Our previous studies found a significant effect of NEs containing BV on improving arthritis in an animal model [13, 14]. We did not know how much BV could have adverse effects after entering the body nor did we know its benefits. Thus, this study aimed to identify the side effects of NEs containing BV on the body by measuring the biochemical and hematological parameters of arthritis rats. We also measured these changes in the CIA animal model on the full inflammation day that were not treated.
The study showed that on day 7 of induction of the CIA model, as the maximum inflammation day, biochemical parameters, including Glu (p ≤ 0.01), Ch (p ≤ 0.001), Ur (p ≤ 0.05), and AST (p ≤ 0.05), had significantly increased, and only, TG (p ≤ 0.05) had significantly decreased. Among the hematological parameters on day 7, only WBCs (p ≤ 0.001) and %Neu (p ≤ 0.001) had significantly increased. The other biochemical and hematological parameter changes in the CIA were insignificant from day 7 until the end of two weeks of the treatment (day 21, see the blank group). Nevertheless, the %Neu was still high compared to day 0 (p ≤ 0.001).
Zainab et al. reported that WBCs and Plts had increased in formaldehyde-induced arthritis, which agrees with our results in the CIA model. Also, she reported that RBCs, Hb, and HCT diminished, which was similar to the results of the present study [22]. Increasing WBCs, especially %Neu in the blood of CIA model rats on day 7, can be due to calling WBCs to the inflammation site in the rat's paw by some inflammatory cytokines after induction of arthritis [23]. In this regard, inflammatory mediators, immune cells, cytokines, and chemokines play a critical role in dyslipidemia, which hence cause metabolic effects in arthritis [24]. An increase in Chol and TG has been reported in formaldehyde-induced arthritis, while in the CIA model, only Chol increased and TG decreased [22]. Thus, there was a disorder of metabolism and dyslipidemia in arthritis and animal models of CIA.
BV has several anti-inflammatory and anti-arthritic properties [11]. It has a variety of components with pharmacological and biochemical activities and can destroy location cells or enter the bloodstream and lyse other blood cells, especially RBCs, as it passes through the skin [7]. Studies have shown that BV can affect the body's metabolic systems to change their function, such as reducing blood glucose [25] and affecting the blood lipids [26]. Although some unfavorable effects of BV can be neutralized by albumin in the bloodstream [27], it can affect organs such as islands of Langerhans of the pancreas [17], liver [15], kidney [16], adrenal glands, and their hormones [18]. Imani et al. showed that BV injection decreased Glu, total Chol, and Tg levels in treated diabetic rats [28]. It has been shown that serum levels of Chol and TG decreased in formaldehyde-induced arthritis male rats receiving injection BV, which was similar to our results only about Chol [22].
The study indicated that the blood levels of Glu decreased at the end of treatment and were similar to the base level on day 0 for all groups, except for the BV control, which was lower than the base level. It suggests that topical BV can reduce the blood Glu, but this effect was not found for Chol and TG. It was reported that BV therapy is associated with multiple causes of kidney injury and hepatotoxicity [29]. Measuring Ur and Cr blood levels is a simple way to assess kidney injuries [30]. Ur and Cr increased in the CIA model on day 7. At the end of treatment on day 21, Ur and Cr dose-dependently dropped in all groups, compared to day 0 and even day 7. Although these increases were not significant, all groups were, especially the blank group. It increased over 21 days, which is more evident for the blank group. AST, ALT, and ALP are the most common tests used to diagnose liver disorders [31, 32]. Our findings express which blood levels these enzymes have increased on day 7 compared to day 0, although only significant for AST (p ≤ 0.05). They decreased in a dose-dependent manner for BV-NEs at the end of treatment. These results align with Nicodim et al.'s investigations about rats with RA induced by Freund complete adjuvant treated with BV [33].
Parameters of Ca and Ph were elevated in CIA rats on day 7, and their reduction in all treatment groups on day 21. The level of Ca significantly decreased dose-dependent manner for all BV-NEs and BV control at the end of treatment. Therefore, BV could reduce the level of Ca and Ph after possible entry into the bloodstream, which was recently reported by Kang [34]. Also, in another study, the reducing effect of melittin of BV on Ca of the equine skeletal muscle has been shown [35] due to the interaction of melittin as a 26-residue peptide with Ca [36].
BV has an anti-inflammatory effect on pro-inflammatory cytokines and is expected to lower WBC and %Neu [19]. A decrease in the total number of WBCs at the end of treatment in all groups compared to day 7 is related to reducing inflammation in the animal. There are more reductions in the positive and BV controls, as well as BV-NEs in comparison with blank and negative control groups, due to BV's immunosuppressive effects [22]. After an increase of %Neu on day 7, it significantly decreased on day 21. The highest %Neu depletion was in the BV-NE-9.37 group. However, this difference was insignificant, and the %Neu did not return to the base level of day 0. In CIA rats treated with BV control and BV-NEs, WBC and the %Neu had diminished, which agrees with the study of formaldehyde-induced arthritis in male rats [22].
On the maximum inflammation day in the CIA rats, anemia could be seen as decreased blood levels of RBCs, Hb, and Hct [19]. After two weeks of treatment, significant changes were not seen by topical BV-NEs, but a dose-dependent increase was observed for BV-NEs (specially BV-NE-9.37 and BV-NE-18.75) about parameters of RBC, Hb, Hct, which could be due to BV improvement of circulation of blood in the micro blood vessels, as well as its role in the stimulation of building erythrocytes [37]. Indices of RBCs, including MCV, MCH, MCHC, and RDW, help elucidate the etiology of anemias [38]. According to the study, there were no crucial changes in these indices on days 7 and 21 which seems that they were less affected by inflammation of CIA and treatment of BV-NEs.
Plts have increased on days 7 and 21; however, these changes were insignificant. An increase of Plts on maximum inflammation day and at the end of treatment in the groups indicates inflammation in RA's early and secondary stages [33]. The critical role of Plts is known in inflammation and immune responses. They actively participate in leukocyte recruitment, especially neutrophils, and host defense regulation in response to exogenous pathogens. This increase in Plts can also be due to the significant effect of cytokines, especially IL-6, which affects the maturation of generated cells of Plts and causes increased production of Plts [39].