in higher total RBC, WBC, Hb, and platelet counts in mice compared to those treated with cyclophosphamide, supporting its anti-anemic, anti-leukopenic, and anti-thrombocytopenic actions. The activation of macrophages, which emit numerous chemicals like colony stimulating factor and interleukin 1, may help to counteract the myelosuppression brought on by cyclophosphamide.

The primary adverse consequence of chemotherapeutic medicines, including cyclophosphamide, is oxidative DNA damage in cells [30]. Through the stimulation of immunity, oxidative stress and the generation of inflammatory cytokines can be controlled, which may aid in the maintenance of a disease-free state [31]. After exposure to cyclophosphamide, the redox balance is said to be disrupted by oxidative stress [32]. It is well recognized that oxidative molecules like MDA and antioxidative enzymes like GR, GSH, and CAT are thought of as common markers of tissue antioxidant state [33].

According to some reports, NAC, NAC + Vit C either functions as a redox regulating agent or a free radical scavenger. Additionally, research is being done on the free radical scavenging activity and antioxidant potential of NAC and Vitamin C on c-radiation-induced protein carbonylation, DNA damage, and liposomal lipid peroxidation.

According to our findings, the injection of NAC and Vitamin C reversed the drop in GR, CAT, and GSH activities as well as the rise of MDA in the cyclophosphamide-treated mouse spleens. Our results show that NAC, NAC + Vit C effectively defended against the oxidative stress injury brought on by cyclophosphamide by increasing antioxidative enzymes and decreasing oxidative enzymes.

A variety of models were used in the current study, each of which offers details on the impact on various immune system components. Against pathogenic organisms, infection of foreign grafts, tumor immunity, and DTH reactions, CMI responses are essential for defense. To assess the impact of NAC, NAC + Vit C in CMI reaction in the current study, DTH model was employed. In order to demonstrate the stimulatory effect of NAC, NAC + Vit C in CMI, mice's increased paw edema in response to T-cell-dependent antigen was observed.

The HA titre test was used to examine how NAC and NAC with Vitamin C affected the humoral immune system. IgG and IgM are the main immunoglobulins that are involved in complement activation, opsonization, the neutralization of poisons, and other humoral immune responses. Antibody molecules, which are produced by B lymphocytes and plasma cells, are essential for humoral immune responses. [34] The findings of the HA titre test demonstrated that both treatments considerably raised the levels of circulating antibodies prior to pretreatment. This model makes it evident that the antioxidant treatments NAC and Vit C together boost the humoral immune system as well.

The neutrophil adhesion test is frequently used to evaluate how various test medications affect cell-mediated immune responses. The migration of cells through blood arteries and the quantity of neutrophils that arrive at the site of inflammation are both indicated by neutrophil adherence to nylon fibres. Neutrophil adherence to the fibre was shown to be improved by both the NAC therapy alone and in combination with vitamin C. This could be as a result of the neutrophils' surface-located 2 integrins, which are upregulated and allow them to firmly attach to the nylon fibres. [35]

NAC functions as an antioxidant by producing glutathione and assisting in the reduction of free radical buildup. Vitamin C's antioxidant characteristics, which enable it to participate in redox reactions and contribute electrons while also becoming oxidised, are thought to be responsible for its immunomodulating effects. Vitamin C is required for the cellular and humoral immune responses at the bare minimum concentration; at higher concentrations, it also has chemotactic, lymphoproliferative, and natural killer cell-inducing properties. As studies show lower vitamin C concentrations in IBD, vitamin C plays a crucial antioxidant role in the gut barrier function and antioxidation.

The study's findings demonstrated the synergistic effects of vitamin C and NAC, which have significant antioxidant characteristics and cause weight growth, a reduction in MDA levels, and an increase in total antioxidant activity and GSH antioxidants in the case of cyclophosphamide. These findings imply that NAC and Vitamin C might be used as ingredients in healthy functional foods to boost immunity and reduce inflammation. We propose for clinical trials to test the benefits, disadvantages, mechanism of action of the use of vitamin C and NAC medically in immunocompromised patients and whether such changes are clinically significant.

Fig. Figure

i.p. intra peritoneal

kg kilogram

mg milligram

ml millilitre

p.o. Per oral

w/w weight/ weight

w/v weight/ volume

NO nitric oxide

GSH reduced glutathione

MDA malondialdehyde

CAT catalase

SOD superoxide dismutase

GPx glutathione peroxidase

TNF-α tumor necrosis factor alpha

Authors’ contributions

BVSL and BKV wrote the manuscript. BVSL and BKV designed and performed the study. All authors participated in data analysis and approved the final manuscript.

Funding

There was no specific fund for this study.

Availability of data and materials

Data analyzed and used for this manuscript are available within the manuscript.

Ethics approval and consent to participate

The Institutional Animal Ethics Committee (Reg no: MRCP/IAEC/2008/1217/05) authorized the protocols for every pharmaceutical trial.

Consent for publication

Not applicable.

Competing interests

The authors declare no conflict of interest.

Author details

1. Department of Pharmacology, School of Allied and Healthcare Sciences, Malla Reddy University, Hyderabad, Telangana, India. 2. Department of Pharmacology, School of Pharmacy, KPJ Healthcare University College, Nilai, Malaysia.

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Table 1: Immunomodulatory effects of NAC, NAC + Vit C in relative change in haematological parameters (Total WBC and DLC) in Cyclophosphamide induced immunosuppression in Balb/C mice.

Group	Total WBC Count(Cells/cu.mm)	Differential Leucocyte Count
Group	Total WBC Count(Cells/cu.mm)	Easinophils	Basophils	Neutrophils	Lymphocytes	Monocytes
Control	6416±216	175±12.3	58.3 ± 3.8	1491.6±71.2	4800±96.6	199±5.8
Cyclophosphamide Treated	4333±135^	120±4.65^	22.1±2.2^	833.3±44.09^	3405±192.7^	72.5±3.09^
N-Acetylcysteine	5333±71*	135±4.83*	33.1±3.0*	1316.6±60.1*	4025±116*	165±6.3*
N-Acetylcysteine + Vitamin C	5550±117**	159±4.16**	47.5±3.8**	1450±76.3**	4316±122**	180±5.5**

Values are shown as mean ± SEM (n = 8). ^ p < 0.01 vs. the NC group, and *p < 0.05 ** p < 0.01 vs. cyclophosphamide treated group.

Table 2: Immunomodulatory effects of NAC, NAC + Vit C in relative change in hematological parameters in Cyclophosphamide induced immunosuppression in Balb/C mice.

Group	Red Blood Cells (Millions/Cu.mm)	Platelets(×10⁴cu.mm)	Haemoglobin (g/dl)
Control (Normal Saline)	7.69±0.19	2.9±0.06	12.08±0.29
Cyclophosphamide Treated	4.7±0.14^	2.0±0.02^	8.1±0.08^
N Acetylcysteine	8.5±0.12*	2.35±0.05*	12.7±0.33*
N Acetylcysteine + Vitamin C	9.1±0.19**	2.7±0.07**	13.5±0.2**

Values are shown as mean ± SEM (n = 8). ^ p < 0.01 vs. the NC group, and *p < 0.05 ** p < 0.01 vs. cyclophosphamide treated group.

No competing interests reported.

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Combined ameliorative effect of N-Acetylcysteine and Vitamin C on Cyclophosphamide-Induced Immunosuppression in Balb/C mice via Immunomodulatory, anti-oxidant and downregulation of pro-inflammatory markers

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Abstract

Figures

Background

Materials and Methods

Experimental animals

Experimental Design

Measurement of physiological parameters

Effect on Mouse Spleen and Thymus Indices

HE Staining of Spleen and Thymus Tissues

Measurement of hematological parameters

Determination of Immunoglobulin and Cytokines in Serum by ELISA

Measurements of Antioxidant Enzyme Activities and the MDA Level

Measurement of immunological parameters

Haemagluttination antibody titre Assay

Determination of Delayed-Type Hypersensitivity Response

Neutrophil Adhesion Test [23]

Statistical analysis

RESULTS

Neutrophil adhesion test

Discussion

Conclusion

Abbreviations

Declarations

References

Tables

Additional Declarations

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