Several studies reported the direct involvement of oxidative stress in the pathogenesis of DN [
2,
36,
37]. Excess generation of ROS leads to the severe cellular oxidative damage viz. LPO derivatives and this weaken the antioxidant machinery system (SOD, CAT, and GPx) [
37,
38] in the kidney of DN rats. Kidney cells are more vulnerable to ROS mediated oxidative damage because of a high rate of oxygen consumptions [
39]. Previously, we have reported that the STZ induced DN rats had clinical characteristics of increase proteinuria, urea and creatinine along with decreased creatinine clearance showed successful induction of DN and PTY-2r treatment significantly ameliorates these changes in a dose-dependent manner [
22]. The present study is a continuous exploration of nephroprotective effect and underlying mechanism of PTY-2r in STZ induced DN rats. We observed the significant increase in oxidative stress markers i.e. ROS & LPO along with decreased ROS scavenging enzymes (SOD, CAT & GPx) in the kidney of DN rats. This is in accordance with the earlier reports, where inverse correlations between oxidative stress and activity of antioxidant enzymes have been reported [
40]. The PTY-2r treatment significantly suppressed the increased production of ROS and LPO as compared to DN control rats and significantly raised the activity of antioxidant enzymes, in a dose-dependent manner, suggesting a significant antioxidant potential of PTY-2r. This antioxidant property of PTY-2r could be due to presence 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (17.08%) and n- hexadecanoic acid which are identified through GC-MS analysis were reported as a strong antioxidant [
41]. PTY-2r extract also rich in flavone, flavonoids, and polyphenols as shown in the phytochemical analysis of PTY-2r extract (Table
1). Thus, PTY-2r showed nephroprotective potential by reducing the oxidative stress through the restoration of antioxidant enzymes similar to other antioxidant agents like curcumin [
42], resveratrol [
43], berbarin [
44] etc.
It has been reported that ROS-mediated oxidative stress also causes the disturbance in the balance between the pro-apoptotic (Bax) and antiapoptotic (Bcl-2) proteins, leading to an excess production of pro-apoptotic protein, which is susceptible to apoptosis [
4]. The ratio of Bax and Bcl-2 protein is an important factor which determines cell survivability versus cell death [
45]. The increased Bax/Bcl-2 ratio may cause damage to the mitochondrial membrane integrity which induces the cytochrome c (Cyt-c) release from the mitochondrial inner membrane (MIM). The leakage of Cyt-c from MIM may lead to activation of active Caspases-3 which may further activate caspase-9 [
46]. Active Caspase-3 belongs to the cysteine proteases family and is involved in various forms of apoptotic pathways. It cleaves several substrates, notably DNA repairing enzymes viz. PARP [
47]. Poly (ADP) ribose polymerase (PARP) is a nuclear enzyme responsible for ribosylation of nuclear enzymes and chromosomal proteins [
48]. The formation of breaks in DNA strands during apoptosis is a stimulus of activation of the PARP-1. Thus PARP-1 plays a critical role in DNA repair and apoptosis. PARP-1 is a substrate for Caspases, particularly Caspase-3 [
49]. Active Caspase-3 induced PARP-1 cleavage induces cellular disassembly, thus it acts as a specific marker for apoptosis [
50]. Oxidative stress-induced apoptosis was evaluated by using the Bcl-2 to Bax ratio, cleaved PARP, and active Caspase-3 in the kidney of DN rats. Here, we found the diminished Bcl-2 expression and increased expression of Bax, active Caspase-3 and cleaved PARP-1 in the kidney of DN rats. However, the treatment with PTY-2r effectively ameliorated the changes in Bax and Bcl-2 family proteins and inhibited the cleavage of PARP-1 and activation of Caspase-3, showed its antiapoptotic potential. The antiapoptotic property of PTY-2r could be attributed to the presence of 5-Hydroxymethylfurfural, because in literature this compound is known for its antiapoptotic potential [
51,
52]. Apoptosis of podocytes is the main contributor for albuminuria [
53], although we have not checked, whether podocyte specifically dying but we are anticipating that podocyte death is because of significant changes in pro-apoptotic and antiapoptotic markers in the glomerular region of the kidney of DN rats. Thus, its prevention has been shown to reduce albumin excretion. Our results also showed that PTY-2r significantly decreased the urinary albumin excretion, which is a clinical characteristic of progression of DN. Previously, there are several reports stating that antioxidants have the ability to suppress the apoptosis [
54,
55]. Cell apoptosis involves ROS as a critical intermediate messenger in its signalling cascade and antioxidants are responsible for scavenging of ROS thus inhibiting apoptosis [
55]. This is also confirmed by our study that the antioxidant potential of PTY-2r is not only capable of suppressing oxidative stress but also inhibits the apoptotic markers in the kidney of DN rats.